Kah‐Leong Lim

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) cause late-onset Parkinson's disease indistinguishable from idiopathic disease. The mechanisms whereby missense alterations in the LRRK2 gene initiate neurodegeneration remain unknown. Here, we demonstrate that seven of 10 suspected familial-linked mutations result in increased kinase activity. Functional and disease-associated mutations in conserved residues reveal the critical link between intrinsic guanosine triphosphatase (GTPase) activity and downstream kinase activity. LRRK2 kinase activity requires GTPase activity, whereas GTPase activity functions independently of kinase activity. Both LRRK2 kinase and GTPase activity are required for neurotoxicity and potentiate peroxide-induced cell death, although LRRK2 does not function as a canonical MAP-kinase-kinase-kinase. These results suggest a link between LRRK2 kinase activity and pathogenic mechanisms relating to neurodegeneration, further supporting a gain-of-function role for LRRK2 mutations.

It is widely accepted that the familial Parkinson's disease (PD)-linked gene product, parkin, functions as a ubiquitin ligase involved in protein turnover via the ubiquitin-proteasome system. Substrates ubiquitinated by parkin are hence thought to be destined for proteasomal degradation. Because we demonstrated previously that parkin interacts with and ubiquitinates synphilin-1, we initially expected synphilin-1 degradation to be enhanced in the presence of parkin. Contrary to our expectation, we found that synphilin-1 is normally ubiquitinated by parkin in a nonclassical, proteasomal-independent manner that involves lysine 63 (K63)-linked polyubiquitin chain formation. Parkin-mediated degradation of synphilin-1 occurs appreciably only at an unusually high parkin to synphilin-1 expression ratio or when primed for lysine 48 (K48)-linked ubiquitination. In addition we found that parkin-mediated ubiquitination of proteins within Lewy-body-like inclusions formed by the coexpression of synphilin-1, alpha-synuclein, and parkin occurs predominantly via K63 linkages and that the formation of these inclusions is enhanced by K63-linked ubiquitination. Our results suggest that parkin is a dual-function ubiquitin ligase and that K63-linked ubiquitination of synphilin-1 by parkin may be involved in the formation of Lewy body inclusions associated with PD.

Mutations in parkin, a ubiquitin ligase, cause early-onset familial Parkinson's disease (AR-JP). How parkin suppresses parkinsonism remains unknown. Parkin was recently shown to promote the clearance of impaired mitochondria by autophagy, termed mitophagy. Here, we show that parkin promotes mitophagy by catalyzing mitochondrial ubiquitination, which in turn recruits ubiquitin-binding autophagic components, HDAC6 and p62, leading to mitochondrial clearance. During the process, juxtanuclear mitochondrial aggregates resembling a protein aggregate-induced aggresome are formed. The formation of these "mito-aggresome" structures requires microtubule motor-dependent transport and is essential for efficient mitophagy. Importantly, we show that AR-JP-causing parkin mutations are defective in supporting mitophagy due to distinct defects at recognition, transportation, or ubiquitination of impaired mitochondria, thereby implicating mitophagy defects in the development of parkinsonism. Our results show that impaired mitochondria and protein aggregates are processed by common ubiquitin-selective autophagy machinery connected to the aggresomal pathway, thus identifying a mechanistic basis for the prevalence of these toxic entities in Parkinson's disease.

Although ubiquitin-enriched protein inclusions represent an almost invariant feature of neurodegenerative diseases, the mechanism underlying their biogenesis remains unclear. In particular, whether the topology of ubiquitin linkages influences the dynamics of inclusions is not well explored. Here, we report that lysine 48 (K48)- and lysine 63 (K63)-linked polyubiquitination, as well as monoubiquitin modification contribute to the biogenesis of inclusions. K63-linked polyubiquitin is the most consistent enhancer of inclusions formation. Under basal conditions, ectopic expression of K63 mutant ubiquitin in cultured cells promotes the accumulation of proteins and the formation of intracellular inclusions in the apparent absence of proteasome impairment. When co-expressed with disease-associated tau and SOD1 mutants, K63 ubiquitin mutant facilitates the formation of tau- and SOD-1-positive inclusions. Moreover, K63-linked ubiquitination was found to selectively facilitate the clearance of inclusions via autophagy. These data indicate that K63-linked ubiquitin chains may represent a common denominator underlying inclusions biogenesis, as well as a general cellular strategy for defining cargo destined for the autophagic system. Collectively, our results provide a novel mechanistic route that underlies the life cycle of an inclusion body. Harnessing this pathway may offer innovative approaches in the treatment of neurodegenerative disorders.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized pathologically by degeneration of catecholaminergic neurons of the substantia nigra pars compacta and locus coeruleus, among other regions. Autosomal-recessive juvenile Parkinsonism (ARJP) is caused by mutations in the PARK2 gene coding for parkin and constitutes the most common familial form of PD. The majority of ARJP-associated parkin mutations are thought to be loss of function-mutations; however, the pathogenesis of ARJP remains poorly understood. Here, we report the generation of parkin null mice by targeted deletion of parkin exon 7. These mice show a loss of catecholaminergic neurons in the locus coeruleus and an accompanying loss of norepinephrine in discrete regions of the central nervous system. Moreover, there is a dramatic reduction of the norepinephrine-dependent startle response. The nigrostriatal dopaminergic system does not show any impairment. This mouse model will help gain a better understanding of parkin function and the mechanisms underlying parkin-associated PD.

Detection of reactive oxygen species (ROS), a hallmark of many pathological processes, is imperative to understanding, detection and treatment of many life-threatening diseases. However, methods capable of real-time in situ imaging of ROS in living animals are still very limited. We herein report the development and optimization of chemiluminescent semiconducting polymer nanoparticles (SPNs) for ultrasensitive in vivo imaging of hydrogen peroxide (H2O2). The chemiluminescence is amplified by adjusting the energy levels between the luminescence reporter and the chemiluminescence substrate to facilitate intermolecular electron transfer in the process of H2O2-activated luminescence. The optimized SPN can emit chemiluminescence with the quantum yield up to 2.30 × 10–2 einsteins/mol and detect H2O2 down to 5 nM, which substantially outperforms the previous probes. Further doping of this SPN with a naphthalocyanine dye creates intraparticle chemiluminescence resonance energy transfer (CRET), leading to the near-infrared (NIR) luminescence responding to H2O2. By virtue of high brightness and ideal NIR optical window, SPN-NIR permits ultrasensitive imaging of H2O2 in the mouse models of peritonitis and neuroinflammation with the minute administration quantity. Thus, this study not only provides a category of optical probes that eliminates the need of external light excitation for imaging of H2O2, but also reveals the underlying principle to enhance the brightness of chemiluminescence systems.

The unusually high MAO-B activity consistently observed in Parkinson’s disease (PD) patients has been proposed as a biomarker; however, this has not been realized due to the lack of probes suitable for MAO-B-specific detection in live cells/tissues. Here we report the first two-photon, small molecule fluorogenic probe (U1) that enables highly sensitive/specific and real-time imaging of endogenous MAO-B activities across biological samples. We also used U1 to confirm the reported inverse relationship between parkin and MAO-B in PD models. With no apparent toxicity, U1 may be used to monitor MAO-B activities in small animals during disease development. In clinical samples, we find elevated MAO-B activities only in B lymphocytes (not in fibroblasts), hinting that MAO-B activity in peripheral blood cells might be an accessible biomarker for rapid detection of PD. Our results provide important starting points for using small molecule imaging techniques to explore MAO-B at the organism level. Monoamine oxidase B is an enzyme that is unusually active in Parkinson’s disease, a feature that makes it an ideal diagnostic biomarker. Here, Li et al. create a highly specific fluorogenic probe that can selectively detect monoamine oxidase B activity in vivoto effectively diagnose Parkinson’s disease.

Defective mitophagy linked to dysfunction in the proteins Parkin and PTEN-induced putative kinase 1 (PINK1) is implicated in the pathogenesis of Parkinson's disease. Although the mechanism by which Parkin mediates mitophagy in a PINK1-dependent manner is becoming clearer, the triggers for this mitophagy pathway remain elusive. Reactive oxygen species (ROS) have been suggested as such triggers, but this proposal remains controversial because ROS scavengers fail to retard mitophagy. Here we demonstrate that the role of ROS in mitophagy has been underappreciated as a result of the inefficiency of ROS scavengers to control ROS bursts after high-dose treatment with carbonyl cyanide m-chlorophenylhydrazone. Supporting this, combinatorial treatment with N-acetyl-l-cysteine and catalase substantially inhibited the ROS upsurge and PINK1-dependent Parkin translocation to mitochondria in response to carbonyl cyanide m-chlorophenylhydrazone treatment. In addition to the chemical mitophagy inducer, overexpression of voltage-dependent anion channel 1 (VDAC1) induced Parkin translocation to mitochondria, presumably by stimulating ROS generation. Similarly, combined N-acetyl-l-cysteine and catalase treatment also suppressed VDAC1-induced redistribution of Parkin. Alongside these observations, we also found that the elevated protein level of PINK1 was not necessary for Parkin translocation to mitochondria. Thus, our data suggest that ROS may act as a trigger for the induction of Parkin/PINK1-dependent mitophagy. In addition, our study casts doubt on the importance of protein quantity of PINK1 in the recruitment of Parkin to mitochondria. Defective mitophagy linked to dysfunction in the proteins Parkin and PTEN-induced putative kinase 1 (PINK1) is implicated in the pathogenesis of Parkinson's disease. Although the mechanism by which Parkin mediates mitophagy in a PINK1-dependent manner is becoming clearer, the triggers for this mitophagy pathway remain elusive. Reactive oxygen species (ROS) have been suggested as such triggers, but this proposal remains controversial because ROS scavengers fail to retard mitophagy. Here we demonstrate that the role of ROS in mitophagy has been underappreciated as a result of the inefficiency of ROS scavengers to control ROS bursts after high-dose treatment with carbonyl cyanide m-chlorophenylhydrazone. Supporting this, combinatorial treatment with N-acetyl-l-cysteine and catalase substantially inhibited the ROS upsurge and PINK1-dependent Parkin translocation to mitochondria in response to carbonyl cyanide m-chlorophenylhydrazone treatment. In addition to the chemical mitophagy inducer, overexpression of voltage-dependent anion channel 1 (VDAC1) induced Parkin translocation to mitochondria, presumably by stimulating ROS generation. Similarly, combined N-acetyl-l-cysteine and catalase treatment also suppressed VDAC1-induced redistribution of Parkin. Alongside these observations, we also found that the elevated protein level of PINK1 was not necessary for Parkin translocation to mitochondria. Thus, our data suggest that ROS may act as a trigger for the induction of Parkin/PINK1-dependent mitophagy. In addition, our study casts doubt on the importance of protein quantity of PINK1 in the recruitment of Parkin to mitochondria.

Mutations in parkin are largely associated with autosomal recessive juvenile parkinsonism. The underlying mechanism of pathogenesis in parkin-associated Parkinson's disease (PD) is thought to be due to the loss of parkin's E3 ubiquitin ligase activity. A subset of missense and nonsense point mutations in parkin that span the entire gene and represent the numerous inheritance patterns that are associated with parkin-linked PD were investigated for their E3 ligase activity, localization and their ability to bind, ubiquitinate and effect the degradation of two substrates, synphilin-1 and aminoacyl-tRNA synthetase complex cofactor, p38. Parkin mutants vary in their intracellular localization, binding to substrates and enzymatic activity, yet they are ultimately deficient in their ability to degrade substrate. These results suggest that not all parkin mutations result in loss of parkin's E3 ligase activity, but they all appear to manifest as loss-of-function mutants due to defects in solubility, aggregation, enzymatic activity or targeting proteins to the proteasome for degradation.

<h2>Abstract</h2> A role for the receptor-like protein tyrosine phosphatase α (PTPα) in regulating the kinase activity of Src family members has been proposed because ectopic expression of PTPα enhances the dephosphorylation and activation of Src and Fyn [1–3]. We have generated mice lacking catalytically active PTPα to address the question of whether PTPα is a physiological activator of Src and Fyn, and to investigate its other potential functions in the context of the whole animal. Mice homozygous for the targeted <i>PTP</i>α allele (<i>PTP</i>α^−/−) and lacking detectable PTPα protein exhibited no gross phenotypic defects. The kinase activities of Src and Fyn were significantly reduced in <i>PTP</i>α^−/− mouse brain and primary embryonic fibroblasts, and this correlated with enhanced phosphorylation of the carboxy-terminal regulatory Tyr527 of Src in <i>PTP</i>α^−/− mice. Thus, PTPα is a physiological positive regulator of the tyrosine kinases Src and Fyn. Increased tyrosine phosphorylation of several unidentified proteins was also apparent in <i>PTP</i>α^−/− mouse brain lysates. These may be PTPα substrates or downstream signaling proteins. Taken together, the results indicate that PTPα has a dual function as a positive and negative regulator of tyrosine phosphorylation events, increasing phosphotyrosyl proteins through activation of Src and Fyn, and directly or indirectly removing tyrosine phosphate from other unidentified proteins.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are currently recognized as the most common genetic cause of parkinsonism. Among the large number of LRRK2 mutations identified to date, the G2019S variant is the most common. In Asia, however, another LRRK2 variant, G2385R, appears to occur more frequently. To better understand the contribution of different LRRK2 variants toward disease pathogenesis, we generated transgenic Drosophila over-expressing various human LRRK2 alleles, including wild type, G2019S, Y1699C, and G2385R LRRK2. We found that transgenic flies harboring G2019S, Y1699C, or G2385R LRRK2 variant, but not the wild-type protein, exhibit late-onset loss of dopaminergic (DA) neurons in selected clusters that is accompanied by locomotion deficits. Furthermore, LRRK2 mutant flies also display reduced lifespan and increased sensitivity to rotenone, a mitochondrial complex I inhibitor. Importantly, coexpression of human parkin in LRRK2 G2019S-expressing flies provides significant protection against DA neurodegeneration that occurs with age or in response to rotenone. Together, our results suggest a potential link between LRRK2, parkin, and mitochondria in the pathogenesis of LRRK2-related parkinsonism.

Inactivation of the tumor suppressor phosphatase and tensin homolog (mutated in multiple advanced cancers 1) (PTEN) is recognized as a major event in the pathogenesis of the brain tumor glioblastoma. However, the mechanisms by which PTEN loss specifically impacts the malignant behavior of glioblastoma cells, including their proliferation and propensity for invasiveness, remain poorly understood. Genetic studies suggest that the transcription factor signal transducers and activators of transcription 3 (STAT3) harbors a PTEN-regulated tumor suppressive function in mouse astrocytes. Here, we report that STAT3 plays a critical tumor suppressive role in PTEN-deficient human glioblastoma cells. Endogenous STAT3 signaling is specifically inhibited in PTEN-deficient glioblastoma cells. Strikingly, reactivation of STAT3 in PTEN-deficient glioblastoma cells inhibits their proliferation, invasiveness, and ability to spread on myelin. We also identify the chemokine interleukin 8 (IL8) as a novel target gene of STAT3 in human glioblastoma cells. Activated STAT3 occupies the endogenous IL8 promoter and directly represses IL8 transcription. Consistent with these results, IL8 is upregulated in PTEN-deficient human glioblastoma tumors. Importantly, IL8 repression mediates STAT3 inhibition of glioblastoma cell proliferation, invasiveness, and spreading on myelin. Collectively, our findings uncover a novel link between STAT3 and IL8, the deregulation of which plays a key role in the malignant behavior of PTEN-deficient glioblastoma cells. These studies suggest that STAT3 activation or IL8 inhibition may have potential in patient-tailored treatment of PTEN-deficient brain tumors.

Mutations in parkin and LRRK2 together account for the majority of familial Parkinson's disease (PD) cases. Interestingly, recent evidence implicates the involvement of parkin and LRRK2 in mitochondrial homeostasis. Supporting this, we show here by means of the Drosophila model system that, like parkin, LRRK2 mutations induce mitochondrial pathology in flies when expressed in their flight muscles, the toxic effects of which can be rescued by parkin coexpression. When expressed specifically in fly dopaminergic neurons, mutant LRRK2 results in the appearance of significantly enlarged mitochondria, a phenotype that can also be rescued by parkin coexpression. Importantly, we also identified in this study that epigallocatechin gallate (EGCG), a green tea-derived catechin, acts as a potent suppressor of dopaminergic and mitochondrial dysfunction in both mutant LRRK2 and parkin-null flies. Notably, the protective effects of EGCG are abolished when AMP-activated protein kinase (AMPK) is genetically inactivated, suggesting that EGCG-mediated neuroprotection requires AMPK. Consistent with this, direct pharmacological or genetic activation of AMPK reproduces EGCG's protective effects. Conversely, loss of AMPK activity exacerbates neuronal loss and associated phenotypes in parkin and LRRK mutant flies. Together, our results suggest the relevance of mitochondrial-associated pathway in LRRK2 and parkin-related pathogenesis, and that AMPK activation may represent a potential therapeutic strategy for these familial forms of PD.

Aggresomes are juxtanuclear inclusion bodies that have been proposed to act as staging grounds for the disposal of protein aggregates via the autophagic route. To examine whether the composition of an aggresome influences its clearance by autophagy, we ectopically expressed a variety of aggregation-prone proteins in cultured cells to generate aggresomes that differ in their protein content. We found that whereas aggresomes generated in cells expressing mutant huntingtin or mutant tau, or co-expressing synphilin-1 and α-synuclein, are amenable to clearance by autophagy, those produced in AIMP2 (p38)- or mutant desmin-expressing cells are apparently resistant to autophagic clearance. Notably, AIMP2 (p38)- and desmin-positive inclusions fail to recruit key components of the autophagic/lysosomal system. However, by altering the composition of inclusions, ‘autophagy-resistant’ aggresomes could be rendered ‘autophagy-susceptible’. Taken together, our results demonstrate that not all aggresomes are efficiently primed for autophagic clearance and highlight a certain degree of selectivity for the supposedly non-discriminative pathway.

Most, if not all, neurodegenerative diseases are marked by the presence of ubiquitin-positive protein inclusions. How proteins within these inclusion bodies escape proteasomal degradation despite being enriched with ubiquitin remains a conundrum. Current evidence suggests a relationship between proteasomal impairment and inclusion formation, a persuasive explanation for the inability of the cell to remove ubiquitinated protein aggregates. Alternatively, the formation of ubiquitin-enriched inclusion may be uncoupled from the proteasome. Supporting this, we recently uncovered a novel, proteasomal-independent, catalytic activity for the Parkinson disease (PD)-linked ubiquitin ligase, parkin, that significantly enhances the formation of Lewy body (LB)-like inclusions generated in cultured cells by the co-expression of α-synuclein and synphilin-1. This unique activity of parkin mediates a non-classical, lysine (K) 63-linked ubiquitin multichain assembly on synphilin-1 that is distinct from the classical, degradation-associated, K48-linked ubiquitination. Interestingly, two other PD-linked gene products, α-synuclein and UCHL1, have recently also been associated with K63-linked ubiquitination. Inclusive of parkin, there are therefore now three PD-related gene products that are known to potentiate K63-linked ubiquitination, thus signalling an important functional relationship between this unique mode of ubiquitin tagging and PD pathogenesis. Mechanistically, the involvement of a “non-degradative” mode of ubiquitination in protein inclusion formation is an attractive explanation for how proteins are seemingly stabilized within inclusions.

Mitophagy is an important type of selective autophagy for specific elimination of damaged mitochondria. PTEN-induced putative kinase protein 1 (PINK1)-catalyzed phosphorylation of ubiquitin (Ub) plays a critical role in the onset of PINK1-Parkin-mediated mitophagy. Phosphatase and tensin homolog (PTEN)-long (PTEN-L) is a newly identified isoform of PTEN, with addition of 173 amino acids to its N-terminus. Here we report that PTEN-L is a novel negative regulator of mitophagy via its protein phosphatase activity against phosphorylated ubiquitin. We found that PTEN-L localizes at the outer mitochondrial membrane (OMM) and overexpression of PTEN-L inhibits, whereas deletion of PTEN-L promotes, mitophagy induced by various mitochondria-damaging agents. Mechanistically, PTEN-L is capable of effectively preventing Parkin mitochondrial translocation, reducing Parkin phosphorylation, maintaining its closed inactive conformation, and inhibiting its E3 ligase activity. More importantly, PTEN-L reduces the level of phosphorylated ubiquitin (pSer65-Ub) in vivo, and in vitro phosphatase assay confirms that PTEN-L dephosphorylates pSer65-Ub via its protein phosphatase activity, independently of its lipid phosphatase function. Taken together, our findings demonstrate a novel function of PTEN-L as a protein phosphatase for ubiquitin, which counteracts PINK1-mediated ubiquitin phosphorylation leading to blockage of the feedforward mechanisms in mitophagy induction and eventual suppression of mitophagy. Thus, understanding this novel function of PTEN-L provides a key missing piece in the molecular puzzle controlling mitophagy, a critical process in many important human diseases including neurodegenerative disorders such as Parkinson's disease.

Mutations in the leucine rich repeat kinase 2 (LRRK2) gene are responsible for autosomal dominant and sporadic Parkinson disease (PD), possibly exerting their effects via a toxic gain of function. A common p.G2019S mutation (rs34637584:A>G) is responsible for up to 30-40% of PD cases in some ethnic populations. Here, we show that LRRK2 interacts with human peroxiredoxin 3 (PRDX3), a mitochondrial member of the antioxidant family of thioredoxin (Trx) peroxidases. Importantly, mutations in the LRRK2 kinase domain significantly increased phosphorylation of PRDX3 compared to wild-type. The increase in PRDX3 phosphorylation was associated with decreased peroxidase activity and increased death in LRRK2-expressing but not in LRRK2-depleted or vector-transfected neuronal cells. LRRK2 mutants stimulated mitochondrial factors involved in apoptosis and induced production of reactive oxygen species (ROS) and oxidative modification of macromolecules. Furthermore, immunoblot and immunohistochemical analysis of postmortem human PD patients carrying the p.G2019S mutation showed a marked increase in phosphorylated PRDX3 (p-PRDX3) relative to normal brain. We showed that LRRK2 mutations increase the inhibition of an endogenous peroxidase by phosphorylation promoting dysregulation of mitochondrial function and oxidative damage. Our findings provide a mechanistic link between the enhanced kinase activity of PD-linked LRRK2 and neuronal cell death.

The design of the first dual-purpose activity-based probe of monoamine oxidase B (MAO-B) is reported. This probe is highly selective towards MAO-B, even at high MAO-A expression levels, and could sensitively report endogenous MAO-B activities by both in situ proteome profiling and live-cell bioimaging. With a built-in imaging module as part of the probe design, the probe was able to accomplish what all previously reported MAO-B imaging probes failed to do thus far: the live-cell imaging of MAO-B activities without encountering diffusion problems.

Reactive oxygen species (ROS) and mitophagy are profoundly implicated in the pathogenesis of neurodegenerative diseases, such as Parkinson's disease (PD). Several studies have suggested that ROS are not involved in mitochondrial translocation of Parkin which primes mitochondria for autophagic elimination. However, whether ROS play a role in the execution of mitophagy is unknown. In the present study, we show that carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment induced both mitochondrial depolarization and generation of ROS that were needed for the mitophagy process. Cells failed to proceed to complete mitophagy if CCCP treatment was discontinued even after recruitment of Parkin and autophagy machinery to mitochondria. Notably, treatment of pro-oxidant was able to replace CCCP treatment to take mitophagy forward, while it alone was insufficient to induce translocation of Parkin to mitochondria or autophagic clearance of mitochondria. In addition, an SOD mimetic that attenuated the superoxide level suppressed mitophagy, while an SOD inhibitor accumulated cellular superoxide and promoted mitophagy. Furthermore, blockage of the p38 signaling pathway inhibited mitophagy induced by ROS, suggesting that it may contribute to the activation of ROS-mediated mitophagy. Together, our study sheds light on the link between ROS and mitophagy at a molecular level, and suggests the therapeutic potential of regulating mitophagy through the superoxide-p38-mitophagy axis.

Parkinson’s disease (PD) is a neurodegenerative disease that devastatingly affects people’s lives. Numerous research studies have shown that peroxynitrite (ONOO–) plays a pivotal role in the pathogenesis of PD. However, a suitable tool that could quickly and sensitively detect ONOO– in various PD models is still lacking. To this end, we designed and synthesized a series of near-infrared probes that could detect ONOO– within seconds by near-infrared fluorescent imaging in an ultrafast and highly selective manner. It is noteworthy that one of those developed probes, NIR-PN1, showed excellent sensing performance and blood–brain barrier penetrating ability. NIR-PN1 was successfully applied for imaging of ONOO– fluxes in multiple PD models including PC12 cell, Drosophila, C. elegans, and mouse brain, indicating its great potential application not only for understanding the biological roles that ONOO– played in PD but also for early PD diagnosis and treatment.

Although a subject of intense research, the etiology of Parkinson disease (PD) remains poorly understood. However, a wide range of studies conducted over the past few decades have collectively implicated aberrant mitochondrial homeostasis as a key contributor to the development of PD. Particularly strong support for this came from the recent demonstration that parkin, a familial PD-linked gene, is a critical regulator of mitochondrial quality control. Indeed, Parkin appears to be involved in all stages of the mitochondrial life cycle (i.e., from biogenesis to its exit from the cell (via mitophagy). Interestingly, the role of Parkin in the biogenesis and clearance of mitochondria is akin to that performed by the energy sensor AMP-activated protein kinase (AMPK), suggesting that the two proteins might act in a functionally converging manner to maintain the quality of cellular mitochondria. In this review, we discuss the contribution of mitochondrial dysfunction to PD pathogenesis and the role of Parkin and AMPK in preserving neuronal mitochondrial homeostasis. Alongside this, we will also articulate our thoughts on the potential alliance between Parkin and AMPK in offering neuroprotection through their ability to maintain energy balance in the brain.

Pruning of unnecessary axons and/or dendrites is crucial for maturation of the nervous system. However, little is known about cell adhesion molecules (CAMs) that control neuronal pruning. In Drosophila, dendritic arborization neurons, ddaCs, selectively prune their larval dendrites. Here, we report that Rab5/ESCRT-mediated endocytic pathways are critical for dendrite pruning. Loss of Rab5 or ESCRT function leads to robust accumulation of the L1-type CAM Neuroglian (Nrg) on enlarged endosomes in ddaC neurons. Nrg is localized on endosomes in wild-type ddaC neurons and downregulated prior to dendrite pruning. Overexpression of Nrg alone is sufficient to inhibit dendrite pruning, whereas removal of Nrg causes precocious dendrite pruning. Epistasis experiments indicate that Rab5 and ESCRT restrain the inhibitory role of Nrg during dendrite pruning. Thus, this study demonstrates the cell-surface molecule that controls dendrite pruning and defines an important mechanism whereby sensory neurons, via endolysosomal pathway, downregulate the cell-surface molecule to trigger dendrite pruning.

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by progressive loss of dopaminergic neurons in the substantia nigra. The precise mechanism underlying pathogenesis of PD is not fully understood, but it has been widely accepted that excessive reactive oxygen species (ROS) are the key mediator of PD pathogenesis. The causative factors of PD such as gene mutation, neuroinflammation, and iron accumulation all could induce ROS generation, and the later would mediate the dopaminergic neuron death by causing oxidation protein, lipids, and other macromolecules in the cells. Obviously, it is of mechanistic and therapeutic significance to understand where ROS are derived and how ROS induce dopaminergic neuron damage. In the present review, we try to summarize and discuss the main source of ROS in PD and the key pathways through which ROS mediate DA neuron death.

Objective We utilized human midbrain‐like organoids (hMLOs) generated from human pluripotent stem cells carrying glucocerebrosidase gene ( GBA1) and α‐synuclein (α‐syn; SNCA) perturbations to investigate genotype‐to‐phenotype relationships in Parkinson disease, with the particular aim of recapitulating α‐syn– and Lewy body–related pathologies and the process of neurodegeneration in the hMLO model. Methods We generated and characterized hMLOs from GBA1 −/− and SNCA overexpressing isogenic embryonic stem cells and also generated Lewy body–like inclusions in GBA1/SNCA dual perturbation hMLOs and conduritol‐b‐epoxide–treated SNCA triplication hMLOs. Results We identified for the first time that the loss of glucocerebrosidase, coupled with wild‐type α‐syn overexpression, results in a substantial accumulation of detergent‐resistant, β‐sheet–rich α‐syn aggregates and Lewy body–like inclusions in hMLOs. These Lewy body–like inclusions exhibit a spherically symmetric morphology with an eosinophilic core, containing α‐syn with ubiquitin, and can also be formed in Parkinson disease patient–derived hMLOs. We also demonstrate that impaired glucocerebrosidase function promotes the formation of Lewy body–like inclusions in hMLOs derived from patients carrying the SNCA triplication. Interpretation Taken together, the data indicate that our hMLOs harboring 2 major risk factors (glucocerebrosidase deficiency and wild‐type α‐syn overproduction) of Parkinson disease provide a tractable model to further elucidate the underlying mechanisms for progressive Lewy body formation. ANN NEUROL 2021;90:490–505

Neurodegenerative diseases (NDDs) such as Alzheimer's disease (AD) and Parkinson's disease (PD) are a heterogeneous group of disorders characterized by progressive degeneration of neurons. NDDs threaten the lives of millions of people worldwide and regretfully remain incurable. It is well accepted that dysfunction of mitochondria underlies the pathogenesis of NDDs. Dysfunction of mitochondria results in energy depletion, oxidative stress, calcium overloading, caspases activation, which dominates the neuronal death of NDDs. Therefore, mitochondria are the preferred target for intervention of NDDs. So far various mitochondria-targeting drugs have been developed and delightfully some of them demonstrate promising outcome, though there are still some obstacles such as targeting specificity, delivery capacity hindering the drugs development. In present review, we will elaborately address 1) the strategy to design mitochondria targeting drugs, 2) the rescue mechanism of respective mitochondria targeting drugs, 3) how to evaluate the therapeutic effect. Hopefully this review will provide comprehensive knowledge for understanding how to develop more effective drugs for the treatment of NDDs.

Abstract Microglia are the resident innate immune cells in the brain with a major role in orchestrating immune responses. They also provide a frontline of host defense in the central nervous system (CNS) through their active phagocytic capability. Being a professional phagocyte, microglia participate in phagocytic and autophagic clearance of cellular waste and debris as well as toxic protein aggregates, which relies on optimal lysosomal acidification and function. Defective microglial lysosomal acidification leads to impaired phagocytic and autophagic functions which result in the perpetuation of neuroinflammation and progression of neurodegeneration. Reacidification of impaired lysosomes in microglia has been shown to reverse neurodegenerative pathology in Alzheimer’s disease. In this review, we summarize key factors and mechanisms contributing to lysosomal acidification impairment and the associated phagocytic and autophagic dysfunction in microglia, and how these defects contribute to neuroinflammation and neurodegeneration. We further discuss techniques to monitor lysosomal pH and therapeutic agents that can reacidify impaired lysosomes in microglia under disease conditions. Finally, we propose future directions to investigate the role of microglial lysosomal acidification in lysosome–mitochondria crosstalk and in neuron–glia interaction for more comprehensive understanding of its broader CNS physiological and pathological implications.

Autosomal recessive juvenile parkinsonism is a movement disorder associated with the degeneration of dopaminergic neurons in substantia nigra pars compacta. The loss of functional parkin caused by <i>parkin</i> gene mutations is the most common single cause of juvenile parkinsonism. Parkin has been shown to aid in protecting cells from endoplasmic reticulum and oxidative stressors presumably due to ubiquitin ligase activity of parkin that targets proteins for proteasomal degradation. However, studies on parkin have been impeded because of limited reagents specific for this protein. Here we report the generation and characterization of a panel of parkin-specific monoclonal antibodies. Biochemical analyses indicate that parkin is present only in the high salt-extractable fraction of mouse brain, whereas it is present in both the high salt-extractable and RIPA-resistant, SDS-extractable fraction in young human brain. Parkin is present at decreased levels in the high salt-extractable fraction and at increased levels in the SDS-extractable fraction from aged human brain. This shift in the extractability of parkin upon aging is seen in humans but not in mice, demonstrating species-specific differences in the biochemical characteristics of murine <i>versus</i> human parkin. Finally, by using these highly specific anti-parkin monoclonal antibodies, it was not possible to detect parkin in α-synuclein-containing lesions in α-synucleinopathies, thereby challenging prior inferences about the role of parkin in movement disorders other than autosomal recessive juvenile parkinsonism.

Mutations in the parkin gene are a predominant cause of familial parkinsonism. Although initially described as a recessive disorder, emerging evidence suggest that single parkin mutations alone may confer increased susceptibility to Parkinson's disease. To better understand the effects of parkin mutations in vivo , we generated transgenic Drosophila overexpressing two human parkin missense mutants, R275W and G328E. Transgenic flies that overexpress R275W, but not wild-type or G328E, human parkin display an age-dependent degeneration of specific dopaminergic neuronal clusters and concomitant locomotor deficits that accelerate with age or in response to rotenone treatment. Furthermore, R275W mutant flies also exhibit prominent mitochondrial abnormalities in their flight muscles. Interestingly, these defects caused by the expression of human R275W parkin are highly similar to those triggered by the loss of endogenous parkin in parkin null flies. Together, our results provide the first in vivo evidence demonstrating that parkin R275W mutant expression mediates pathogenic outcomes and suggest the interesting possibility that select parkin mutations may directly exert neurotoxicity in vivo .

Mutations in parkin are associated with various inherited forms of Parkinson's disease (PD). Parkin is a ubiquitin ligase enzyme that catalyzes the covalent attachment of ubiquitin moieties onto substrate proteins destined for proteasomal degradation. The substrates of parkin-mediated ubiquitination have yet to be completely identified. Using a yeast two-hybrid screen, we isolated the septin, human SEPT5_v2 (also known as cell division control-related protein 2), as a putative parkin-binding protein. SEPT5_v2 is highly homologous to another septin, SEPT5, which was recently identified as a target for parkin-mediated ubiquitination. SEPT5_v2 binds to parkin at the amino terminus and in the ring finger domains. Several lines of evidence have validated the putative link between parkin and SEPT5_v2. Parkin co-precipitates with SEPT5_v2 from human substantia nigra lysates. Parkin ubiquitinates SEPT5_v2 in vitro, and both SEPT5_v1 and SEPT5_v2 accumulate in brains of patients with ARJP, suggesting that parkin is essential for the normal metabolism of these proteins. These findings suggest that an important relationship exists between parkin and septins.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. Although a subject of intense research, the etiology of PD remains poorly understood. Recently, several lines of evidence have implicated an intimate link between aberrations in the ubiquitin proteasome system (UPS) and PD pathogenesis. Derangements of the UPS, which normally functions as a type of protein degradation machinery, lead to alterations in protein homeostasis that could conceivably promote the toxic accumulation of proteins detrimental to neuronal survival. Not surprisingly, various cellular and animal models of PD that are based on direct disruption of UPS function reproduce the most prominent features of PD. Although persuasive, new developments in the past few years have in fact raised serious questions about the link between the UPS and PD. Here I review current thoughts and controversies about their relationship and discuss whether strategies aimed at mitigating UPS dysfunction could represent rational ways to intervene in the disease. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).

Protein aggregation as a result of misfolding is a common theme underlying neurodegenerative diseases. In Parkinson's disease (PD), research on protein misfolding and aggregation has taken center stage following the association of α-synuclein gene mutations with familial forms of the disease, and importantly, the identification of the protein as a major component of Lewy bodies, a pathological hallmark of PD. Fueling this excitement is the subsequent identification of another PD-linked gene, parkin, as a ubiquitin ligase associated with the proteasome, a major intracellular protein degradation machinery that destroys unwanted, albeit mainly soluble, proteins. Notably, a role for parkin in the clearance of insoluble protein aggregates via macroautophagy has also been implicated by more recent studies. Paradoxically, like α-synuclein, parkin is also prone to misfolding, especially in the presence of age-related stress. Similarly, protein misfolding can also affect the function of other key PD-linked genes such as DJ-1, PINK1, and perhaps also LRRK2. Here, we discuss the role of protein misfolding and aggregation in PD, and how impairments of the various cellular protein quality systems could precipitate these events and lead to neuronal demise. Towards the end of our discussion, we also revisited the role of Lewy body formation in PD. Antioxid. Redox Signal. 11, 2119–2134.

Although mutations in the parkin gene are frequently associated with familial Parkinsonism, emerging evidence suggests that parkin also plays a role in cancers as a putative tumor suppressor. Supporting this, we show here that parkin expression is dramatically reduced in several breast cancer-derived cell lines as well as in primary breast cancer tissues. Importantly, we found that ectopic parkin expression in parkin-deficient breast cancer cells mitigates their proliferation rate both in vitro and in vivo, as well as reduces the capacity of these cells to migrate. Cell cycle analysis revealed the arrestment of a significant percentage of parkin-expressing breast cancer cells at the G1-phase. However, we did not observe significant changes in the levels of the G1-associated cyclin D1 and E. On the other hand, the level of cyclin-dependent kinase 6 (CDK6) is dramatically and selectively elevated in parkin-expressing breast cancer cells, the extent of which correlates well with the expression of parkin. Interestingly, a recent study demonstrated that CDK6 restrains the proliferation of breast cancer cells. Taken together, our results support a negative role for parkin in tumorigenesis and provide a potential mechanism by which parkin exerts its suppressing effects on breast cancer cell proliferation.

Parkinson disease (PD), a prevalent neurodegenerative motor disorder, is characterized by the rather selective loss of dopaminergic neurons and the presence of α-synuclein-enriched Lewy body inclusions in the substantia nigra of the midbrain. Although the etiology of PD remains incompletely understood, emerging evidence suggests that dysregulated iron homeostasis may be involved. Notably, nigral dopaminergic neurons are enriched in iron, the uptake of which is facilitated by the divalent metal ion transporter DMT1. To clarify the role of iron in PD, we generated SH-SY5Y cells stably expressing DMT1 either singly or in combination with wild type or mutant α-synuclein. We found that DMT1 overexpression dramatically enhances Fe(2+) uptake, which concomitantly promotes cell death. This Fe(2+)-mediated toxicity is aggravated by the presence of mutant α-synuclein expression, resulting in increased oxidative stress and DNA damage. Curiously, Fe(2+)-mediated cell death does not appear to involve apoptosis. Instead, the phenomenon seems to occur as a result of excessive autophagic activity. Accordingly, pharmacological inhibition of autophagy reverses cell death mediated by Fe(2+) overloading. Taken together, our results suggest a role for iron in PD pathogenesis and provide a mechanism underlying Fe(2+)-mediated cell death.

Abstract Mutations in the parkin gene, which encodes a ubiquitin ligase, are a major genetic cause of parkinsonism. Interestingly, parkin also plays a role in cancer as a putative tumor suppressor, and the gene is frequently targeted by deletion and inactivation in human malignant tumors. Here, we investigated a potential tumor suppressor role for parkin in gliomas. We found that parkin expression was dramatically reduced in glioma cells. Restoration of parkin expression promoted G1 phase cell-cycle arrest and mitigated the proliferation rate of glioma cells in vitro and in vivo. Notably, parkin-expressing glioma cells showed a reduction in levels of cyclin D1, but not cyclin E, and a selective downregulation of Akt serine-473 phosphorylation and VEGF receptor levels. In accordance, cells derived from a parkin-null mouse model exhibited increased levels of cyclin D1, VEGF receptor, and Akt phosphorylation, and divided significantly faster when compared with wild-type cells, with suppression of these changes following parkin reintroduction. Clinically, analysis of parkin pathway activation was predictive for the survival outcome of patients with glioma. Taken together, our study provides mechanistic insight into the tumor suppressor function of parkin in brain tumors and suggests that measurement of parkin pathway activation may be used clinically as a prognostic tool in patients with brain tumor. Cancer Res; 72(10); 2543–53. ©2012 AACR.

Mutations in PARKIN (PARK2), an ubiquitin ligase, cause early onset Parkinson disease. Parkin was shown to bind, ubiquitinate, and target depolarized mitochondria for destruction by autophagy. This process, mitophagy, is considered crucial for maintaining mitochondrial integrity and suppressing Parkinsonism. Here, we report that under moderate mitochondrial stress, parkin does not translocate to mitochondria to induce mitophagy; rather, it stimulates mitochondrial connectivity. Mitochondrial stress-induced fusion requires PINK1 (PARK6), mitofusins, and parkin ubiquitin ligase activity. Upon exposure to mitochondrial toxins, parkin binds α-synuclein (PARK1), and in conjunction with the ubiquitin-conjugating enzyme Ubc13, stimulates K63-linked ubiquitination. Importantly, α-synuclein inactivation phenocopies parkin overexpression and suppresses stress-induced mitochondria fission, whereas Ubc13 inactivation abrogates parkin-dependent mitochondrial fusion. The convergence of parkin, PINK1, and α-synuclein on mitochondrial dynamics uncovers a common function of these PARK genes in the mitochondrial stress response and provides a potential physiological basis for the prevalence of α-synuclein pathology in Parkinson disease.Background: Parkin is proposed to maintain mitochondrial QC through promoting mitophagy.Results: Under moderate mitochondrial stress conditions, parkin stimulates mitochondrial fusion instead of mitophagy by catalyzing K63-linked ubiquitination and inactivating α-synuclein.Conclusion: Parkin, PINK1, and α-synuclein form a regulatory circuit to regulate mitochondrial stress response.Significance: This study provides a physiological context to functionally connect key PARK genes in the pathogenesis of Parkinson disease. Mutations in PARKIN (PARK2), an ubiquitin ligase, cause early onset Parkinson disease. Parkin was shown to bind, ubiquitinate, and target depolarized mitochondria for destruction by autophagy. This process, mitophagy, is considered crucial for maintaining mitochondrial integrity and suppressing Parkinsonism. Here, we report that under moderate mitochondrial stress, parkin does not translocate to mitochondria to induce mitophagy; rather, it stimulates mitochondrial connectivity. Mitochondrial stress-induced fusion requires PINK1 (PARK6), mitofusins, and parkin ubiquitin ligase activity. Upon exposure to mitochondrial toxins, parkin binds α-synuclein (PARK1), and in conjunction with the ubiquitin-conjugating enzyme Ubc13, stimulates K63-linked ubiquitination. Importantly, α-synuclein inactivation phenocopies parkin overexpression and suppresses stress-induced mitochondria fission, whereas Ubc13 inactivation abrogates parkin-dependent mitochondrial fusion. The convergence of parkin, PINK1, and α-synuclein on mitochondrial dynamics uncovers a common function of these PARK genes in the mitochondrial stress response and provides a potential physiological basis for the prevalence of α-synuclein pathology in Parkinson disease. Background: Parkin is proposed to maintain mitochondrial QC through promoting mitophagy. Results: Under moderate mitochondrial stress conditions, parkin stimulates mitochondrial fusion instead of mitophagy by catalyzing K63-linked ubiquitination and inactivating α-synuclein. Conclusion: Parkin, PINK1, and α-synuclein form a regulatory circuit to regulate mitochondrial stress response. Significance: This study provides a physiological context to functionally connect key PARK genes in the pathogenesis of Parkinson disease.

Intensive research over the last 15 years has led to the identification of several autosomal recessive and dominant genes that cause familial Parkinson's disease (PD). Importantly, the functional characterization of these genes has shed considerable insights into the molecular mechanisms underlying the etiology and pathogenesis of PD. Collectively; these studies implicate aberrant protein and mitochondrial homeostasis as key contributors to the development of PD, with oxidative stress likely acting as an important nexus between the two pathogenic events. Interestingly, recent genome-wide association studies (GWAS) have revealed variations in at least two of the identified familial PD genes (i.e. α-synuclein and LRRK2) as significant risk factors for the development of sporadic PD. At the same time, the studies also uncovered variability in novel alleles that is associated with increased risk for the disease. Additionally, in-silico meta-analyses of GWAS data have allowed major steps into the investigation of the roles of gene-gene and gene-environment interactions in sporadic PD. The emergent picture from the progress made thus far is that the etiology of sporadic PD is multi-factorial and presumably involves a complex interplay between a multitude of gene networks and the environment. Nonetheless, the biochemical pathways underlying familial and sporadic forms of PD are likely to be shared.

Parkin is a unique, multifunctional ubiquitin ligase whose various roles in the cell, particularly in neurons, are widely thought to be protective. The pivotal role that Parkin plays in maintaining neuronal survival is underscored by our current recognition that Parkin dysfunction represents not only a predominant cause of familial parkinsonism but also a formal risk factor for the more common, sporadic form of Parkinson's disease (PD). Accordingly, keen research on Parkin over the past decade has led to an explosion of knowledge regarding its physiological roles and its relevance to PD. However, our understanding of Parkin is far from being complete. Indeed, surprises emerge from time to time that compel us to constantly update the paradigm of Parkin function. For example, we now know that Parkin's function is not confined to mere housekeeping protein quality control (QC) roles but also includes mitochondrial homeostasis and stress-related signaling. Furthermore, emerging evidence also suggest a role for Parkin in several other major neurodegenerative diseases including Alzheimer's disease (AD) and Amyotrophic Lateral Sclerosis (ALS). Yet, it remains truly amazing to note that a single enzyme could serve such multitude of functions and cellular roles. Clearly, its activity has to be tightly regulated. In this review, we shall discuss this and how dysregulated Parkin function may precipitate neuronal demise in various neurodegenerative disorders.

Abstract The cellular-level effects of low/high frequency oscillating magnetic field on excitable cells such as neurons are well established. In contrast, the effects of a homogeneous, static magnetic field (SMF) on Central Nervous System (CNS) glial cells are less investigated. Here, we have developed an in vitro SMF stimulation set-up to investigate the genomic effects of SMF exposure on oligodendrocyte differentiation and neurotrophic factors secretion. Human oligodendrocytes precursor cells (OPCs) were stimulated with moderate intensity SMF (0.3 T) for a period of two weeks (two hours/day). The differential gene expression of cell activity marker (c-fos), early OPC (Olig1, Olig2. Sox10), and mature oligodendrocyte markers (CNP, MBP) were quantified. The enhanced myelination capacity of the SMF stimulated oligodendrocytes was validated in a dorsal root ganglion microfluidics chamber platform. Additionally, the effects of SMF on the gene expression and secretion of neurotrophic factors- BDNF and NT3 was quantified. We also report that SMF stimulation increases the intracellular calcium influx in OPCs as well as the gene expression of L-type channel subunits-CaV1.2 and CaV1.3. Our findings emphasize the ability of glial cells such as OPCs to positively respond to moderate intensity SMF stimulation by exhibiting enhanced differentiation, functionality as well as neurotrophic factor release.

A common genetic form of Parkinson's disease (PD) is caused by mutations in LRRK2. We identify WSB1 as a LRRK2 interacting protein. WSB1 ubiquitinates LRRK2 through K27 and K29 linkage chains, leading to LRRK2 aggregation and neuronal protection in primary neurons and a Drosophila model of G2019S LRRK2. Knocking down endogenous WSB1 exacerbates mutant LRRK2 neuronal toxicity in neurons and the Drosophila model, indicating a role for endogenous WSB1 in modulating LRRK2 cell toxicity. WSB1 is in Lewy bodies in human PD post-mortem tissue. These data demonstrate a role for WSB1 in mutant LRRK2 pathogenesis, and suggest involvement in Lewy body pathology in sporadic PD. Our data indicate a role in PD for ubiquitin K27 and K29 linkages, and suggest that ubiquitination may be a signal for aggregation and neuronal protection in PD, which may be relevant for other neurodegenerative disorders. Finally, our study identifies a novel therapeutic target for PD.

The concept of neuroinflammation has come a full circle; from being initially regarded as a controversial viewpoint to its present day acceptance as an integral component of neurodegenerative processes. A closer look at the etiopathogenesis of many neurodegenerative conditions will reveal a patho-symbiotic relationship between neuroinflammation and neurodegeneration, where the two liaise with each other to form a self-sustaining vicious cycle that facilitates neuronal demise. Here, we focus on damage associated molecular patterns or DAMPs as a potentially important nexus in the context of this lethal neuroinflammation-neurodegeneration alliance. Since their nomenclature as “DAMPs” about a decade ago, these endogenous moieties have consistently been reported as novel players in sterile (non-infective) inflammation. However, their roles in inflammatory responses in the central nervous system (CNS), especially during chronic neurodegenerative disorders are still being actively researched. The aim of this review is to first provide a general overview of the neuroimmune response in the CNS within the purview of DAMPs, its receptors and downstream signaling. This is then followed by discussions on some of the DAMP-mediated neuroinflammatory responses involved in chronic neurodegenerative diseases. Along the way, we also highlighted some important gaps in our existing knowledge regarding the role of DAMPs in neurodegeneration, the clarification of which we believe would aid in the prospects of developing treatment or screening strategies directed at these molecules.

The orphan nuclear receptor Nurr1 is critical for the development, maintenance and protection of midbrain dopaminergic (mDA) neurons. Here we show that prostaglandin E1 (PGE1) and its dehydrated metabolite, PGA1, directly interact with the ligand-binding domain (LBD) of Nurr1 and stimulate its transcriptional function. We also report the crystallographic structure of Nurr1-LBD bound to PGA1 at 2.05 Å resolution. PGA1 couples covalently to Nurr1-LBD by forming a Michael adduct with Cys566, and induces notable conformational changes, including a 21° shift of the activation function-2 helix (H12) away from the protein core. Furthermore, PGE1/PGA1 exhibit neuroprotective effects in a Nurr1-dependent manner, prominently enhance expression of Nurr1 target genes in mDA neurons and improve motor deficits in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse models of Parkinson’s disease. Based on these results, we propose that PGE1/PGA1 represent native ligands of Nurr1 and can exert neuroprotective effects on mDA neurons, via activation of Nurr1’s transcriptional function. Prostaglandins PGE1 and PGA1 have neuroprotective effects by enhancing the transcriptional activity of Nurr1 by directly binding to its ligand-binding domain and upregulating their target genes implicated in Parkinson’s disease.

The blood-brain barrier (BBB) represents a key challenge in developing brain-penetrating therapeutic molecules. BBB dysfunction is also associated with the onset and progression of various brain diseases. The BBB-on-a-chip (μBBB), an organ-on-chip technology, has emerged as a powerful in vitro platform that closely mimics the human BBB microenvironments. While the μBBB technology has seen wide application in the study of brain cancer, its utility in other brain disease models ("μBBB+") is less appreciated. Based on the advances of the μBBB technology and the evolution of in vitro models for brain diseases over the last decade, we propose the concept of a "μBBB+" system and summarize its major promising applications in pathological studies, personalized medical research, drug development, and multi-organ-on-chip approaches. We believe that such a sophisticated "μBBB+" system is a highly tunable and promising in vitro platform for further advancement of the understanding of brain diseases.

Loss of parkin function is a predominant cause of familial Parkinsonism. Emerging evidence also suggests that parkin expression variability may confer a risk for sporadic Parkinson disease. We have recently demonstrated that a wide variety of Parkinson disease-linked stressors, including dopamine (DA), induce parkin solubility alterations and promote its aggregation within the cell, a phenomenon that may underlie the progressive susceptibility of the brain to degeneration. The vulnerability of parkin to stress-induced modification is likely due to its abundance of cysteine residues. Here, we performed a comprehensive mutational analysis and demonstrate that Cys residues residing both within and outside of the RING-IBR (in between RING fingers)-RING domain of parkin are important in maintaining its solubility. The majority of these Cys residues are highly conserved in parkin across different species and potentially fulfil important structural roles. Further, we found that both parkin and HHARI (human homologue of Drosophila ariadne), another RING-IBR-RING-type ubiquitin ligase, are comparably more susceptible to solubility alterations induced by oxidative and nitrosative stress when compared with other non-RING-IBR-RING Cys-containing enzymes. However, parkin appears to be uniquely sensitive to DA-mediated stress, the specificity of which is likely due to DA modification of 2 Cys residues on parkin (Cys-268 and Cys-323) that are distinct from other RING-IBR-RING members. Loss of parkin function is a predominant cause of familial Parkinsonism. Emerging evidence also suggests that parkin expression variability may confer a risk for sporadic Parkinson disease. We have recently demonstrated that a wide variety of Parkinson disease-linked stressors, including dopamine (DA), induce parkin solubility alterations and promote its aggregation within the cell, a phenomenon that may underlie the progressive susceptibility of the brain to degeneration. The vulnerability of parkin to stress-induced modification is likely due to its abundance of cysteine residues. Here, we performed a comprehensive mutational analysis and demonstrate that Cys residues residing both within and outside of the RING-IBR (in between RING fingers)-RING domain of parkin are important in maintaining its solubility. The majority of these Cys residues are highly conserved in parkin across different species and potentially fulfil important structural roles. Further, we found that both parkin and HHARI (human homologue of Drosophila ariadne), another RING-IBR-RING-type ubiquitin ligase, are comparably more susceptible to solubility alterations induced by oxidative and nitrosative stress when compared with other non-RING-IBR-RING Cys-containing enzymes. However, parkin appears to be uniquely sensitive to DA-mediated stress, the specificity of which is likely due to DA modification of 2 Cys residues on parkin (Cys-268 and Cys-323) that are distinct from other RING-IBR-RING members. Parkinson disease (PD) 4The abbreviations used are: PD, Parkinson disease; DA, dopamine; HRP, horseradish peroxidase; HA, hemagglutinin; HMW, high molecular weight; CHIP, carboxyl terminus of the Hsc70-interacting protein; HHARI, human homologue of Drosophila ariadne; UBL, ubiquitin-like; IBR, in between RING fingers. is the most common neurodegenerative movement disorder characterized pathologically by the rather selective loss of midbrain dopaminergic neurons in the substantia nigra pars compacta and the presence of intraneuronal protein inclusions known as Lewy bodies. Although most cases of PD occur in a sporadic manner, a subset of PD cases is inheritable and attributable to mutations in specific genes. These familial PD-linked genes include α-synuclein, parkin, DJ-1, PINK1, and LRRK2 (1Savitt J.M. Dawson V.L. Dawson T.M. J. Clin. Investig. 2006; 116: 1744-1754Crossref PubMed Scopus (526) Google Scholar, 2Lim K.L. Dawson V.L. Dawson T.M. Ann. N. Y. Acad. Sci. 2003; 991: 80-92Crossref PubMed Scopus (34) Google Scholar). Of these, mutations in the parkin gene are currently recognized to be a predominant cause of familial, early onset PD (3Klein C. Hedrich K. Wellenbrock C. Kann M. Harris J. Marder K. Lang A.E. Schwinger E. Ozelius L.J. Vieregge P. Pramstaller P.P. Kramer P.L. Ann. Neurol. 2003; 54: 415-417Crossref Scopus (27) Google Scholar, 4Lucking C.B. Durr A. Bonifati V. Vaughan J. De Michele G. Gasser T. Harhangi B.S. Meco G. Denefle P. Wood N.W. Agid Y. Brice A. N. Engl. J. Med. 2000; 342: 1560-1567Crossref PubMed Scopus (1288) Google Scholar, 5Oliveira S.A. Scott W.K. Martin E.R. Nance M.A. Watts R.L. Hubble J.P. Koller W.C. Pahwa R. Stern M.B. Hiner B.C. Ondo W.G. Allen Jr., F.H. Scott B.L. Goetz C.G. Small G.W. Mastaglia F. Stajich J.M. Zhang F. Booze M.W. Winn M.P. Middleton L.T. Haines J.L. Pericak-Vance M.A. Vance J.M. Ann. Neurol. 2003; 53: 624-629Crossref PubMed Scopus (178) Google Scholar). Further, emerging evidence also suggests that parkin expression variability may confer a risk for the development of the more common, sporadic form of PD (6West A. Periquet M. Lincoln S. Lucking C.B. Nicholl D. Bonifati V. Rawal N. Gasser T. Lohmann E. Deleuze J.F. Maraganore D. Levey A. Wood N. Durr A. Hardy J. Brice A. Farrer M. Am. J. Med. Genet. 2002; 114: 584-591Crossref PubMed Scopus (192) Google Scholar, 7West A.B. Maraganore D. Crook J. Lesnick T. Lockhart P.J. Wilkes K.M. Kapatos G. Hardy J.A. Farrer M.J. Hum. Mol. Genet. 2002; 11: 2787-2792Crossref PubMed Scopus (110) Google Scholar). The importance of functional parkin to dopaminergic neuronal survival is probably related to the multitude of neuroprotective roles it serves (8Cookson M.R. Curr. Biol. 2003; 13: R522-R524Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 9Feany M.B. Pallanck L.J. Neuron. 2003; 38: 13-16Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Parkin functions as a ubiquitin ligase associated with protein homeostasis and apparently confers protection to neurons against a diverse range of cellular insults (8Cookson M.R. Curr. Biol. 2003; 13: R522-R524Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 9Feany M.B. Pallanck L.J. Neuron. 2003; 38: 13-16Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Recently, we have demonstrated that a wide variety of PD-linked stressors, including dopamine (DA), induce parkin solubility alterations and promote its aggregation within the cell (10Wang C. Ko H.S. Thomas B. Tsang F. Chew K.C. Tay S.P. Ho M.W. Lim T.M. Soong T.W. Pletnikova O. Troncoso J. Dawson V.L. Dawson T.M. Lim K.L. Hum. Mol. Genet. 2005; 14: 3885-3897Crossref PubMed Scopus (187) Google Scholar). Our observations corroborated with a similar study conducted by LaVoie et al. (11LaVoie M.J. Ostaszewski B.L. Weihofen A. Schlossmacher M.G. Selkoe D.J. Nat. Med. 2005; 11: 1214-1221Crossref PubMed Scopus (607) Google Scholar), who further showed that DA covalently modifies parkin via its Cys residues, although the number and location of the Cys targeted by DA remain unknown. Since parkin functions as a broad spectrum neuroprotectant, the effects on parkin brought about by these oxidative stressors could deplete the availability of soluble parkin in the brain, and as such, may underlie the progressive susceptibility of the brain to degeneration. We have previously speculated that enzymes whose structure and function are dependent on catalytic Cys are more susceptible to the consequence of oxidative modification (10Wang C. Ko H.S. Thomas B. Tsang F. Chew K.C. Tay S.P. Ho M.W. Lim T.M. Soong T.W. Pletnikova O. Troncoso J. Dawson V.L. Dawson T.M. Lim K.L. Hum. Mol. Genet. 2005; 14: 3885-3897Crossref PubMed Scopus (187) Google Scholar). Although the active site Cys-dependent tyrosine phosphatase family of enzymes provides one example (12Tonks N.K. Cell. 2005; 121: 667-670Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar), the RING finger-containing ubiquitin ligases, all of which are characterized by their Cys-rich catalytic moieties, potentially represent another. In particular, the abundance of Cys residues residing on RING-IBR-RING-containing proteins, such as parkin, conceivably could present a ready source of targets for oxidative modification. As the majority of these highly conserved RING finger Cys residues are thought to fulfil important structural roles (13Capili A.D. Edghill E.L. Wu K. Borden K.L. J. Mol. Biol. 2004; 340: 1117-1129Crossref PubMed Scopus (85) Google Scholar, 14Zheng N. Wang P. Jeffrey P.D. Pavletich N.P. Cell. 2000; 102: 533-539Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar), it is conceivable that their modification by oxidation could disrupt the overall structural integrity of the protein, leading to alterations in their biochemical properties. In this study, we systematically mutate almost all of parkin's Cys residues and demonstrate that conserved Cys residues residing both within and outside of the RING-IBR-RING domain of parkin are important for maintaining its solubility, although modification of Cys residues within parkin RING-IBR-RING domain also resulted in a significantly higher tendency for the protein to form aggresome-like structures within the cell. Further, we found that both parkin and HHARI, another RING-IBR-RING ubiquitin ligase, are comparably more susceptible to solubility alterations induced by oxidative and nitrosative stress when compared with other non-RING-IBR-RING Cys-containing enzymes examined. However, parkin appears to be uniquely sensitive to DA-mediated stress. Further mutational analysis suggests the involvement of 2 nonconserved Cys residues on parkin, Cys-268 and Cys-323, in DA-mediated parkin insolubility. As these residues are distinct from other RING-IBR-RING members, including HHARI, they could potentially explain for the enhanced sensitivity of parkin to modification by DA. Antibodies and Reagents—The following antibodies were used: monoclonal anti-FLAG-HRP (Sigma), monoclonal anti-Myc-HRP (Roche Applied Science), monoclonal anti-HA-HRP (Roche Applied Science), monoclonal anti-β actin (Sigma), rhodamine-conjugated anti-mouse IgG (Molecular Probes), monoclonal anti-UCH-L1 (Dako), polyclonal anti-HHARI (Abcam), polyclonal anti-c-Cbl (Cell Signaling), and polyclonal anti-CHIP (gift from L. Petrucelli). The FLAG-tagged wild-type parkin and Myc-tagged CHIP expression constructs were gifts from R. Takahashi, whereas HA-tagged HHARI and c-Cbl expression constructs were gifts from G. Guy. All other chemicals, unless otherwise stated, were purchased from Sigma. DA stock solution (100 mm in boiled water) was freshly prepared prior to use, whereas NOC-18 stock solution (50 mm in water) was prepared and stored at -20 °C. Bioinformatics—Amino acid sequences of parkin from human (O60260), rat (NP064478), mouse (NP057903), Fugu (AAS79348), zebrafish (NP001017635), Drosophila (AAM43930), Anopheles (XP316606), and Caenorhabditis elegans (CAB04599) were retrieved from National Center for Biotechnology Information (NCBI) and aligned using CLUSTALW (15Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56668) Google Scholar). The structural coordinates for the RING domain of c-Cbl (1FBV) and HHARI (1WD2) and the UBL domain of parkin (1IYF) were obtained from Protein Data Base (PDB). Parkin RING1 and RING2 were modeled against the templates of c-Cbl and HHARI, respectively, using the alignment interface of the SWISS-MODEL program (16Schwede T. Kopp J. Guex N. Peitsch M.C. Nucleic Acids Res. 2003; 31: 3381-3385Crossref PubMed Scopus (4624) Google Scholar). All structures were viewed and manipulated (aligned structure analysis, superimpositions, computation of molecular surface, and on-screen mutations) using Swiss-PdbViewer v3.7b2 (SPdbV) (17Guex N. Diemand A. Peitsch M.C. Trends Biochem. Sci. 1999; 24: 364-367Abstract Full Text Full Text PDF PubMed Google Scholar). Site-directed Mutagenesis and Generation of Parkin Deletion Constructs—All the Cys → Ala (C → A) parkin mutants were constructed using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions and verified via DNA sequencing. Parkin 1-137 and 1-237 deletion constructs were generated by means of PCR amplification of the designated regions using wild-type FLAG-parkin cDNA as a template and subcloned into pCDNA3 plasmid. For parkin Δ77-237, the UBL and RING-IBR-RING regions of parkin were amplified separately from wild-type FLAG-parkin cDNA by means of PCR and subsequently cloned in-frame into pCDNA3. Cell Culture, Treatment with Various Stressors, and Western Blot Analysis—SH-SY5Y neuroblastoma cells were seeded at 2 × 105 cell density for all transfections. pCDNA3 plasmid bearing FLAG-tagged wild-type parkin or various C → A parkin mutants were transfected using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions. At 48 h after transfection, the cells were harvested for sequential fractionation of the cell lysates into Triton-X-soluble and SDS-soluble fractions as described previously (18Wang C. Tan J.M. Ho M.W. Zaiden N. Wong S.H. Chew C.L. Eng P.W. Lim T.M. Dawson T.M. Lim K.L. J. Neurochem. 2005; 93: 422-431Crossref PubMed Scopus (110) Google Scholar). An equivalent amount of proteins among different Triton-X-soluble and SDS-soluble fractions was resolved by means of SDS-PAGE, and the levels of various proteins were analyzed by means of Western blotting procedures using ECL detection reagents (Amersham Biosciences). For stress treatments, plasmids containing FLAG-parkin species, HA-HHARI, HA-c-Cbl, Myc-CHIP, or Myc-UCH-L1 were transfected using Lipofectamine 2000 reagent (Invitrogen). At 24 h after transfection, transfected cells were treated with 20 mm H2O2 for 30 min, 0.5-1 mm DA for 12 h, or 0.25-0.5 mm NOC-18 for 24 h. Immunocytochemistry and Confocal Microscopy—5 × 104 SH-SY5Y cells were seeded on coverslips for subsequent transfection with FLAG-tagged wild-type parkin or selected C → A parkin mutants using Lipofectamine Plus reagent (Invitrogen). At 48 h after transfection, the cells were fixed with 3% paraformaldehyde (Sigma) for 1 h at 4 °C. Cellular distributions of wild-type and mutant parkin were examined by means of immunocytochemistry and confocal microscopy as described previously (4Lucking C.B. Durr A. Bonifati V. Vaughan J. De Michele G. Gasser T. Harhangi B.S. Meco G. Denefle P. Wood N.W. Agid Y. Brice A. N. Engl. J. Med. 2000; 342: 1560-1567Crossref PubMed Scopus (1288) Google Scholar). At least 100 transfected cells were counted to quantify the incidence of inclusions. Human Tissues and Statistical Analysis—Aliquots of previously described (10Wang C. Ko H.S. Thomas B. Tsang F. Chew K.C. Tay S.P. Ho M.W. Lim T.M. Soong T.W. Pletnikova O. Troncoso J. Dawson V.L. Dawson T.M. Lim K.L. Hum. Mol. Genet. 2005; 14: 3885-3897Crossref PubMed Scopus (187) Google Scholar) human brain lysates prepared from postmortem brains of control and PD individuals that were stored at -80 °C were used in this study. Statistical significance for all the quantitative data obtained was analyzed using Student's t test (*, p < 0.05, **, p < 0.001) unless otherwise stated. Conservation and Structural Implication of Parkin's Cysteine Residues—Inspection of the amino acid sequence of human parkin reveals a total of 35 Cys residues, the majority of which (23 out of 35) reside within the RING-IBR-RING domain of the protein (Fig. 1A). Further, comparison of human parkin protein sequence with orthologous sequences from rodent, fish, insect, and worm reveals that most of the parkin's Cys residues are absolutely conserved across these diverse species, a feature that suggests their importance to the protein's structure and/or function (Fig. 1A and supplemental Fig. S1). Although the invariant Cys residues in parkin across different species are largely found within the catalytically important RING-IBR-RING domain, a number of such highly conserved Cys residues, such as Cys-150, Cys-166, Cys-212, and Cys-457, are notably also found along the length of the protein outside of this domain (Fig. 1A). To gain insights into the importance of individual Cys residue on parkin to its overall tertiary architecture, it is essential to elucidate the three-dimensional structure of the protein, information of which is currently lacking. However, the structure of both RING1 and RING2 in related proteins, c-Cbl and HHARI, respectively, have previously been reported (13Capili A.D. Edghill E.L. Wu K. Borden K.L. J. Mol. Biol. 2004; 340: 1117-1129Crossref PubMed Scopus (85) Google Scholar, 14Zheng N. Wang P. Jeffrey P.D. Pavletich N.P. Cell. 2000; 102: 533-539Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar). Accordingly, we used the structure of c-Cbl RING1 and HHARI RING2 as templates to model parkin RING1 and RING2 domains, respectively. Homology models of parkin RING1 and RING2 so obtained reveal the coordination of Cys-238, Cys-241, Cys-260, and Cys-263 to a zinc atom in RING1 and the coordination of Cys-418, Cys-421, Cys-436, and Cys-441 to another zinc atom in RING2 (Fig. 1B). Although our program failed to model a small stretch of parkin sequence containing Cys-289 and Cys-293 residues accurately, these residues, together with Cys-253 and His-257, should coordinate a second zinc atom in RING1 in view of their high sequence homology to c-Cbl RING1 (14Zheng N. Wang P. Jeffrey P.D. Pavletich N.P. Cell. 2000; 102: 533-539Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar) (Fig. 1B). Not surprisingly, all of these structurally important Cys residues are absolutely conserved in parkin across different species (Fig. 1A and supplemental Fig. S1). On the other hand, Cys-268, which resides on a solvent-exposed surface (supplemental Fig. S2A) and does not appear to have a critical structural role in RING1 (Fig. 1B), is replaced by a leucine (Leu) in C. elegans parkin (supplemental Fig. S1) and by other amino acid residues in related proteins such as RBCK1 (isoleucine) and ariadne-2 (phenylalanine) (19Morett E. Bork P. Trends Biochem. Sci. 1999; 24: 229-231Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Similarly, the presumably nonstructural Cys-451 residue proximal to RING 2 (Fig. 1B) is poorly conserved among parkin from different species (supplemental Fig. S1). Since the structure of parkin's UBL domain is known (20Sakata E. Yamaguchi Y. Kurimoto E. Kikuchi J. Yokoyama S. Yamada S. Kawahara H. Yokosawa H. Hattori N. Mizuno Y. Tanaka K. Kato K. EMBO Rep. 2003; 4: 301-306Crossref PubMed Scopus (230) Google Scholar), we also inspected the structural position of Cys-59 and found that this Cys residue, like Cys-268, is located at a solvent-exposed loop on the surface of the UBL domain and not within its core (Fig. 1C), thereby offering some structural flexibility. Notably, in C. elegans parkin, Cys-59 is substituted with a Leu (supplemental Fig. S1). Taken together, the degree of Cys conservation in parkin across different species appears to correlate with their structural importance. Conceivably, modification of any of the numerous highly conserved Cys residues on parkin is likely to influence its structural topology, and thereby, its biochemical properties. Conserved Cysteine Residues on Parkin Residing Both Within and Outside the RING-IBR-RING Domain Are Important in Maintaining Its Solubility—To examine whether the modification of parkin's Cys residues would influence its solubility, we generated a large series of parkin Cys → Ala (C → A) point mutants that cover the length of the protein via site-directed mutagenesis and expressed each of these mutants in SH-SY5Y neuroblastoma cells (Fig. 2A). When cells transfected with these mutants were subjected to sequential detergent extraction, we found that all the C → A mutations occurring on Cys residues that are invariant in parkin across different species, except C431A, show preferential localization to the detergent-insoluble (P) fraction relative to control, wild-type parkin (Fig. 2A). Conversely, Cys residues on parkin such as Cys-59, Cys-95, Cys-268, Cys-323, Cys-431, and Cys-451 that are either not absolutely conserved among different species or otherwise appear structurally unimportant, or both, do not significantly alter parkin solubility when mutated (Fig. 2A). Consistent with our in silico sequence and structural prediction, our results suggest the importance of conserved parkin's Cys residues in maintaining the structure and thus solubility of the protein and that alteration of parkin solubility via the modification of its Cys residues is not limited to those residing within the RING-IBR-RING motif. We have previously demonstrated an association between altered parkin solubility and its propensity to form intracellular aggregates (18Wang C. Tan J.M. Ho M.W. Zaiden N. Wong S.H. Chew C.L. Eng P.W. Lim T.M. Dawson T.M. Lim K.L. J. Neurochem. 2005; 93: 422-431Crossref PubMed Scopus (110) Google Scholar). Since mutations of conserved and non-conserved Cys residues on parkin produce different effects on the protein's solubility, we were interested to know their respective influence in promoting parkin aggregation within the cell. For this purpose, representative pairs of mutants containing C → A substitutions of either an invariant or a non-invariant Cys at different regions of parkin were examined (Fig. 2B). Between the paired mutants, we found that aggresome-like structures occur more frequently in cells expressing the one substituting for the absolutely conserved Cys and vice versa (Fig. 2B). This is consistent with their respective solubility profile as described above (Fig. 2A). Quantitatively, C → A mutations occurring on invariant Cys residues located on the RING-IBR-RING domain or the C-terminal tail of the protein show the highest propensity to generate intracellular inclusions when compared with wild-type parkin as well as mutants bearing similar mutations at the N-terminal region of parkin (Fig. 2C). It thus appears that Cys modification occurring on the RING-IBR-RING domain or C-terminal end of parkin result in more significant alterations of the protein (i.e. solubility changes and higher tendency to aggregate) when compared with analogous modification occurring at other regions of the protein. Susceptibility of Parkin and Other Cysteine-containing Enzymes to Stress-induced Solubility Alterations—Given the demonstrated importance of parkin's Cys residues in maintaining its structural and biochemical properties, one could appreciate the reported alteration of parkin function via modification of its Cys residues (11LaVoie M.J. Ostaszewski B.L. Weihofen A. Schlossmacher M.G. Selkoe D.J. Nat. Med. 2005; 11: 1214-1221Crossref PubMed Scopus (607) Google Scholar). Since parkin and HHARI contain a comparable number of Cys residues (35 and 32, respectively), and the majority of these Cys are found within their RING-IBR-RING domains, it is likely that they share comparable sensitivities to modification by various stress-inducing agents. Thus, we were intrigued by recent findings showing that parkin, but not HHARI, is selectively vulnerable to DA-mediated modification (11LaVoie M.J. Ostaszewski B.L. Weihofen A. Schlossmacher M.G. Selkoe D.J. Nat. Med. 2005; 11: 1214-1221Crossref PubMed Scopus (607) Google Scholar). To examine the relative susceptibility of parkin and other cysteine-containing enzymes to stress-induced solubility alterations, we subjected SH-SY5Y cells ectopically expressing parkin, HHARI, c-Cbl (RING domain ubiquitin-protein isopeptide ligase (E3) with 23 Cys residues), CHIP (U box protein with 7 Cys residues), or UCH-L1 (ubiquitin hydrolase with 6 Cys residues) to a variety of stresses, including hydrogen peroxide (H2O2), NOC-18 (a nitrogen oxide donor), and DA, all of which have been previously reported to induce parkin insolubility (10Wang C. Ko H.S. Thomas B. Tsang F. Chew K.C. Tay S.P. Ho M.W. Lim T.M. Soong T.W. Pletnikova O. Troncoso J. Dawson V.L. Dawson T.M. Lim K.L. Hum. Mol. Genet. 2005; 14: 3885-3897Crossref PubMed Scopus (187) Google Scholar, 11LaVoie M.J. Ostaszewski B.L. Weihofen A. Schlossmacher M.G. Selkoe D.J. Nat. Med. 2005; 11: 1214-1221Crossref PubMed Scopus (607) Google Scholar). Consistent with their similar structure and abundance of Cys, we found that both parkin and HHARI are more or less equally susceptible to solubility alterations promoted by the treatment of cells with H2O2 and NOC-18 (Fig. 3A). On the other hand, H2O2-mediated stress has no apparent effects on the solubility of c-Cbl, CHIP, and UCH-L1 (Fig. 3A). Although NOC-18 treatment of transfected cells at 0.25 mm markedly altered the solubility of parkin and HHARI, its effects on c-Cbl, CHIP, and UCH-L1 are more apparent only at a higher dose of 0.5 mm (Fig. 3A). For DA-mediated stress, we used a similar treatment paradigm to that reported by LaVoie et al. (11LaVoie M.J. Ostaszewski B.L. Weihofen A. Schlossmacher M.G. Selkoe D.J. Nat. Med. 2005; 11: 1214-1221Crossref PubMed Scopus (607) Google Scholar). Interestingly, we found that DA treatment of cells ectopically expressing parkin promotes a significant increase in the amount of monomeric and high molecular weight (HMW) parkin species in the detergent-insoluble fraction without a corresponding decrease in the levels of soluble parkin (Fig. 3B). Surprisingly, this phenomenon is specific to parkin as all the other Cys-containing enzymes examined, including HHARI, remain unaffected, suggesting that parkin is uniquely sensitive to DA-mediated modification (Fig. 3B). Although our findings on the effect of DA on parkin are consistent with that reported earlier (11LaVoie M.J. Ostaszewski B.L. Weihofen A. Schlossmacher M.G. Selkoe D.J. Nat. Med. 2005; 11: 1214-1221Crossref PubMed Scopus (607) Google Scholar), HHARI, despite sharing similar RING-IBR-RING structure and comparable sensitivity to parkin toward the other stress paradigms examined, is spared from DA-mediated effects. Nonetheless, when taken together, our results suggest that RING-IBR-RING proteins are more sensitive than other cysteine-containing enzymes to stress-induced modification but parkin is selectively vulnerable to DA-induced alterations. DA Modifies Residues Residing on the RING-IBR-RING Motif as well as the Linker Region of Parkin—To map the region on parkin that confers its unique susceptibility to DA-induced modification, we generated deletion mutants of FLAG-tagged parkin devoid either of its linker region (parkin Δ77-237) or its RING-IBR-RING domain (parkin 1-237 and parkin 1-137) (Fig. 4A). We then treated cells expressing these various parkin deletion mutants with the same concentrations of DA as described above. Although DA treatment of cells expressing parkin 1-237 and parkin Δ77-237 promotes an accumulation of detergent-insoluble parkin species in a manner similar to that observed with full-length parkin, parkin 1-137 is apparently spared from DA-mediated modification, even at the higher concentration of DA dose used (Fig. 4B). Thus, the region on parkin stretching from amino acids 138-237 appears to be as susceptible to modification by DA as the RING-IBR-RING domain. Interestingly, this stretch of parkin sequence contains several Cys residues that are not found in HHARI. Given the apparent selective sensitivity of parkin to DA-mediated modification, it is tempting to suggest from our results that DA preferentially modifies Cys residues that are located either at the unique linker region and/or at the C-terminal portion of protein involving residues such as Cys-268, Cys-323, and Cys-451 that are also unique to parkin. DA Preferentially Modifies Cys-268 and Cys-323 on Parkin—Since DA modification of parkin promotes the formation of insoluble monomeric and HMW parkin species without causing a corresponding decrease in the levels of soluble parkin (Fig. 3B), it is reasonable to assume that DA targets either non-conserved Cys residues or those that are structurally less important. Although four of such Cys residues (Cys-268, Cys-323, Cys-431, and Cys-451) are present at the C-terminal region of parkin, only 1 (Cys-182) is found between amino acids 138 and 237. Accordingly, we repeated the above experiment with a compendium of parkin mutants containing C → A substitution at position Cys-182, Cys-268, Cys-323, Cys-431, or Cys-451. We also included an insoluble mutant, C441A, as a control. Surprisingly, we found that DA modifies the parkin C182A mutant in a similar fashion to that observed with the wild-type protein (Fig. 5A). Parkin C441A, found predominantly in the detergent-insoluble fraction, also appears to be modified by DA (Fig. 5A). On the other hand, two parkin mutants, C268A and C323A, residing on RING1 and IBR domain of parkin, respectively, are apparently significantly more resistant to DA-mediated modification when compared with their counterparts (Fig. 5A). Although DA treatment of cells expressing wild-type, C431A, or C451A parkin produce robust amounts of insoluble monomeric and HMW parkin species, the amounts of both these insoluble parkin species are dramatically reduced in the case of the C268A and C323A mutants (Fig. 5A). Notably, the levels of insoluble parkin C323A species generated by DA treatment are so modest that they compare well with untreated wild-type parkin control (Fig. 5A). We next examined the effects of DA on the cellular localization of wild-type, Cys-268, and Cys-323 parkin. Consistent with its susceptibility to DA-mediated modification, we found that cells expressing wild-type parkin exhibit a high tendency to form parkin-positive inclusions following DA treatment. In contrast, such inclusions occur rarely in DA-treated cells expressing C268A and C323A parkin mutant (Fig. 5B). Taken together, our results demonstrate that mutation of Cys-268 and Cys-323, respectively, to alanine render parkin less susceptible to insolubility induced by DA treatment, suggesting that DA modification of parkin's Cys residues takes place predominantly at these Cys residues. Distribution of Various Cysteine-containing Enzymes in Normal and PD Human Brains—We and others have previously demonstrated a significant increase in the amount of detergent-insoluble parkin in the caudate region of idiopathic PD brains when compared with those in control brains (10Wang C. Ko H.S. Thomas B. Tsang F. Chew K.C. Tay S.P. Ho M.W. Lim T.M. Soong T.W. Pletnikova O. Troncoso J. Dawson V.L. Dawson T.M. Lim K.L. Hum. Mol. Genet. 2005; 14: 3885-3897Crossref PubMed Scopus (187) Google Scholar, 11LaVoie M.J. Ostaszewski B.L. Weihofen A. Schlossmacher M.G. Selkoe D.J. Nat. Med. 2005; 11: 1214-1221Crossref PubMed Scopus (607) Google Scholar). To examine whether a similar phenomenon occurs with other cysteine-containing enzymes, particularly HHARI, in PD and control brains, we performed anti-HHARI, anti-UCH-L1, anti-c-Cbl, and anti-CHIP immunoblotting on fractionated detergent-insoluble lysates prepared from post-mortem normal and PD brains (Fig. 6A). In contrast to parkin, as reported previously (10Wang C. Ko H.S. Thomas B. Tsang F. Chew K.C. Tay S.P. Ho M.W. Lim T.M. Soong T.W. Pletnikova O. Troncoso J. Dawson V.L. Dawson T.M. Lim K.L. Hum. Mol. Genet. 2005; 14: 3885-3897Crossref PubMed Scopus (187) Google Scholar, 11LaVoie M.J. Ostaszewski B.L. Weihofen A. Schlossmacher M.G. Selkoe D.J. Nat. Med. 2005; 11: 1214-1221Crossref PubMed Scopus (607) Google Scholar), we did not observe an accumulation of any of the enzymes examined in the detergent-insoluble fraction of PD over control brain samples (Fig. 6B). Quantitatively, we only recorded a significant difference in the levels of CHIP between PD and control brains (Fig. 6B). However, the amount of CHIP decreased, instead of increased, in PD brains when compared with controls (Fig. 6B). Taken together, our results demonstrate that unlike parkin, HHARI, UCH-L1, c-Cbl, and CHIP do not accumulate in the detergent-insoluble fractions of PD brains, suggesting that parkin is uniquely sensitive to stress-induced modification in the caudate region of the brain. In this report, we demonstrate that the modification of any of the numerous highly conserved Cys residues on parkin (except Cys-431), both within and outside its RING-IBR-RING motif, leads to alterations of the protein biochemical and cellular properties, and thereby, its function. As several of these structurally important Cys on parkin are not found in other related proteins, our findings suggest an increased vulnerability of parkin to stress-induced modification. Indeed, parkin appears to be more susceptible to oxidative and nitrosative stress and uniquely sensitive to DA-mediated stress. Given parkin's neuroprotective roles and the oxidative environment of dopaminergic neurons, the enhanced sensitivity of parkin to intracellular stress may underlie the progressive susceptibility of an individual to PD. Of the 20 naturally occurring amino acids found in proteins, cysteines are recognized to be exceptionally susceptible to oxidative modification due to the presence of sulfhydryl groups (21Stokes A.H. Hastings T.G. Vrana K.E. J. Neurosci. Res. 1999; 55: 659-665Crossref PubMed Scopus (392) Google Scholar). Sulfhydryl groups are the strongest nucleophile in the cell at physiological pH and thus represent ideal targets for nucleophilic attack by oxidants or nitrosative agents. Accordingly, the abundance of Cys residues on a protein should, in part, contribute to the tendency for the protein to be modified by cellular oxidants. We found that this appears to be the case when we subjected enzymes with different cysteine content to the effects of oxidative/nitrosative stress agents. Although parkin and HHARI, both containing over 30 Cys residues, are comparably susceptible to H2O2- or NOC-18-mediated effects, c-Cbl, CHIP, and UCH-L1, containing 23, 7, and 6 Cys residues, respectively, either remain inert to these stress agents or otherwise require higher concentrations of these agents to produce a similar effect observed with parkin and HHARI. The extent of a protein's alteration via its Cys modification is obviously also related to the importance of the targeted Cys residue to the overall tertiary structure of the protein. We have demonstrated that the large majority of Cys residues residing on parkin (23 out of 28 examined), both within and outside the RING-IBR-RING domain, are important in maintaining its solubility. With the notable exception of Cys-431, all the Cys residues of parkin found to be invariant across diverse species resulted in parkin insolubility when they are mutated to alanine, suggesting their importance in fulfilling critical structural roles. Although the Zn2+-coordinating Cys residues in RING1 and RING2 are obviously structurally important, it is interesting that almost all the Cys residues located at the IBR, a domain whose function remains unclear, appears to be critically important for the native folding of parkin. On the other hand, despite being an absolutely conserved residue, Cys-431 does not appear to play an important role in maintaining parkin's RING2 structure, which probably explains its lack of effects on parkin solubility when mutated. Nonetheless, our result with the C431A mutant in this study appears to contradict our previous finding that the C431F mutant promotes parkin insolubilty and intracellular aggregation (18Wang C. Tan J.M. Ho M.W. Zaiden N. Wong S.H. Chew C.L. Eng P.W. Lim T.M. Dawson T.M. Lim K.L. J. Neurochem. 2005; 93: 422-431Crossref PubMed Scopus (110) Google Scholar). In the latter case, it is conceivable that the substitution of Cys with the bulky Phe could result in marked steric hindrance that might indirectly affect parkin's RING2 structure. Accordingly, we generated a homology model of parkin's RING2 structure with Phe substituting for Cys at position 431 and found that the benzene ring of Phe may interfere sterically with the RING structure (supplemental Fig. S2B). We speculated that substitution of Cys-431 with the equally bulky Tyr residue would bring about similar effects to that observed with the C431F mutant. Using both homology modeling and in vitro experiments similar to the ones described above, we were able to confirm our speculation with the C431Y mutant (supplemental Fig. S2, B and C). Thus, parkin solubility alteration mediated by C431F mutation is likely to arise from steric rather than overt structural aberrations. Interestingly, all the invariant Cys residues residing within the parkin RING-IBR-RING motif are also conserved among members of the RING-IBR-RING family (19Morett E. Bork P. Trends Biochem. Sci. 1999; 24: 229-231Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). This feature probably explains the comparable susceptibility of HHARI and parkin to H2O2 or NOC-18-mediated solubility alterations, and at the same time, suggests the vulnerability of other RING-IBR-RING members to stress-induced modifications. The implication of this is that oxidative stress occurring in the brain, particularly in dopaminergic neurons, could promote the dysfunction of not just parkin but potentially all RING-IBR-RING family members as well. It is well known that dopaminergic neurons in the brain are particularly exposed to oxidative stress because the metabolism of DA produces various reactive oxygen species (peroxide, superoxide, and hydroxyl radicals) (21Stokes A.H. Hastings T.G. Vrana K.E. J. Neurosci. Res. 1999; 55: 659-665Crossref PubMed Scopus (392) Google Scholar, 22Lotharius J. Brundin P. Nat. Rev. Neurosci. 2002; 3: 932-942Crossref PubMed Scopus (988) Google Scholar). If not handled properly, the reactive oxygen species generated could create a considerable damaging environment. Further, DA could auto-oxidize to DA-quinone, a reactive species that has been demonstrated to covalently modify cellular macromolecules, including parkin, and contribute to DA-induced neurotoxicity (11LaVoie M.J. Ostaszewski B.L. Weihofen A. Schlossmacher M.G. Selkoe D.J. Nat. Med. 2005; 11: 1214-1221Crossref PubMed Scopus (607) Google Scholar, 21Stokes A.H. Hastings T.G. Vrana K.E. J. Neurosci. Res. 1999; 55: 659-665Crossref PubMed Scopus (392) Google Scholar, 22Lotharius J. Brundin P. Nat. Rev. Neurosci. 2002; 3: 932-942Crossref PubMed Scopus (988) Google Scholar). However, unlike our previous observation with parkin (10Wang C. Ko H.S. Thomas B. Tsang F. Chew K.C. Tay S.P. Ho M.W. Lim T.M. Soong T.W. Pletnikova O. Troncoso J. Dawson V.L. Dawson T.M. Lim K.L. Hum. Mol. Genet. 2005; 14: 3885-3897Crossref PubMed Scopus (187) Google Scholar), we did not observe an accumulation of HHARI in the detergent-insoluble fraction from the caudate region of PD brains relative to controls. It would appear that parkin is uniquely vulnerable in this region of the brain, at least when compared with HHARI, to PD-linked stress. Conceivably, the modification of parkin in this case must involve regions or residues of the protein that are distinct from HHARI. Interestingly, DA modification of parkin appears to fit this requirement nicely, as suggested by our following findings in this study. 1) DA-mediated promotion of insoluble parkin species is apparently a specific phenomenon as neither HHARI nor the other cysteine-containing enzymes examined are modified by DA in such a manner, even at the higher concentration of DA used. 2) DA could modify a truncated form of parkin bearing its unique linker region but devoid of the RING-IBR-RING motif in a similar manner to that observed with the wild-type protein. However, we failed to pinpoint the Cys residue involved in this DA-mediated effect and therefore cannot exclude alternative possibilities. 3) Importantly, DA appears to predominantly target 2 Cys residues, Cys-268 and Cys-323, on RING1 and IBR domain, respectively, which are unique to parkin and thus not found in HHARI. It is noteworthy that C → A substitution of these 2 non-conserved Cys residues does not produce markedly insoluble proteins. This might explain why DA-mediated accumulation of detergent-insoluble parkin species occurs without apparent depletion of corresponding detergent-soluble parkin species, a milder effect when compared with the solubility alterations of parkin produced by H2O2 or NOC-18 as described above or by various other stressors that we have previously reported (10Wang C. Ko H.S. Thomas B. Tsang F. Chew K.C. Tay S.P. Ho M.W. Lim T.M. Soong T.W. Pletnikova O. Troncoso J. Dawson V.L. Dawson T.M. Lim K.L. Hum. Mol. Genet. 2005; 14: 3885-3897Crossref PubMed Scopus (187) Google Scholar). Consistent with this, both C268A and C323A fail to protect parkin against H2O2-induced solubility alterations (data not shown). Given the selective vulnerability of brain parkin to stress-induced modifications, as shown in this study, it is tempting to suggest that the principal culprit promoting brain parkin insolubility is likely DA, DA-related, or otherwise one that influences parkin in a similar manner to that brought about by DA. Whether and how detergent-insoluble monomeric and HMW parkin species influence soluble parkin function and cellular survivability remain to be characterized. However, their association with PD brains (10Wang C. Ko H.S. Thomas B. Tsang F. Chew K.C. Tay S.P. Ho M.W. Lim T.M. Soong T.W. Pletnikova O. Troncoso J. Dawson V.L. Dawson T.M. Lim K.L. Hum. Mol. Genet. 2005; 14: 3885-3897Crossref PubMed Scopus (187) Google Scholar, 11LaVoie M.J. Ostaszewski B.L. Weihofen A. Schlossmacher M.G. Selkoe D.J. Nat. Med. 2005; 11: 1214-1221Crossref PubMed Scopus (607) Google Scholar) would suggest pathogenic roles. In conclusion, our results here offer significant insights into the components important for parkin solubility, and at the same time, provide some structural basis for the solubility alterations of the protein produced by cellular stress. Further, we have mapped 2 Cys residues on parkin that render the protein less susceptible to insolubility induced by DA treatment, thus potentially representing the major target sites of DA-mediated modification. Notably, both these residues are unique to parkin and might therefore explain the selective vulnerability of parkin to DA-induced changes. Future experiments should clarify the potential pathogenecity of the insoluble parkin species promoted by DA modification. We thank Michelle Ho for technical assistance. We thank Juan Troncoso and Olga Pletinkova and the Morris K. Udall Parkison's Disease Center Neuropathology Core for the human postmortem brain material. Download .pdf (.14 MB) Help with pdf files

Mutations in the genes coding for α-synuclein and parkin cause autosomal-dominant and autosomal-recessive forms of Parkinson's disease (PD), respectively. α-Synuclein is a major component of Lewy bodies, the proteinaceous cytoplasmic inclusions that are the pathological hallmark of idiopathic PD. Lewy bodies appear to be absent in cases of familial PD associated with mutated forms of parkin. Parkin is an ubiquitin E3 ligase, and it may be involved in the processing and/or degradation of α-synuclein, as well as in the formation of Lewy bodies. Here we report the behavioral, biochemical, and histochemical characterization of double-mutant mice overexpressing mutant human A53T α-synuclein on a parkin null background. We find that the absence of parkin does not have an impact on the onset and progression of the lethal phenotype induced by overexpression of human A53T α-synuclein. Furthermore, all major behavioral, biochemical, and morphological characteristics of A53T α-synuclein-overexpressing mice are not altered in parkin null α-synuclein-overexpressing double-mutant mice. Our results demonstrate that mutant α-synuclein induces neurodegeneration independent of parkin-mediated ubiquitin E3 ligase activity in nondopaminergic systems and suggest that PD caused by α-synuclein and parkin mutations may occur via independent mechanisms.

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. Although a subject of intense research, the etiology of PD remains poorly understood. Over the last decade, the ubiquitin–proteasome system (UPS) has emerged as a compelling player in PD pathogenesis. Disruption of the UPS, which normally identifies and degrades intracellular proteins, is thought to promote the toxic accumulation of proteins detrimental to neuronal survival, thereby contributing to their demise. Support for this came from a broad range of studies, including genetics, gene profiling and post-mortem analysis, as well as in vitro and in vivo modeling. Notably, various cellular and animal models of PD based on direct disruption of UPS function reproduce the salient features of PD. However, several gaps remain in our current knowledge regarding the precise role of UPS dysfunction in the pathogenesis of the disease. Current thoughts regarding their relationship are reviewed here and some major unresolved questions, the clarification of which would considerably advance our understanding of the implicated role of the UPS in PD pathogenesis, are discussed.

The proteasome, which identifies and destroys unwanted proteins rapidly, plays a vital role in maintaining cellular protein homeostasis. Proteins that are destined for proteasome-mediated degradation are usually tagged with a chain of ubiquitin linked via lysine (K) 48 that targets them to the proteolytic machinery. However, when the proteasome becomes compromised in its function, it is attractive to think that the cell may switch to an alternative, non-proteolytic form of ubiquitination that could help divert cargo proteins away from an otherwise overloaded proteasome. Of the several types of ubiquitin chain topologies, K63-linked ubiquitination is the only one known to fulfil diverse proteasome-independent roles, including DNA repair, endocytosis and NFκB signaling. By virtue of its apparent dissociation from the proteasome, we have originally proposed that K63-linked ubiquitination may be involved in cargo diversion during proteasomal stress and accordingly, in the biogenesis of inclusion bodies associated with neurodegenerative diseases. Here, we provide an overview of this non-classic form of ubiquitin modification, and discuss current evidence and controversies surrounding our proposed role for K63 polyubiquitin as a key regulator of inclusion dynamics that is relevant to neurodegeneration. This article is part of a Special Issue entitled “Autophagy and protein degradation in neurological diseases.”

AbstractAlthough protein inclusions associated with neurodegenerative diseases are typically enriched with ubiquitin, it is currently unclear whether the topology of ubiquitin linkage plays a role in their biogenesis. In an attempt to clarify this, our recent work identified K63-linked polyubiquitin as a key regulator of inclusion dynamics. We found in the setting of ectopic overexpression of different ubiquitin species in cultured cells that K63-linked ubiquitination promotes the formation and autophagic clearance of protein inclusions linked to several major neurodegenerative diseases. Further supporting this, we report here a similar phenomenon in cells co-expressing Ubc13 and Uev1a but not those expressing UbcH7 or UbcH8. Notably, Ubc13 in association with Uev1a is known to promote K63-linked ubiquitination. In exploring how K63-linked ubiquitination could promote the clearance of inclusions by autophagy, we also found in our current study that K63-linked polyubiquitin interacts with p62, a ubiquitin-binding protein previously demonstrated by others to facilitate autophagy-mediated clearance of inclusions. Further, K63 ubiquitin-positive inclusions were found to be enriched with p62. Given the observed intimate relationship between p62 and K63 polyubiquitin, our results suggest that p62 and K63-linked polyubiquitin may function as key partners involved in directing clearance of protein inclusions by autophagy.Addendum to: Tan JMM, Wong ESP, Kirkpatrick DS, Pletnikova O, Ko HS, Tay S-P, Ho M.W.L., Troncoso J, Gygi SP, Lee MK, Dawson VL, Dawson TM, Lim K-L. Lysine 63-linked ubiquitination promotes the formation and autophagic clearance of protein inclusions associated with neurodegenerative diseases. Human Mol Genet; In press.

Significance: Parkinson’s disease (PD) is a prevalent neurodegenerative disease affecting millions of individuals worldwide. Despite intensive efforts devoted to drug discovery, the disease remains incurable. To provide more effective medical therapy for PD, better understanding of the underlying causes of the disease is clearly necessary. Recent Advances: A broad range of studies conducted over the past few decades have collectively implicated aberrant mitochondrial homeostasis as a key contributor to the development of PD. Supporting this, mutations in several PD-linked genes are directly or indirectly linked to mitochondrial dysfunction. In particular, recent discoveries have identified parkin, whose mutations are causative of recessive parkinsonism, as a key regulator of mitochondrial homeostasis. Critical Issues: Parkin appears to be involved in the entire spectrum of mitochondrial dynamics, including organelle biogenesis, fusion/fission, and clearance via mitophagy. How a single protein can regulate such diverse mitochondrial events is as intriguing as it is amazing; the mechanism underlying this is currently under intense research. Here, we provide an overview of mitochondrial dynamics and its relationship with neurodegenerative diseases and discuss current evidence and controversies surrounding the role of parkin in mitochondrial quality control and its relevance to PD pathogenesis. Future Directions: Although the emerging field of parkin-mediated mitochondrial quality control has proven to be exciting, it is important to recognize that PD pathogenesis is likely to involve an intricate network of interacting pathways. Elucidating the reciprocity of pathways, particularly how other PD-related pathways potentially influence mitochondrial homeostasis, may hold the key to therapeutic development. Antioxid. Redox Signal. 16, 935–949.

Although a subject of intense research, the mechanisms underlying dopaminergic neurodegeneration in Parkinson's disease (PD) remains poorly understood. However, a broad range of studies conducted over the past few decades, including epidemiological, genetic, and post-mortem analysis, as well as in vitro and in vivo modeling, have contributed significantly to our understanding of the pathogenesis of the disease. In particular, the recent identification and functional characterization of several genes, including α-synuclein, parkin, DJ-1, PINK1, and LRRK2, whose mutations are causative of rare familial forms of PD have provided tremendous insights into the molecular pathways underlying dopaminergic neurodegeneration. Collectively, these studies implicate aberrant mitochondrial and protein homeostasis as key contributors to the development of PD, with oxidative stress likely acting as an important nexus between the two pathogenic events. Aberrations in homeostatic processes leading to protein aggregation and mitochondrial dysfunction may arise intrinsically in substantia nigra pars compacta dopaminergic neurons as a result of impairments in the ubiquitin-proteasome system, failure in autophagy-mediated clearance, alterations of mitochondrial dynamics, redox imbalance, iron mishandling, dopamine dysregulation, or simply from the chronic pace-making activity of nigra-localized L-type calcium channels, or extrinsically from non-autonomous sources of stress. Given the myriad of culprits implicated, the pathogenesis of PD necessarily involves an intricate network of interwoven pathways rather than a linear sequence of events. Obviously, understanding how the various disease-associated pathways interact with and influence each other is of mechanistic and therapeutic importance. Here, we shall discuss some key PD-related pathways and how they are interwoven together into a tapestry of events.

Current pharmacological strategies for Parkinson's disease (PD), the most common neurological movement disorder worldwide, are predominantly symptom relieving and are often plagued with undesirable side effects after prolonged treatment. Despite this, they remain as the mainstay treatment for PD due to the lack of better alternatives. Nutraceuticals are compounds derived from natural food sources that have certain therapeutic value and the advent of which has opened doors to the use of alternative strategies to tackle neurodegenerative diseases such as PD. Notably, nutraceuticals are able to position themselves as a "safer" strategy due to the fact that they are naturally derived compounds, therefore possibly having less side effects. Significant efforts have been put into better comprehending the role of nutraceuticals in PD, and we will look at some of them in this review. Broadly speaking, these compounds execute their positive effects via modulating signalling pathways, inhibiting oxidative stress, inflammation and apoptosis, as well as regulating mitochondrial homoeostasis. Importantly, we will highlight how a component of green tea, epigallocatechin-3-gallate (EGCG), confers neuroprotection in PD via its ability to activate AMP kinase and articulate how its beneficial effects in PD are possibly due to enhancing mitochondrial quality control.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are common causes of familial Parkinson's disease (PD). LRRK2 has been shown to bind peroxiredoxin-3 (PRDX3), the most important scavenger of hydrogen peroxide in the mitochondria, in vitro. Here, we examined the interactions of LRRK2 and PRDX3 in Drosophila models by crossing transgenic LRRK2 and PRDX3 flies. As proof of principle experiments, we subsequently challenged LRRK2 and LRRK2/PRDX3 flies with a peroxidase mimic, Ebselen. We demonstrated that co-expression of PRDX3 with the LRRK2 kinase mutant G2019S in bigenic Drosophila ameliorated the G2019S mutant-induced reduction in peroxidase capacity, loss of dopaminergic neurons, shortened lifespan and mitochondrial defects of flight muscles in monogenic flies expressing the G2019S alone. Challenges with Ebselen recapitulated similar rescue of these phenotypic features in mutant-expressing Drosophila. The peroxidase mimic preserved neuronal and mitochondrial and neuronal integrity and improved mobility and survival in mutant-expressing Drosophila. Taken together, our study provides the first in vivo evidence to suggest that phosphoinhibition of endogenous peroxidases could be a mechanism in LRRK2-induced oxidant-mediated neurotoxicity. Our therapeutic experiments also highlight the potential of thiol peroxidases as neuroprotective agents in PD patients carrying LRRK2 mutations.

Mutations in LRRK2, which encodes leucine-rich repeat kinase 2, are the most common genetic cause of familial and sporadic Parkinson's disease (PD), a degenerative disease of the central nervous system that causes impaired motor function and, in advanced stages, dementia. Dementia is a common symptom of another neurodegenerative disease, Alzheimer's disease, and research suggests that there may be pathophysiological and genetic links between the two diseases. Aggregates of β amyloid [a protein produced through cleavage of amyloid precursor protein (APP)] are seen in both diseases and in PD patients carrying G2019S-mutant LRRK2. Using patient-derived cells, brain tissue, and PD model mice, we found that LRRK2 interacted with and phosphorylated APP at Thr668 within its intracellular domain (AICD). Phosphorylation of APP at Thr668 promoted AICD transcriptional activity and correlated with increased nuclear abundance of AICD and decreased abundance of a dopaminergic neuron marker in cultures and brain tissue. The AICD regulates the transcription of genes involved in cytoskeletal dynamics and apoptosis. Overexpression of AICD, but not a phosphodeficient mutant (AICDT668A), increased the loss of dopaminergic neurons in older mice expressing LRRK2G2019S Moreover, the amount of Thr668-phosphorylated APP was substantially greater in postmortem brain tissue and dopaminergic neurons (generated by reprogramming skin cells) from LRRK2G2019S patients than in those from healthy individuals. LRRK2 inhibitors reduced the phosphorylation of APP at Thr668 in the patient-derived dopaminergic neurons and in the midbrains of LRRK2G2019S mice. Thus, APP is a substrate of LRRK2, and its phosphorylation promotes AICD function and neurotoxicity in PD.

Despite intensive research, the etiology of Parkinson's disease (PD) remains poorly understood and the disease remains incurable. However, compelling evidence gathered over decades of research strongly support a role for mitochondrial dysfunction in PD pathogenesis. Related to this, PGC-1α, a key regulator of mitochondrial biogenesis, has recently been proposed to be an attractive target for intervention in PD. Here, we showed that silencing of expression of the Drosophila PGC-1α ortholog spargel results in PD-related phenotypes in flies and also seem to negate the effects of AMPK activation, which we have previously demonstrated to be neuroprotective, that is, AMPK-mediated neuroprotection appears to require PGC-1α. Importantly, we further showed that genetic or pharmacological activation of the Drosophila PGC-1α ortholog spargel is sufficient to rescue the disease phenotypes of Parkin and LRRK2 genetic fly models of PD, thus supporting the proposed use of PGC-1α-related strategies for neuroprotection in PD.

As the main driver of energy production in eukaryotes, mitochondria are invariably implicated in disorders of cellular bioenergetics. Given that dopaminergic neurons affected in Parkinson's disease (PD) are particularly susceptible to energy fluctuations by their high basal energy demand, it is not surprising to note that mitochondrial dysfunction has emerged as a compelling candidate underlying PD. A recent approach towards forestalling dopaminergic neurodegeneration in PD involves near-infrared (NIR) photobiomodulation (PBM), which is thought to enhance mitochondrial function of stimulated cells through augmenting the activity of cytochrome C oxidase. Notwithstanding this, our understanding of the neuroprotective mechanism of PBM remains far from complete. For example, studies focusing on the effects of PBM on gene transcription are limited, and the mechanism through which PBM exerts its effects on distant sites (i.e., its "abscopal effect") remains unclear. Also, the clinical application of NIR in PD proves to be challenging. Efficacious delivery of NIR light to the substantia nigra pars compacta (SNpc), the primary site of disease pathology in PD, is fraught with technical challenges. Concerted efforts focused on understanding the biological effects of PBM and improving the efficiency of intracranial NIR delivery are therefore essential for its successful clinical translation. Nonetheless, PBM represents a potential novel therapy for PD. In this review, we provide an update on the role of mitochondrial dysfunction in PD and how PBM may help mitigate the neurodegenerative process. We also discussed clinical translation aspects of this treatment modality using intracranially implanted NIR delivery devices.

Aberrant aggregation of α-synuclein (α-syn) is a key pathological feature of Parkinson's disease (PD), but the precise role of intestinal α-syn in the progression of PD is unclear. In a number of genetic Drosophila models of PD, α-syn was frequently ectopically expressed in the neural system to investigate the pathobiology.We investigated the potential role of intestinal α-syn in PD pathogenesis using a Drosophila model. Human α-syn was overexpressed in Drosophila guts, and life span, survival, immunofluorescence and climbing were evaluated. Immunofluorescence, Western blotting and reactive oxygen species (ROS) staining were performed to assess the effects of intestinal α-syn on intestinal dysplasia. High-throughput RNA and 16S rRNA gene sequencing, quantitative RT-PCR, immunofluorescence, and ROS staining were performed to determine the underlying molecular mechanism.We found that the intestinal α-syn alone recapitulated many phenotypic and pathological features of PD, including impaired life span, loss of dopaminergic neurons, and progressive motor defects. The intestine-derived α-syn disrupted intestinal homeostasis and accelerated the onset of intestinal ageing. Moreover, intestinal expression of α-syn induced dysbiosis, while microbiome depletion was efficient to restore intestinal homeostasis and ameliorate the progression of PD. Intestinal α-syn triggered ROS, and eventually led to the activation of the dual oxidase (DUOX)-ROS-Jun N-terminal Kinase (JNK) pathway. In addition, α-syn from both the gut and the brain synergized to accelerate the progression of PD.The intestinal expression of α-syn recapitulates many phenotypic and pathologic features of PD, and induces dysbiosis that aggravates the pathology through the DUOX-ROS-JNK pathway in Drosophila. Our findings provide new insights into the role of intestinal α-syn in PD pathophysiology.

Objective: Vascular dementia (VaD) is the second most common cause of dementia worldwide. The increasing contribution of lifestyle-associated risk factors to VaD has pointed towards gene-environment interactions (i.e. epigenetics). This study thus aims to investigate the DNA methylation landscape in a chronic cerebral hypoperfusion (CCH) mouse model of VaD. As a nexus between the gene-environment interaction, intermittent fasting (IF) was introduced as a prophylactic intervention. Methods: Bilateral common carotid artery stenosis (BCAS) was used to induce CCH by placing micro-coils of 0.18 mm in each common carotid artery of the mice. The coils were left in the mice for 7, 15 and 30 days to study temporal differences. IF was introduced for 16 h daily for 4 months prior to BCAS. Reduced Representation Bisulfite Sequencing (RRBS) was used to study the DNA methylation landscape. Cognitive impairment was measured using Barnes Maze Test. White matter lesions (WML) and neuronal loss were measured using Luxol fast blue staining and cresyl violet staining respectively. Results: IF mice subjected to CCH displayed significantly better cognitive learning ability and memory, improved neuropathological alterations with reduced WMLs and neuronal loss. Modulation of DNA methylation patterns in the cortex of AL CCH mice was re-modelled and signs of reversal was observed in IF CCH mice across all three timepoints. Conclusions: These findings provide an understanding of how IF may protect the brain against damage caused by CCH and show promise in offering potential beneficial effects in mitigating the neuropathology and cognitive deficits in VaD.

Parkinson's Disease (PD) is a prevalent neurodegenerative disorder that is characterized pathologically by the loss of A9-specific dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) of the midbrain. Despite intensive research, the etiology of PD is currently unresolved, and the disease remains incurable. This, in part, is due to the lack of an experimental disease model that could faithfully recapitulate the features of human PD. However, the recent advent of induced pluripotent stem cell (iPSC) technology has allowed PD models to be created from patient-derived cells. Indeed, DA neurons from PD patients are now routinely established in many laboratories as monolayers as well as 3D organoid cultures that serve as useful toolboxes for understanding the mechanism underlying PD and also for drug discovery. At the same time, the iPSC technology also provides unprecedented opportunity for autologous cell-based therapy for the PD patient to be performed using the patient's own cells as starting materials. In this review, we provide an update on the molecular processes underpinning the development and differentiation of human pluripotent stem cells (PSCs) into midbrain DA neurons in both 2D and 3D cultures, as well as the latest advancements in using these cells for drug discovery and regenerative medicine. For the novice entering the field, the cornucopia of differentiation protocols reported for the generation of midbrain DA neurons may seem daunting. Here, we have distilled the essence of the different approaches and summarized the main factors driving DA neuronal differentiation, with the view to provide a useful guide to newcomers who are interested in developing iPSC-based models of PD.

We have examined the in vivo activity of receptor-like protein-tyrosine phosphatase α (PTPα) toward p59fyn, a widely expressed Src family kinase. In a coexpression system, PTPα effected a dose-dependent tyrosine dephosphorylation and activation of p59fyn, where maximal dephosphorylation correlated with a 5-fold increase in kinase activity. PTPα expression resulted in increased accessibility of the p59fyn SH2 domain, consistent with a PTPα-mediated dephosphorylation of the regulatory C-terminal tyrosine residue of p59fyn. No p59fyn dephosphorylation was observed with an enzymatically inactive mutant form of PTPα or with another receptor-like PTP, CD45. Many enzyme-linked receptors are complexed with their substrates, and we examined whether PTPα and p59fyn underwent association. Reciprocal immunoprecipitations and assays detected p59fyn and an appropriate kinase activity in PTPα immunoprecipitates and PTPα and PTP activity in p59fyn immunoprecipitates. No association between CD45 and p59fyn was detected in similar experiments. The PTPα-mediated activation of p59fyn is not prerequisite for association since wild-type and inactive mutant PTPα bound equally well to p59fyn. Endogenous PTPα and p59fyn were also found in association in mouse brain. Together, these results demonstrate a physical and functional interaction of PTPα and p59fyn that may be of importance in PTPα-initiated signaling events. We have examined the in vivo activity of receptor-like protein-tyrosine phosphatase α (PTPα) toward p59fyn, a widely expressed Src family kinase. In a coexpression system, PTPα effected a dose-dependent tyrosine dephosphorylation and activation of p59fyn, where maximal dephosphorylation correlated with a 5-fold increase in kinase activity. PTPα expression resulted in increased accessibility of the p59fyn SH2 domain, consistent with a PTPα-mediated dephosphorylation of the regulatory C-terminal tyrosine residue of p59fyn. No p59fyn dephosphorylation was observed with an enzymatically inactive mutant form of PTPα or with another receptor-like PTP, CD45. Many enzyme-linked receptors are complexed with their substrates, and we examined whether PTPα and p59fyn underwent association. Reciprocal immunoprecipitations and assays detected p59fyn and an appropriate kinase activity in PTPα immunoprecipitates and PTPα and PTP activity in p59fyn immunoprecipitates. No association between CD45 and p59fyn was detected in similar experiments. The PTPα-mediated activation of p59fyn is not prerequisite for association since wild-type and inactive mutant PTPα bound equally well to p59fyn. Endogenous PTPα and p59fyn were also found in association in mouse brain. Together, these results demonstrate a physical and functional interaction of PTPα and p59fyn that may be of importance in PTPα-initiated signaling events. Members of the Src family of tyrosine kinases have been implicated in a variety of physiological and pathophysiological processes. These include mediating mitogenic responses initiated by growth factor receptors, control of cellular architecture through cytoskeletal reorganization, the UV and stress response, mitotic functions, and the induction of tumors (for review, see Ref. 1Erpel T. Courtneidge S.A. Curr. Opin. Cell Biol. 1995; 7: 176-182Crossref PubMed Scopus (283) Google Scholar). While the biological roles of the Src family kinases are not known, it is well established that the activities of these kinases are regulated, in part, by the phosphorylation state of the negative regulatory tyrosine residue corresponding to Tyr-527 of p60c-src (reviewed in Refs. 2Cooper J.A. Howell B. Cell. 1993; 73: 1051-1054Abstract Full Text PDF PubMed Scopus (495) Google Scholar and 3Taylor S.J. Shalloway D. Curr. Opin. Genet. Dev. 1993; 3: 26-34Crossref PubMed Scopus (64) Google Scholar). 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O'Dell T.J. Karl K.A. Stein P.L. Soriano P. Kandel E.R. Science. 1992; 258: 1903-1910Crossref PubMed Scopus (965) Google Scholar)). Second, together with pp60c-src, p59fyn is well defined in terms of its cellular actions. While studies in mutant mice show that Src and Fyn kinases have a high degree of functional redundancy (37Stein P.L. Vogel H. Soriano P. Genes Dev. 1994; 8: 1999-2007Crossref PubMed Scopus (269) Google Scholar), nevertheless, they also have specific and distinct functions (for example, in cytoskeletal organization (38Thomas S.M. Soriano P. Imamoto A. Nature. 1995; 376: 267-271Crossref PubMed Scopus (304) Google Scholar) and adhesion molecule-directed axonal growth (33Ignelzi M.A. Miller D.R. Soriano P. Maness P.F. Neuron. 1994; 12: 873-884Abstract Full Text PDF PubMed Scopus (276) Google Scholar, 34Beggs H.E. Soriano P. Maness P.F. J. Cell Biol. 1994; 127: 825-833Crossref PubMed Scopus (247) Google Scholar)). It is thus important to define which specific intermediate signaling molecules may mediate a spectrum of PTPα-directed cellular events. Numbering of the PTPα amino acid sequence is according to Krueger et al. (17Krueger N.X. Streuli M. Saito H. EMBO J. 1990; 9: 3241-3252Crossref PubMed Scopus (372) Google Scholar). The expression vector pXJ41-PTPα-neo, encoding full-length PTPα, has been described (20Zheng X.M. Wang Y. Pallen C.J. Nature. 1992; 359: 336-339Crossref PubMed Scopus (388) Google Scholar). The mutagenesis altering the essential cysteine residues to serine residues in the active sites of PTPα (C414S/C704S) has been described (39Lim K.L. Lai D.S.Y. Kalousek M.B. Wang Y. Pallen C.J. Eur. J. Biochem. 1997; 245: 693-700Crossref PubMed Scopus (35) Google Scholar), and a fragment of this cDNA was used to replace the corresponding piece of wild-type PTPα cDNA to produce pXJ41-PTPα(C414S/C704S)-neo. Vectors expressing VSVG-tagged versions of PTPα were constructed as follows. Primers (with PacI sites added) corresponding to the amino acid sequences RVGIHL and MNRLGK found at either end of a 29-amino acid C-terminal fragment of VSVG (40Rose J.K. Welch W.J. Sefton B.M. Esch F.S. Ling N.C. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3884-3888Crossref PubMed Scopus (88) Google Scholar) were used in a polymerase chain reaction with VSVG cDNA (a gift from Dr. S. H. Wong) as template. The primer sequences were 5′-GCGGTTAATTAACCGAGTTGGTATTTATCTT-3′ (forward) and 5′-GCGGTTAATTAACTTTCCAAGTCGGTTCAT-3′ (reverse). The amplified fragment was inserted into a unique PacI site in the PTPα cDNAs of pXJ41-neo, permitting the expression of PTPα proteins with a VSVG tag at amino acid 16 in the extracellular region. Plasmid containing CD45 cDNA (pAW-HCLA) was a gift of Dr. G. Koretzky. The CD45 cDNA insert was removed with HindIII and subcloned into the HindIII site of pXJ41-Hy (pXJ41 containing the gene conferring hygromycin resistance). fyn cDNA was isolated from a human fetal brain cDNA library in λgt11 (CLONTECH, Palo Alto, CA) and subcloned into theEcoRI site of pXJ41-neo. Murine neuronal c-srccDNA (NcoI fragment in pGEM5Z(+); Promega) was a gift of Dr. P. Bello. The c-src cDNA insert was removed with SphI and SacI and blunt end-ligated into theEcoRI site of pXJ41-neo. COS-1 cells were obtained from American Type Culture Collection (Rockville, MD). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and penicillin/streptomycin in an atmosphere of 5% CO2 at 37 °C. COS-1 cells at 50–70% confluency (100-mm dishes) were transfected with plasmid DNA by liposome-mediated transfection with 30 μl (1 mg/ml) of Lipofectin™ or LipofectAMINE™ reagent (Life Technologies, Inc.) for 5–6 h as described by the manufacturer and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum for an additional 18–40 h prior to harvesting. The empty expression plasmid pXJ41-neo was used to normalize the amount of DNA in each transfection. In experiments that did not involve the association of PTPα (or CD45) and p59fyn, cell extracts were prepared by lysing cells either in buffer A (50 mm Tris-Cl (pH 7.2), 150 mm NaCl, 0.2 mmNa3VO4, 1% Triton X-100, 10 μg/ml aprotinin, and 0.1 mm phenylmethylsulfonyl fluoride) (see Figs. 2 and4 C) or in modified radioimmune precipitation assay buffer (10 mm sodium phosphate (pH 7.0), 150 mm NaCl, 1 mm EDTA, 50 mm NaF, 0.1 mmNa3VO4, 1% Nonidet P-40, 0.1% SDS, 1% sodium deoxycholate, 10 μg/ml aprotinin, and 0.1 mmphenylmethylsulfonyl fluoride) (see Figs. 1 and 3) for 60 min at 4 °C. Cytosol and Triton X-100-solubilized membrane fractions of cells were obtained as described (20Zheng X.M. Wang Y. Pallen C.J. Nature. 1992; 359: 336-339Crossref PubMed Scopus (388) Google Scholar), essentially involving initial lysis of cells by sonication in buffer A without Triton X-100. In experiments involving co-immunoprecipitation of PTPα (or CD45) and p59fyn, cell lysates were prepared in 10 mm Tris-Cl (pH 7.2), 150 mm NaCl, 1 mm EDTA, 1% Brij 96, 10 μg/ml aprotinin, and 0.1 mm phenylmethylsulfonyl fluoride (see Figs. Figure 5, Figure 6, Figure 7). Lysates were clarified by centrifugation, and protein content was determined by Bradford analysis (41Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). Protein extracts were separated by SDS-polyacrylamide gel electrophoresis on a 7 or 9% gel and electrophoretically transferred to polyvinylidene difluoride membranes. Membranes were immunoblotted with anti-PTPα antiserum 3897 (raised against a glutathioneS-transferase/PTPα-D2 fusion protein (39Lim K.L. Lai D.S.Y. Kalousek M.B. Wang Y. Pallen C.J. Eur. J. Biochem. 1997; 245: 693-700Crossref PubMed Scopus (35) Google Scholar) containing the second catalytic domain and C-terminal tail region of PTPα) followed by goat anti-rabbit IgG conjugated to peroxidase (Sigma) or with anti-VSVG (Sigma), anti-p59fyn (Transduction Laboratories), anti-pp60c-src (Oncogene Science Inc.), or anti-CD45 9.4 (Dr. G. Koretzky) monoclonal antibodies followed by goat anti-mouse IgG conjugated to peroxidase (Sigma) or peroxidase-conjugated anti-phosphotyrosine antibody (Transduction Laboratories). Immunoblots were developed using the ECL system (Amersham Pharmacia Biotech). Immunoprecipitations of cell lysates were carried out using 250–600 μg of protein. For immunoprecipitation of p59fyn, either anti-Fyn antiserum (a gift of C. Rudd; see Fig. 1) or anti-Fyn polyclonal antibody (FYN3-G, Santa Cruz Biotechnology, Inc.) was added to the cell lysates and incubated for 60–120 min at 4 °C. For pp60c-src and VSVG-PTPα immunoprecipitations, anti-pp60c-src or anti-VSVG monoclonal antibodies were added to the cell lysates and incubated for 60 min at 4 °C, followed by incubation with rabbit anti-mouse IgG (Dako Corp.) for another 60 min at 4 °C. Protein A cell suspension (Sigma) was then added and mixed at 4 °C for 1 h. The immunoprecipitates were washed three times each in the respective cell lysis buffer (see above) and once in either 2× kinase assay buffer or phosphatase assay buffer (see below). Immunoblot analysis of the immunoprecipitated proteins was as described above.Figure 4PTPα (but not CD45) expression induces dephosphorylation of p59fyn. A, cells were transfected with 4 μg of p59fyn cDNA and 4 μg of PTPα or CD45 cDNA, with the total amount of DNA in each transfection equalized by the addition of empty plasmid. Membrane (M;lanes 1, 3, and 5) and cytosol (C; lanes 2, 4, and 6) fractions were prepared from lysates of COS-1 cells expressing p59fyn alone or together with PTPα or CD45 as indicated and immunoblotted with anti-p59fyn (top panel), anti-PTPα (middle panel), and anti-CD45 (bottom panel) antibodies. B, cell fractions (0.5 μg of protein) were assayed for phosphatase activity toward phosphotyrosyl-RR-Src peptide as described under “Experimental Procedures.” Activity of the membrane fraction of the PTPα-expressing lysate was taken as 100%. C, immunoprecipitates of p59fyn from 500 μg of total cell lysates were probed for phosphotyrosine (top panel) and p59fyn (bottom panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1Coexpression of PTPα with p59fynand p60c-src results in dephosphorylation of p59fyn and p60c-src. COS-1 cells were transfected with empty plasmid (mock), 3 μg each of p59fyn and pp60c-src cDNAs together, or 3 μg each of p59fyn, pp60c-src, and PTPα cDNAs together, with the total amount of DNA in each transfection equalized by the addition of empty plasmid. Immunoprecipitates of p59fyn (A) or pp60c-src (B) from 250 μg of the cell lysates were probed with anti-phosphotyrosine (top panels) and anti-p59fyn (A) or pp60c-src(B) (bottom panels) antibodies. HC, heavy chain antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Accessibility of the p59fyn SH2 domain is increased upon coexpression with PTPα. COS-1 cells were transfected with 4 μg of p59fyn cDNA alone or together with 4 μg of PTPα cDNA as indicated, with the total amount of DNA in each transfection equalized by the addition of empty plasmid. Lysates of cells expressing p59fyn in the absence (lane 1) or presence (lane 2) of PTPα were immunoblotted with anti-PTPα (A), anti-p59fyn(B), and anti-phosphotyrosine (C) antibodies, respectively. The cell lysates (300 μg) were incubated with unphosphorylated Src Tyr-527 peptide (D, top panel) or Src Tyr-527 phosphopeptide (bottom panel) coupled to Sepharose beads as described under “Experimental Procedures.” The washed precipitates were immunoblotted with anti-p59fyn antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Whole brain from an adult BALB/c mouse was homogenized in a hand-held Wheaton homogenizer in 6–8 ml of 10 mm Tris-Cl (pH 7.2), 150 mm NaCl, 1 mm EDTA, 1% Brij 96, 10 μg/ml aprotinin, and 0.1 mm phenylmethylsulfonyl fluoride. After incubation at 4 °C for 90 min, the lysate was clarified by centrifugation, and the protein content was determined. For immunoprecipitations, 1 mg of lysate was diluted a further 4-fold in homogenization buffer and then incubated with anti-PTPα (3680, raised against a peptide comprising the C-terminal 18 amino acids of PTPα), anti-p59fyn (FYN3, Santa Cruz Biotechnology, Inc.), or anti-Csk (C-20, Santa Cruz Biotechnology, Inc.) polyclonal antibodies. In some experiments, anti-PTPα antibody 3680 was blocked before immunoprecipitation by preincubation with recombinant purified PTPα-D2 polypeptide (amino acids 485–774) for 45 min at 4 °C. The brain lysate was incubated with the above antibodies for 16 h at 4 °C, and then Protein G PLUS/Protein A-agarose (Calbiochem) was added, and incubation was continued for 60 min at 4 °C. The immunoprecipitates were washed three times each in lysis buffer containing Brij 96 and once in lysis buffer without detergent, resolved by 8.5% SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. Membranes were immunoblotted with anti-PTPα antibody 3897 or 3680 followed by goat anti-rabbit IgG conjugated to peroxidase. Immunoblots were developed using the SuperSignal chemiluminescence system (Pierce). Synthetic peptides with the sequence TSTEPQYQPGENL, representing the sequence surrounding Tyr-527 of pp60c-src, were made using either phosphotyrosine or tyrosine at the appropriate step and were purified by high pressure liquid chromatography (Biotechnology Center, National University of Singapore). The phosphopeptide or peptide was covalently coupled to CNBr-activated Sepharose-4B (Amersham Pharmacia Inc.) and added to 300 μg of whole cell lysates (prepared with radioimmune precipitation assay buffer as described above) of COS-1 cells transfected with p59fyn cDNA alone or in combination with PTPα cDNA. After incubation for 2 h at 4 °C, the Sepharose beads were washed twice each with radioimmune precipitation assay buffer in the presence and absence of SDS/sodium deoxycholate, respectively, and resolved by electrophoresis on a 10% SDS-polyacrylamide gel. Immunoblot analysis was as described above. The kinase assays were performed in 20-μl reactions containing 10 mm Pipes (pH 7.0), 5 mmMnCl2, 0.5 mm dithiothreitol, 0.25 mm Na3VO4, and 5 μCi of [γ-32P]ATP at 37 °C for 10 min. The reactions were stopped with sample loading buffer, heated at 100 °C for 5 min, resolved by 9% SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and autoradiographed. In experiments where PTP activity was measured, Na3VO4 was omitted from the cell lysis buffer. PTP activity was measured at 30 °C in reactions containing 50 mm Mes (pH 6.0), 0.5 mg/ml bovine serum albumin, 0.5 mm dithiothreitol, and 2.5–5 μmphosphotyrosyl-RR-Src peptide (RRLIEDAEY(P)AARG, corresponding to the sequence encompassing Tyr-416 of pp60c-src and phosphorylated as described (39Lim K.L. Lai D.S.Y. Kalousek M.B. Wang Y. Pallen C.J. Eur. J. Biochem. 1997; 245: 693-700Crossref PubMed Scopus (35) Google Scholar)). The specific activity of the substrate ranged between 3000 and 4000 cpm/pmol of RR-Src. The reaction was carried out for 3 min unless otherwise indicated. The ability of PTPα to dephosphorylate pp60c-src and p59fyn was assessed in COS-1 cells cotransfected with both kinases. Anti-phosphotyrosine probing of p59fyn and pp60c-src immunoprecipitates demonstrated that PTPα expression resulted in tyrosine dephosphorylation of both kinases (Fig. 1, A and B). Blotting of these cell lysates with anti-cst.1, an antibody that recognizes both p59fyn and pp60c-srcequally well (42Kypta R.M. Goldberg Y. Ulug E.T. Courtneidge S.A. Cell. 1990; 62: 481-492Abstract Full Text PDF PubMed Scopus (480) Google Scholar), showed that similar amounts of these kinases were expressed with PTPα (data not shown). As p59fyn represents a novel potential substrate for PTPα, we characterized the catalytic action of PTPα toward p59fyn in more detail. The extent of p59fyn dephosphorylation increased as increasing amounts of PTPα cDNA were transfected, reaching a plateau of 80–90% tyrosine dephosphorylation (Fig. 2 A). Dephosphorylation was completely dependent on the catalytic activity of PTPα since a PTPα mutant, PTPα(C414S/C704S), in which the essential cysteine residues in the active sites of both catalytic domains of PTPα were mutated to serine residues, was unable to effect p59fyn dephosphorylation (Fig. 2 A). Besides tyrosine dephosphorylation of p59fyn, the coexpression of PTPα resulted in kinase activation of p59fyn. As shown in Fig. 2 B, when p59fyn immunoprecipitates from cells coexpressing PTPα were used in an immunocomplex kinase assay, increasing p59fynautophosphorylation was observed with increasing expression of PTPα. A maximum 5-fold increase in kinase activity was obtained (Fig. 2 B, lanes 5 and 6) at the same PTPα/p59fyn cDNA ratio observed to give maximal p59fyn dephosphorylation (Fig. 2 A). No increase in the kinase activity of p59fyn was measured when catalytically inactive PTPα was expressed with p59fyn (data not shown). Dephosphorylation of the C-terminal tyrosine residue of Src family kinases is linked to kinase activation (5Cartwright C.A. Kaplan P.L. Cooper J.A. Hunter T. Eckhart W. Mol. Cell. Biol. 1986; 6: 1562-1570Crossref PubMed Scopus (71) Google Scholar, 6Kmiecik T.E. Shalloway D. 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These observations support a model of kinase activation where disruption of an intramolecular association between the C-terminal phosphotyrosyl peptide and the SH2 domain of the kinase results in catalytic activation as well as novel interactions of the SH2 domain with other phosphotyrosyl proteins (2Cooper J.A. Howell B. Cell. 1993; 73: 1051-1054Abstract Full Text PDF PubMed Scopus (495) Google Scholar, 3Taylor S.J. Shalloway D. Curr. Opin. Genet. Dev. 1993; 3: 26-34Crossref PubMed Scopus (64) Google Scholar). The effect of PTPα on p59fyn SH2 domain accessibility was examined by determining the ability of p59fyn to bind to a synthetic phosphopeptide representing the C-terminal Tyr-527 peptide of pp60c-src. This peptide is identical to the C-terminal sequence surrounding Tyr-531 of p59fyn except for the replacement of alanine in position 2 with serine (45Kawakami T. Pennington C.Y. Robbins K. Mol. Cell. Biol. 1986; 6: 4195-4201Crossref PubMed Scopus (83) Google Scholar). As shown in Fig. 3, PTPα-induced dephosphorylation of p59fyn (Fig. 3 C) correlated with a 3-fold increase in the amount of Fyn protein precipitated from cell lysates with the Src Tyr-527 phosphopeptide coupled to Sepharose beads (Fig. 3 D, bottom panel). A control precipitation using an unphosphorylated Src Tyr-527 peptide-Sepharose conjugate contained barely detectable but equivalent amounts of Fyn protein from p59fyn- and PTPα/p59fyn-expressing cells (Fig. 3 D, top panel). While the site(s) of p59fyn dephosphorylation remain to be mapped, the increased p59fyn catalytic activity and SH2 availability for binding are consistent with a PTPα-mediated dephosphorylation of the C-terminal Tyr-531 of p59fyn. To examine whether the PTPα-mediated p59fyn dephosphorylation was a specif

Mutations in the parkin gene cause autosomal recessive familial Parkinson's disease (PD). Parkin-deficient mouse models fail to recapitulate nigrostriatal dopaminergic neurodegeneration as seen in PD, but produce deficits in dopaminergic neurotransmission and noradrenergic-dependent behavior. Since sporadic PD is thought to be caused by a combination of genetic susceptibilities and environmental factors, we hypothesized that neurotoxic insults from catecholaminergic toxins would render parkin knockout mice more vulnerable to neurodegeneration. Accordingly, we investigated the susceptibility of catecholaminergic neurons in parkin knockout mice to the potent dopaminergic and noradrenergic neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) respectively. We report that nigrostriatal dopaminergic neurons in parkin knockout mice do not show increased susceptibility to the parkinsonian neurotoxin, MPTP, in acute, subacute and chronic dose regimens of the neurotoxin. Additionally, parkin knockout mice do not show increased vulnerability to the noradrenergic neurotoxin, DSP-4, regarding levels of norepinephrine in cortex, brain stem and spinal cord. These findings suggest that absence of parkin in mice does not increase susceptibility to the loss of catecholaminergic neurons upon exposure to both dopaminergic and noradrenergic neurotoxins.

To date, a truly representative animal model of Parkinson disease (PD) remains a critical unmet need. Although toxin-induced PD models have served many useful purposes, they have generally failed to recapitulate accurately the progressive process as well as the nature and distribution of the human pathology. During the last decade or so, the identification of several genes whose mutations are causative of rare familial forms of PD has heralded in a new dawn for PD modelling. Numerous mammalian as well as non mammalian models of genetically-linked PD have since been created. However, despite initial optimism, none of these models turned out to be a perfect replica of PD. Meanwhile, genetic and toxin-induced models alike continue to evolve towards mimicking the disease more faithfully. Notwithstanding this, current genetic models have collectively illuminated several important pathways relevant to PD pathogenesis. Here, we have attempted to provide a comprehensive discussion on existing genetic models of PD.

Background Mutations in the parkin gene, which encodes a ubiquitin ligase (E3), are a major cause of autosomal recessive parkinsonism. Although parkin-mediated ubiquitination was initially linked to protein degradation, accumulating evidence suggests that the enzyme is capable of catalyzing multiple forms of ubiquitin modifications including monoubiquitination, K48- and K63-linked polyubiquitination. In this study, we sought to understand how a single enzyme could exhibit such multifunctional catalytic properties. Methods and Findings By means of in vitro ubiquitination assays coupled with mass spectrometry analysis, we were surprised to find that parkin is apparently capable of mediating E2-independent protein ubiquitination in vitro, an unprecedented activity exhibited by an E3 member. Interestingly, whereas full length parkin catalyzes solely monoubiquitination regardless of the presence or absence of E2, a truncated parkin mutant containing only the catalytic moiety supports both E2-independent and E2-dependent assembly of ubiquitin chains. Conclusions Our results here suggest a complex regulation of parkin's activity and may help to explain how a single enzyme like parkin could mediate diverse forms of ubiquitination.

We developed a facile and feasible fluorescent nanoswitch assay for reversible recognition of glutamate (Glu) and Al3+ in human serum and living cell. The proposed nanoswitch assay is based on our recently developed method for controlled synthesis of fluorescent polydopamine dots (PDADs) at room temperature with dopamine as the sole precursor. The fluorescence of nanoswitch assay could be quickly and efficiently quenched by Glu (turn-Off), and the addition of Al3+ could recover the fluorescence of the PDADs–Glu system (turn-On). Meanwhile, the reversible recognition of Glu and Al3+ in this nanoswitch system was stable after three cycles. Additionally, the system displayed excellent performance for Glu and Al3+ determination with a low detection limit of 0.12 and 0.2 μM, respectively. Moreover, PDADs are successfully applied to determine Glu and monitor Al3+ in human serum. Noteworthy, the nanoswitch assay is transported into HepG2 cells and realized “Off” detection of Glu and “On” sensing Al3+ in the living cells. Therefore, this PDADs-based nanoswitch assay provides a strategy to develop reversible recognition biosensors for intracellular and external molecular analysis.

Mutations in leucine-rich repeat kinase-2 are the most common cause of familial Parkinson's disease. The prevalent G2019S mutation increase oxidative, kinase and toxic activity and inhibit endogenous peroxidases. We initially screened a library of 84 antioxidants and identified seven phenolic compounds that inhibited kinase activity on leucine-rich repeat kinase-2 substrates. The representative antioxidants (piceatannol, thymoquinone, and esculetin) with strong kinase inhibitor activity, reduced loss in dopaminergic neurons, oxidative dysfunction, and locomotor defects in G2019S-expressing neuronal and Drosophila models compared to weak inhibitors. We provide proof of principle that natural antioxidants with dual antioxidant and kinase inhibitor properties could be useful for leucine-rich repeat kinase-2-linked Parkinson's disease.

Abstract Newly synthesized proteins constitute an important subset of the proteome involved in every cellular process, yet existing chemical tools used to study them have major shortcomings. Herein we report a suite of cell‐permeable puromycin analogues capable of being metabolically incorporated into newly synthesized proteins in different mammalian cells, including neuronal cells. Subsequent labeling with suitable bioorthogonal reporters, in both fixed and live cells, enabled direct imaging and enrichment of these proteins. By taking advantage of the mutually orthogonal reactivity of these analogues, we showed multiplexed labeling of different protein populations, as well as quantitative measurements of protein dynamics by fluorescence correlation spectroscopy, could be achieved in live‐cell environments.

Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by progressive loss of dopaminergic neurons in the substantia nigra of the midbrain. The etiology of PD has yet to be elucidated, and the disease remains incurable. Increasing evidence suggests that oxidative stress is the key causative factor of PD. Due to their capacity to alleviate oxidative stress, antioxidants hold great potential for the treatment of PD. Vitamins are essential organic substances for maintaining the life of organisms. Vitamin deficiency is implicated in the pathogenesis of various diseases, such as PD. In the present study, we investigated whether administration of vitamin B12 (VB12) could ameliorate PD phenotypes in vitro and in vivo. Our results showed that VB12 significantly reduced the generation of reactive oxygen species (ROS) in the rotenone-induced SH-SY5Y cellular PD model. In a Parkin gene knockout C. elegans PD model, VB12 mitigated motor dysfunction. Moreover, in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse PD model, VB12 also displayed protective effects, including the rescue of mitochondrial function, dopaminergic neuron loss, and movement disorder. In summary, our results suggest that vitamin supplementation may be a novel method for the intervention of PD, which is safer and more feasible than chemical drug treatment.

Transplantation of stem cell-derived retinal pigment epithelial (RPE) cells is considered a viable therapeutic option for age-related macular degeneration (AMD). Several landmark Phase I/II clinical trials have demonstrated safety and tolerability of RPE transplants in AMD patients, albeit with limited efficacy. Currently, there is limited understanding of how the recipient retina regulates the survival, maturation, and fate specification of transplanted RPE cells. To address this, we transplanted stem cell-derived RPE into the subretinal space of immunocompetent rabbits for 1 mo and conducted single-cell RNA sequencing analyses on the explanted RPE monolayers, compared to their age-matched in vitro counterparts. We observed an unequivocal retention of RPE identity, and a trajectory-inferred survival of all in vitro RPE populations after transplantation. Furthermore, there was a unidirectional maturation toward the native adult human RPE state in all transplanted RPE, regardless of stem cell resource. Gene regulatory network analysis suggests that tripartite transcription factors (FOS, JUND, and MAFF) may be specifically activated in posttransplanted RPE cells, to regulate canonical RPE signature gene expression crucial for supporting host photoreceptor function, and to regulate prosurvival genes required for transplanted RPE's adaptation to the host subretinal microenvironment. These findings shed insights into the transcriptional landscape of RPE cells after subretinal transplantation, with important implications for cell-based therapy for AMD.

Manganese (Mn(2+)) neurotoxicity from occupational exposure is well documented to result in a Parkinson-like syndrome. Although the understanding of Mn(2+) cytotoxicity is still incomplete, both Mn(2+) and Fe(2+) can be transported via the divalent metal transporter 1 (DMT1), suggesting that competitive uptake might disrupt Fe(2+) homeostasis. Here, we found that DMT1 overexpression significantly enhanced Mn(2+) cytoplasmic accumulation and JNK phosphorylation, leading to a reduction in cell viability. Although a robust activation of autophagy was observed alongside these changes, it did not trigger autophagic cell death, but was instead shown to be essential for the degradation of ferritin, which normally sequesters labile Fe(2+). Inhibition of ferritin degradation through the neutralization of lysosomal pH resulted in increased ferritin and enhanced cytoplasmic Fe(2+) depletion. Similarly, direct Fe(2+) chelation also resulted in aggravated Mn(2+)-mediated JNK phosphorylation, while Fe(2+) repletion protected cells, and this occurs via the ASK1-thioredoxin pathway. Taken together, our study presents the novel findings that Mn(2+) cytotoxicity involves the depletion of the cytoplasmic Fe(2+) pool, and the increase in autophagy-lysosome activity is important to maintain Fe(2+) homeostasis. Thus, Fe(2+) supplementation could have potential applications in the prevention and treatment of Mn(2+)-mediated toxicity.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic neurons in the substantia nigra of the human brain, leading to depletion of dopamine production. Dopamine replacement therapy remains the mainstay for attenuation of PD symptoms. Nonetheless, the potential benefit of current pharmacotherapies is mostly limited by adverse side effects, such as drug-induced dyskinesia, motor fluctuations and psychosis. Non-dopaminergic receptors, such as human A2A adenosine receptors, have emerged as important therapeutic targets in potentiating therapeutic effects and reducing the unwanted side effects. In this study, new chemical entities targeting both human A2A adenosine receptor and dopamine D2 receptor were designed and evaluated. Two computational methods, namely support vector machine (SVM) models and Tanimoto similarity-based clustering analysis, were integrated for the identification of compounds containing indole-piperazine-pyrimidine (IPP) scaffold. Subsequent synthesis and testing resulted in compounds 5 and 6, which acted as human A2A adenosine receptor binders in the radioligand competition assay (Ki = 8.7–11.2 μM) as well as human dopamine D2 receptor binders in the artificial cell membrane assay (EC50 = 22.5–40.2 μM). Moreover, compound 5 showed improvement in movement and mitigation of the loss of dopaminergic neurons in Drosophila models of PD. Furthermore, in vitro toxicity studies on compounds 5 and 6 did not reveal any mutagenicity (up to 100 μM), hepatotoxicity (up to 30 μM) or cardiotoxicity (up to 30 μM).

Parkin E3 ubiquitin-ligase activity and its role in mitochondria homeostasis are thought to play a role in Parkinson's disease (PD). We now report that AF-6 is a novel parkin interacting protein that modulates parkin ubiquitin-ligase activity and mitochondrial roles. Parkin interacts with the AF-6 PDZ region through its C-terminus. This leads to ubiquitination of cytosolic AF-6 and its degradation by the proteasome. On the other hand, endogenous AF-6 robustly increases parkin translocation and ubiquitin-ligase activity at the mitochondria. Mitochondrial AF-6 is not a parkin substrate, but rather co-localizes with parkin and enhances mitochondria degradation through PINK1/parkin-mediated mitophagy. On the other hand, several parkin and PINK1 juvenile disease-mutants are insensitive to AF-6 effects. AF-6 is present in Lewy bodies and its soluble levels are strikingly decreased in the caudate/putamen and substantia nigra of sporadic PD patients, suggesting that decreased AF-6 levels may contribute to the accumulation of dysfunctional mitochondria in the disease. The identification of AF-6 as a positive modulator of parkin translocation to the mitochondria sheds light on the mechanisms involved in PD and underscores AF-6 as a novel target for future therapeutics.

Elevated iron deposition has been reported in Parkinson's disease (PD). However, the route of iron uptake leading to high deposition in the substantia nigra is unresolved. Here, we show a mechanism in enhanced Fe 2+ uptake via S-nitrosylation of divalent metal transporter 1 (DMT1). While DMT1 could be S-nitrosylated by exogenous nitric oxide donors, in human PD brains, endogenously S-nitrosylated DMT1 was detected in postmortem substantia nigra. Patch-clamp electrophysiological recordings and iron uptake assays confirmed increased Mn 2+ or Fe 2+ uptake through S-nitrosylated DMT1. We identified two major S-nitrosylation sites, C23 and C540, by mass spectrometry, and DMT1 C23A or C540A substitutions abolished nitric oxide (NO)-mediated DMT1 current increase. To evaluate in vivo significance, lipopolysaccharide (LPS) was stereotaxically injected into the substantia nigra of female and male mice to induce inflammation and production of NO. The intranigral LPS injection resulted in corresponding increase in Fe 2+ deposition, JNK activation, dopaminergic neuronal loss and deficit in motoric activity, and these were rescued by the NO synthase inhibitor l -NAME or by the DMT1-selective blocker ebselen. Lentiviral knockdown of DMT1 abolished LPS-induced dopaminergic neuron loss. SIGNIFICANCE STATEMENT Neuroinflammation and high cytoplasmic Fe 2+ levels have been implicated in the initiation and progression of neurodegenerative diseases. Here, we report the unexpected enhancement of the functional activity of transmembrane divalent metal transporter 1 (DMT1) by S-nitrosylation. We demonstrated that S-nitrosylation increased DMT1-mediated Fe 2+ uptake, and two cysteines were identified by mass spectrometry to be the sites for S-nitrosylation and for enhanced iron uptake. One conceptual advance is that while DMT1 activity could be increased by external acidification because the gating of the DMT1 transporter is proton motive, we discovered that DMT1 activity could also be enhanced by S-nitrosylation. Significantly, lipopolysaccharide-induced nitric oxide (NO)-mediated neuronal death in the substantia nigra could be ameliorated by using l -NAME, a NO synthase inhibitor, or by ebselen, a DMT1-selective blocker.

Abstract The design of the first dual‐purpose activity‐based probe of monoamine oxidase B (MAO‐B) is reported. This probe is highly selective towards MAO‐B, even at high MAO‐A expression levels, and could sensitively report endogenous MAO‐B activities by both in situ proteome profiling and live‐cell bioimaging. With a built‐in imaging module as part of the probe design, the probe was able to accomplish what all previously reported MAO‐B imaging probes failed to do thus far: the live‐cell imaging of MAO‐B activities without encountering diffusion problems.

Although the Parkin/PINK1 pathway has received considerable attention in recent years as a key regulator of mitophagy in mammals, it is important to recognize that multiple mitophagy receptors like BNIP3, NIX, and FUNDC1 exist that can promote the selective clearance of mitochondria in the absence of Parkin. In this issue, Bhujabal et al expand the repertoire of Parkin-independent mitophagy receptors to include the anti-apoptotic protein, FKBP8. The authors demonstrate that FKBP8 interacts preferentially with LC3A via its LIR motif to destroy damaged mitochondria. During the process, FKBP8 escapes from the destruction presumably to prevent apoptosis during mitophagy [1].

Axonal injury is a common feature of central nervous system insults. Upregulation of amyloid precursor protein (APP) is observed following central nervous system neurotrauma and is regarded as a marker of central nervous system axonal injury. However, the underlying mechanism by which APP mediates neuronal death remains to be elucidated. Here, we used mouse optic nerve axotomy (ONA) to model central nervous system axonal injury replicating aspects of retinal ganglion cell (RGC) death in optic neuropathies. APP and APP intracellular domain (AICD) were upregulated in retina after ONA and APP knockout reduced Tuj1+ RGC loss. Pathway analysis of microarray data combined with chromatin immunoprecipitation and a luciferase reporter assay demonstrated that AICD interacts with the JNK3 gene locus and regulates JNK3 expression. Moreover, JNK3 was found to be upregulated after ONA and to contribute to Tuj1+ RGC death. APP knockout reduced the ONA-induced enhanced expression of JNK3 and phosphorylated JNK (pJNK). Gamma-secretase inhibitors prevented production of AICD, reduced JNK3 and pJNK expression similarly, and protected Tuj1+ RGCs from ONA-induced cell death. Together these data indicate that ONA induces APP expression and that gamma-secretase cleavage of APP releases AICD, which upregulates JNK3 leading to RGC death. This pathway may be a novel target for neuronal protection in optic neuropathies and other forms of neurotrauma.

Abstract Mutations in LRRK2 cause autosomal dominant and sporadic Parkinson’s disease, but the mechanisms involved in LRRK2 toxicity in PD are yet to be fully understood. We found that LRRK2 translocates to the nucleus by binding to seven in absentia homolog (SIAH-1), and in the nucleus it directly interacts with lamin A/C, independent of its kinase activity. LRRK2 knockdown caused nuclear lamina abnormalities and nuclear disruption. LRRK2 disease mutations mostly abolish the interaction with lamin A/C and, similar to LRRK2 knockdown, cause disorganization of lamin A/C and leakage of nuclear proteins. Dopaminergic neurons of LRRK2 G2019S transgenic and LRRK2 −/− mice display decreased circularity of the nuclear lamina and leakage of the nuclear protein 53BP1 to the cytosol. Dopaminergic nigral and cortical neurons of both LRRK2 G2019S and idiopathic PD patients exhibit abnormalities of the nuclear lamina. Our data indicate that LRRK2 plays an essential role in maintaining nuclear envelope integrity. Disruption of this function by disease mutations suggests a novel phosphorylation-independent loss-of-function mechanism that may synergize with other neurotoxic effects caused by LRRK2 mutations.

Highly efficient neurogenic differentiation, maturation as well as spontaneous amplification of pathogenic amyloid-beta 42 (Aβ42) and phospho-tau expression were achieved on interfacing iPSC-derived neurons with 3D PLGA microfiber scaffolds.

Alzheimer's disease (AD) is the most common cause of dementia that affects millions of predominantly elderly individuals worldwide. Despite intensive research over several decades, controversies still surround the etiology of AD and the disease remains incurable. Meanwhile, new molecular players of the central amyloid cascade hypothesis have emerged and among these is a protease known as β-site APP cleavage enzyme 2 (BACE2). Unlike BACE1, BACE2 cleaves the amyloid-β protein precursor within the Aβ domain that accordingly prevents the generation of Aβ42 peptides, the aggregation of which is commonly regarded as the toxic entity that drives neurodegeneration in AD. Given this non-amyloidogenic role of BACE2, it is attractive to position BACE2 as a therapeutic target for AD. Indeed, several groups including ours have demonstrated a neuroprotective role for BACE2 in AD. In this review, we discuss emerging evidence supporting the ability of BACE2 in mitigating AD-associated pathology in various experimental systems including human pluripotent stem cell-derived cerebral organoid disease models. Alongside this, we also provide an update on the identification of single nucleotide polymorphisms occurring in the BACE2 gene that are linked to increased risk and earlier disease onset in the general population. In particular, we highlight a recently identified point mutation on BACE2 that apparently leads to sporadic early-onset AD. We believe that a better understanding of the role of BACE2 in AD would provide new insights for the development of viable therapeutic strategies for individuals with dementia.

Weight loss is the most effective treatment for non-alcoholic fatty liver disease (NAFLD). There is evidence that Mediterranean diets rich in unsaturated fatty acids and fiber have beneficial effects on weight homeostasis and metabolic risk factors in individuals with NAFLD. Studies have also shown that higher circulating concentrations of pentadecanoic acid (C15:0) are associated with lower risk for NAFLD. To examine the effects of a Mediterranean-like, culturally contextualized Asian diet rich in fiber and unsaturated fatty acids, with or without C15:0 supplementation, in Chinese females with NAFLD. In a double-blinded, parallel-design, randomized controlled trial, 88 Chinese females with NAFLD were randomized to one of three groups for 12-weeks: diet with C15:0 supplementation (n=31), diet without C15:0 supplementation (n=28), or control (habitual diet and no C15:0 supplementation, n=29). At baseline and after the intervention, body fat percentage, intrahepatic lipid content, muscle and abdominal fat, liver enzymes, cardiometabolic risk factors and gut microbiome were assessed. In intention-to-treat analysis, weight reductions of 4.0±0.5 kg (5.3%), 3.4±0.5 kg (4.5%), and 1.5±0.5 kg (2.1%) were achieved in the diet-with-C15:0, diet-without-C15:0, and the control groups, respectively. The proton density fat fraction (PDFF) of the liver decreased by 33%, 30% and 10%, respectively. Both diet groups achieved significantly greater reductions in body weight, liver PDFF, total cholesterol, gamma-glutamyl transferase, and triglyceride concentrations compared to the control group. C15:0 supplementation reduced LDL-cholesterol further, and increased abundance of Bifidobacterium adolescentis. Fat mass, visceral adipose tissue, subcutaneous abdominal adipose tissue (deep and superficial), insulin, glycated hemoglobin and blood pressure decreased significantly in all groups, in parallel with weight loss. Mild weight loss induced by a Mediterranean-like diet adapted for Asians has multiple beneficial health effects in women with NAFLD. C15:0 supplementation lowers LDL-cholesterol and may cause beneficial shifts in gut microbiome. clinicaltrials.gov, NCT05259475

The two tandem homologous catalytic domains of PTPα possess different kinetic properties, with the membrane proximal domain (D1) exhibiting much higher activity than the membrane distal (D2) domain. Sequence alignment of PTPα-D1 and -D2 with the D1 domains of other receptor-like PTPs, and modeling of the PTPα-D1 and -D2 structures, identified two non-conserved amino acids in PTPα-D2 that may account for its low activity. Mutation of each residue (Val-536 or Glu-671) to conform to its invariant counterpart in PTPα-D1 positively affected the catalytic efficiency of PTPα-D2 toward the in vitro substratespara-nitrophenylphosphate and the phosphotyrosyl-peptide RR-src. Together, they synergistically transformed PTPα-D2 into a phosphatase with catalytic efficiency forpara-nitrophenylphosphate equal to PTPα-D1 but not approaching that of PTPα-D1 for the more complex substrate RR-src.In vivo, no gain in D2 activity toward p59fyn was effected by the double mutation. Alteration of the two corresponding invariant residues in PTPα-D1 to those in D2 conferred D2-like kinetics toward all substrates. Thus, these two amino acids are critical for interaction with phosphotyrosine but not sufficient to supply PTPα-D2 with a D1-like substrate specificity for elements of the phosphotyrosine microenvironment present in RR-src and p59fyn. Whether the structural features of D2 can uniquely accommodate a specific phosphoprotein substrate or whether D2 has an alternate function in PTPα remains an open question. The two tandem homologous catalytic domains of PTPα possess different kinetic properties, with the membrane proximal domain (D1) exhibiting much higher activity than the membrane distal (D2) domain. Sequence alignment of PTPα-D1 and -D2 with the D1 domains of other receptor-like PTPs, and modeling of the PTPα-D1 and -D2 structures, identified two non-conserved amino acids in PTPα-D2 that may account for its low activity. Mutation of each residue (Val-536 or Glu-671) to conform to its invariant counterpart in PTPα-D1 positively affected the catalytic efficiency of PTPα-D2 toward the in vitro substratespara-nitrophenylphosphate and the phosphotyrosyl-peptide RR-src. Together, they synergistically transformed PTPα-D2 into a phosphatase with catalytic efficiency forpara-nitrophenylphosphate equal to PTPα-D1 but not approaching that of PTPα-D1 for the more complex substrate RR-src.In vivo, no gain in D2 activity toward p59fyn was effected by the double mutation. Alteration of the two corresponding invariant residues in PTPα-D1 to those in D2 conferred D2-like kinetics toward all substrates. Thus, these two amino acids are critical for interaction with phosphotyrosine but not sufficient to supply PTPα-D2 with a D1-like substrate specificity for elements of the phosphotyrosine microenvironment present in RR-src and p59fyn. Whether the structural features of D2 can uniquely accommodate a specific phosphoprotein substrate or whether D2 has an alternate function in PTPα remains an open question. protein-tyrosine phosphatase glutathioneS-transferase para-nitrophenylphosphate receptor protein-tyrosine phosphatase 1,4-piperazinediethanesulfonic acid. Protein tyrosine phosphorylation status is a key determinant of nearly all eukaryotic cell processes and is controlled by the protein-tyrosine kinases and phosphatases (PTPs).1 Phosphotyrosine hydrolysis is catalyzed by members of the large and diverse PTP superfamily, and although the specific roles of most of these enzymes have yet to be determined, they can positively or negatively regulate cellular signaling pathways (1Tonks N.K. Neel B.G. Cell. 1996; 87: 365-368Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 2Streuli M. Curr. Opin. Cell Biol. 1996; 8: 182-188Crossref PubMed Scopus (165) Google Scholar). The PTPs include enzymes with absolute specificity for phosphotyrosine as well as dual specificity enzymes that can also dephosphorylate serine and threonine residues. Most do not share significant sequence identity, with the exception of the phosphotyrosine-specific receptor and non-receptor-like PTPs, which have stretches of amino acid identity throughout their catalytic domains. Nevertheless, despite dissimilarities in primary sequence, PTPs from diverse subgroups have a remarkably conserved tertiary structure and predicted catalytic mechanism (3Denu J.M. Stuckey J.A. Saper M.A. Dixon J.E. Cell. 1996; 87: 361-364Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 4Fauman E.B. Saper M.A. Trends Biochem. Sci. 1996; 21: 413-417Abstract Full Text PDF PubMed Scopus (319) Google Scholar). Enzymological and mutational studies have elucidated several features of the catalytic mechanism of PTPs (5Zhang Z.-Y. Crit. Rev. Biochem. Mol. Biol. 1998; 33: 1-52Crossref PubMed Scopus (252) Google Scholar). All have an absolutely conserved CX5R motif in the active site and nucleophilic attack on the phosphate ester by this essential cysteine results in the formation of a covalent thiophosphate intermediate (6Guan K.L. Dixon J.E. J. Biol. Chem. 1991; 266: 17026-17030Abstract Full Text PDF PubMed Google Scholar, 7Cho H.J. Krishnaraj R. Kitas E. Bannwarth W. Walsh C.T. Anderson K.S. J. Am. Chem. Soc. 1992; 114: 7206-7298Google Scholar, 8Zhang Z.-Y. Wang Y. Wu L. Fauman E.B. Stuckey J.A. Schubert H.L. Saper M.A. Dixon J.E. Biochemistry. 1994; 33: 15266-15270Crossref PubMed Scopus (171) Google Scholar). This phosphate transfer is facilitated by proton donation to the phenolic oxygen of phosphotyrosine from a general acid, identified in the tyrosine-specific PTP1 and Yop51 from Yersinia as the aspartate residue within a conserved WPD motif about 30–40 residues N-terminal to the active site sequence (9Zhang Z.-Y. Wang Y. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1624-1627Crossref PubMed Scopus (255) Google Scholar, 10Denu J.M. Lohse D.L. Vijayalakshmi J. Saper M.A. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2493-2498Crossref PubMed Scopus (252) Google Scholar). Subsequent transfer of the phosphate to water is likely aided by the same aspartate residue acting as a general base (10Denu J.M. Lohse D.L. Vijayalakshmi J. Saper M.A. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2493-2498Crossref PubMed Scopus (252) Google Scholar). The crystal structures of several PTPs support this mechanism and have further shown that the CX5R lies at the base of a phosphotyrosine-binding pocket, with substrate binding inducing the movement of a loop containing the WPD sequence so that the aspartate residue is brought into the catalytic site and in proximity to the leaving group oxygen (11Barford D. Flint A.J. Tonks N.K. Science. 1994; 263: 1397-1404Crossref PubMed Scopus (687) Google Scholar, 12Stuckey J.A. Schubert H.L. Fauman E.B. Zhang Z.-Y. Dixon J.E. Saper M.A. Nature. 1994; 370: 571-575Crossref PubMed Scopus (378) Google Scholar, 13Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (558) Google Scholar). Many receptor-like PTPs (RPTPs) have the structural distinction of possessing two homologous tandem catalytic domains in the intracellular region, raising the intriguing question of the functional roles of each repeat. The first, or membrane proximal domains (D1) of LAR, CD45, PTPμ, and PTPε are catalytically active, whereas the second or membrane distal domains (D2) have either no detectable or extremely lowin vitro activity, usually less than 0.1% of the activity of D1 (14Streuli M. Krueger N.X. Thai T. Tang M. Saito H. EMBO J. 1990; 9: 2399-2407Crossref PubMed Scopus (268) Google Scholar, 15Johnson P. Ostergaard H.L. Wasden C. Trowbridge I.S. J. Biol. Chem. 1992; 267: 8035-8041Abstract Full Text PDF PubMed Google Scholar, 16Gebbink M.F.B.G. Verheijen M.H.G. Zondag G.C.M. van Etten I. Moolenaar W.H. Biochemistry. 1993; 32: 13516-13522Crossref PubMed Scopus (34) Google Scholar, 17Streuli M. Krueger N.X. Tsai A.Y.M. Saito H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8698-8702Crossref PubMed Scopus (246) Google Scholar, 18Pot D.A. Woodford T.A. Remboutsika E. Haun R.S. Dixon J.E. J. Biol. Chem. 1991; 266: 19688-19696Abstract Full Text PDF PubMed Google Scholar, 19Lim K.L. Lai D.S.Y. Kalousek M.B. Wang Y. Pallen C.J. Eur. J. Biochem. 1997; 245: 693-700Crossref PubMed Scopus (35) Google Scholar). This points to a non-redundant function of the tandem domains, with D2 possibly playing a regulatory role. Where studied, there is also no evidence for an in vivo catalytic action of D2, as the inactivation of D2 of CD45 does not detectably affect the action of CD45 in T cell activation (20Desai D.M. Sap J. Silvennoinen O. Schlessinger J. Weiss A. EMBO J. 1994; 13: 4002-4010Crossref PubMed Scopus (91) Google Scholar). The RPTPs CD45, LAR, PTPμ, and PTPα all display altered D1 activity or substrate specificityin vitro in the absence of D2 (14Streuli M. Krueger N.X. Thai T. Tang M. Saito H. EMBO J. 1990; 9: 2399-2407Crossref PubMed Scopus (268) Google Scholar, 15Johnson P. Ostergaard H.L. Wasden C. Trowbridge I.S. J. Biol. Chem. 1992; 267: 8035-8041Abstract Full Text PDF PubMed Google Scholar, 16Gebbink M.F.B.G. Verheijen M.H.G. Zondag G.C.M. van Etten I. Moolenaar W.H. Biochemistry. 1993; 32: 13516-13522Crossref PubMed Scopus (34) Google Scholar, 19Lim K.L. Lai D.S.Y. Kalousek M.B. Wang Y. Pallen C.J. Eur. J. Biochem. 1997; 245: 693-700Crossref PubMed Scopus (35) Google Scholar, 21Cho H. Ramer S.E. Itoh M. Kitas E. Bannwarth W. Burn P. Saito H. Walsh C.T. Biochemistry. 1992; 31: 133-138Crossref PubMed Scopus (53) Google Scholar). A novel interaction of D2 of PTPδ with D1 of PTPς inhibits PTPς-D1 activity in in vitro assays, suggesting a role for D2 in regulating the formation and activity of receptor heterodimers (22Wallace M. Fladd C. Batt J. Rotin D. Mol. Cell. Biol. 1998; 18: 2608-2616Crossref PubMed Google Scholar). On the other hand, the D2 domain of PTPα is exceptional in exhibitingin vitro catalytic activity of up to about 10% of D1 activity toward certain substrates (19Lim K.L. Lai D.S.Y. Kalousek M.B. Wang Y. Pallen C.J. Eur. J. Biochem. 1997; 245: 693-700Crossref PubMed Scopus (35) Google Scholar, 23Wang Y. Pallen C.J. EMBO J. 1991; 10: 3231-3237Crossref PubMed Scopus (102) Google Scholar, 24Wu L. Buist A. den Hertog J. Zhang Z.-Y. J. Biol. Chem. 1997; 272: 6994-7002Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) and cannot be ruled out as a direct contributor to cellular PTPα activity. The relatively high activity of PTPα-D2 is due to two factors, the higher intrinsic activity of PTPα-D2 compared with other D2 domains and the lower activity of PTPα-D1 compared with other D1 domains (19Lim K.L. Lai D.S.Y. Kalousek M.B. Wang Y. Pallen C.J. Eur. J. Biochem. 1997; 245: 693-700Crossref PubMed Scopus (35) Google Scholar). Structure-function studies of PTPα-D2 present a unique opportunity to assess the minimal sequence requirements that might distinguish the characteristic catalytic properties of the homologous D1 and D2 domains. Most of the highly conserved and invariant residues among the tyrosine-specific PTPs are found within or near the catalytic cleft and are involved in interaction with phosphotyrosine or in actual hydrolysis (12Stuckey J.A. Schubert H.L. Fauman E.B. Zhang Z.-Y. Dixon J.E. Saper M.A. Nature. 1994; 370: 571-575Crossref PubMed Scopus (378) Google Scholar, 13Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (558) Google Scholar). Mutation of many of these residues impairs PTP activity (8Zhang Z.-Y. Wang Y. Wu L. Fauman E.B. Stuckey J.A. Schubert H.L. Saper M.A. Dixon J.E. Biochemistry. 1994; 33: 15266-15270Crossref PubMed Scopus (171) Google Scholar, 14Streuli M. Krueger N.X. Thai T. Tang M. Saito H. EMBO J. 1990; 9: 2399-2407Crossref PubMed Scopus (268) Google Scholar, 25Johnson P. Ostergaard H.L. Wasden C. Trowbridge I.S. J. Biol. Chem. 1992; 267: 8035-8041Abstract Full Text PDF PubMed Google Scholar, 26Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (685) Google Scholar). The lack of certain of these apparently critical residues in PTPα-D2 suggests that its low activity may simply be due to defective substrate binding or catalysis. If so, this would imply a non-catalytic role for D2 rather than an enzymatic function. To investigate this possibility, we have mutated two atypical residues in PTPα-D2 to conform to the corresponding amino acid found in all other tyrosine-specific PTPs with activity. One such residue is the putative general acid of D2 necessary for formation of the thiophosphate intermediate. The other is a residue that, in PTP1B, is located at the top of the catalytic cleft where it interacts with phosphotyrosine of the substrate (13Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (558) Google Scholar). The in vitro and in vivo activities of the D2 single and double mutants, as well as those of D1 single and double mutants possessing wild-type D2 residues in these positions, have been analyzed. Modeling of structures was performed using LOOK (Molecular Applications Group). Sequences of D1 and D2 were initially aligned to the target sequence of PTP1B (sequence identities of 47 and 41%, respectively), and the structure was subsequently modeled based on the algorithm of Lee and Subbiah (the algorithm uses self-consistent ensemble optimization to determine the global minimum structure resulting in the location of side chains with high accuracy) (27Lee C. Subbiah S. J. Mol. Biol. 1991; 217: 373-388Crossref PubMed Scopus (249) Google Scholar). The target structure was the complexed form of PTP1B(C215S) with phosphotyrosyl-hexapeptide (13Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (558) Google Scholar). Numbering of the PTPα amino acid sequence is according to Krueger et al. (28Krueger N.X. Streuli M. Saito H. EMBO J. 1990; 9: 3241-3252Crossref PubMed Scopus (370) Google Scholar). The bacterial expression plasmids pGEX-KG containing PTPα-D1 or -D2 have been described (19Lim K.L. Lai D.S.Y. Kalousek M.B. Wang Y. Pallen C.J. Eur. J. Biochem. 1997; 245: 693-700Crossref PubMed Scopus (35) Google Scholar), and they served as template for polymerase chain reaction site-directed mutagenesis. For the WPD mutants, the forward mutant primer sequences were 5′-ACCAGCTGGCCAGAATTTGGGGTG-3′ for D1(D382E), 5′-ACCAGCTGGCCAGCCTTTGGGGTG-3′ for D1(D382A), 5′-CATGGCTGGCCTGACGTGGGCATC-3′ for D2(E671D), and 5′-CATGGCTGGCCTGCAGTGGGCATC-3′ for D2(E671A). For the KNRY mutants, the reverse primer sequences were 5′-CAGGTGGACTCTAGAGTGGTCATAAGGCAAGATGTTTACAACTCGATTTTTTTC-3′ for D1(Y243V) and 5′-CACTCTGTTGAATTCATATGGAATGATCTGTAAATAACGGTTCTTCTT-3′ for D2(V536Y). The pGEX-KG-PTPα-D1(Y243V/D382E) was constructed by removing appropriate restriction fragments from pGEX-KG-PTPα-D1(D382E) and pGEX-KG-PTPα-D1(Y243V) and assembling them together so that they contained the double mutation. A similar strategy was used in the construction of pGEX-KG-PTPα-D2(V536Y/E671D). All mutations introduced by polymerase chain reaction were confirmed by DNA sequencing, and no extraneous mutations were found. Restricted fragments of these mutants encompassing the desired mutations and most of the catalytic domain were used to replace homologous fragments of the full-length PTPα in the expression vector pXJ41-neo, where PTPα already contained an inactivating Cys to Ser mutation in D1 (C414S) and D2 (C704S) (30Bhandari V. Lim K.L. Pallen C.J. J. Biol. Chem. 1998; 273: 8691-8698Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The plasmids pXJ41-PTPα-D1(C414S)D2-neo and pXJ41PTPα-D1D2(C704S)-neo were constructed by replacing D1 or D2 within pXJ41-PTPα-neo with a corresponding restriction fragment encompassing D1(C414S) or D2(C704S) that was derived from pXJ41-PTPα-D1(C414S)D2(C704S)-neo. The Cys to Ser mutations are denoted in the figure legends as a subscript S following the domain containing the mutation. These plasmids and those containing wild-type PTPα (pXJ41-PTPα-neo) (29Zheng X.M. Wang Y. Pallen C.J. Nature. 1992; 359: 336-339Crossref PubMed Scopus (387) Google Scholar) or PTPα-D1(C414S)D2(C704S) (19Lim K.L. Lai D.S.Y. Kalousek M.B. Wang Y. Pallen C.J. Eur. J. Biochem. 1997; 245: 693-700Crossref PubMed Scopus (35) Google Scholar) were subsequently used for cotransfection studies with p59fyn in COS-1 cells. The expression vector pXJ41-neo-p59fyn has been described (30Bhandari V. Lim K.L. Pallen C.J. J. Biol. Chem. 1998; 273: 8691-8698Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The expression, purification, quantitation, and storage of GST-PTPα fusion proteins have been previously described (19Lim K.L. Lai D.S.Y. Kalousek M.B. Wang Y. Pallen C.J. Eur. J. Biochem. 1997; 245: 693-700Crossref PubMed Scopus (35) Google Scholar). Phosphatase activity toward RR-src was measured in 30-μl reactions containing 50 mm Mes (pH 6.0), 0.5 mg/ml bovine serum albumin, and 0.5 mmdithiothreitol. Dephosphorylation of pNPP was measured in 450-μl reactions containing 50 mm sodium acetate (pH 5.5), 0.5 mg/ml bovine serum albumin, and 0.5 mm dithiothreitol. ForK m and V max determinations, RR-src concentrations generally ranged from 2.0 to 25 μm, and pNPP concentrations ranged from 0.5 to 10 mm. All reactions were carried out at 30 °C and terminated during the linear portion of the reaction. Released 32P orp-nitrophenol was quantitated as described previously (23Wang Y. Pallen C.J. EMBO J. 1991; 10: 3231-3237Crossref PubMed Scopus (102) Google Scholar). Phosphatase activity was plotted against substrate concentration in the form of a Lineweaver-Burk plot and manually extrapolated to determineK m and V max values. COS-1 cells were obtained from American Type Culture Collection (Rockville, MD). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and penicillin/streptomycin in an atmosphere of 5% CO2 at 37 °C. Prior to transfection, confluent monolayers of COS-1 cells were trypsinized and replated in 60- or 100-mm tissue culture dishes and incubated for 16 h until 50–70% confluency. Cells were transfected with 2–4 μg of plasmid DNA by liposome-mediated transfection with 10 μl (1 mg/ml) (60-mm dishes) or 30 μl (1 mg/ml) (100-mm dishes) of Lipofectin or LipofectAMINE reagent (Life Technologies, Inc.) for 6 h as described by the manufacturer and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum for an additional 18 h prior to harvesting. The empty expression plasmid pXJ41neo was used to normalize the amount of DNA in each transfection. Equivalent amount of the various forms of PTPα were expressed. The preparation of cell extracts and subsequent Western blot procedures have been described (30Bhandari V. Lim K.L. Pallen C.J. J. Biol. Chem. 1998; 273: 8691-8698Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Membranes were immunoblotted with anti-PTPα-D1 antiserum (no. 2205, raised against a GST-PTPα fusion protein containing the first catalytic domain of PTPα) (1:1000) and followed by goat anti-rabbit IgG conjugated to peroxidase (Sigma) (1:2500), anti-p59fyn monoclonal antibody (Transduction Laboratories) (1:300) followed by goat anti-mouse IgG conjugated to peroxidase (1:2500), or peroxidase-conjugated anti-phosphotyrosine antibody (Transduction Laboratories) (1:2000). Immunoblots were developed using the ECL system (Amersham Pharmacia Biotech). For immunoprecipitation of p59fyn, anti-p59fyn (FYN3, Santa Cruz) was added to the cell lysates (1 μl per 100 μg of protein) and incubated for 2 h at 4 °C. Protein A cell suspension (Sigma) was then added and mixed at 4 °C for 2 h. After low speed centrifugation, the immunoprecipitates were washed twice each with lysis buffer and once in 2× kinase assay buffer containing 10 mm Pipes (pH 7.0), 5 mm MnCl2, and 0.5 mmdithiothreitol. Part of the immunoprecipitates were used in kinase assays to measure p59fyn autophosphorylation as described previously (30Bhandari V. Lim K.L. Pallen C.J. J. Biol. Chem. 1998; 273: 8691-8698Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Other portions of the immunoprecipitates were probed for p59fyn as described above. The p59fyn level, phosphotyrosine content, and kinase activity were quantitated using a GS700 Bio-Rad densitometer. Alignment of the amino acid sequences of the catalytic D1 domains of 16 active mammalian receptor-like PTPs shows that they possess 42 invariant residues, highlighted in the amino acid sequence of PTPα-D1 (Fig. 1 A). No D2 domain of these RPTPs possesses all 42 invariant residues, although PTPα-D2 is only lacking 3 of them: a tyrosine at position 536, a leucine at position 549, and an aspartate at position 671 (Fig. 1 A). In fact, all of the D2 domains examined are lacking the corresponding tyrosine and aspartate residues, suggesting that the substitution of these residues may be a common denonimator that, in the absence of other obvious defects (for example, the substitution of the essential cysteine residue in the active site of PTPγ, PTPζ, and PTP-OST), accounts for low D2 activity. Furthermore, the counterpart Tyr in the KNRY motif and the Asp in the WPD motif of non-receptor PTP1B are involved in interactions with the substrate. The crystal structures of PTP1B complexed with phosphopeptide shows that the corresponding invariant tyrosine (Tyr-46) interacts with the phenyl ring of phosphotyrosine of the substrate (13Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (558) Google Scholar). This tyrosine is one of several hydrophobic, conserved residues that form the recognition site for phosphotyrosine. In the PTP1B structure, the invariant aspartate (Asp-181) is found in the movable WPD loop and is brought into the catalytic site upon substrate binding, where it acts as a general acid to facilitate phosphoester hydrolysis. The involvement of these residues in substrate binding and catalysis suggests that their altered nature in PTPα-D2 could have profound effects on phosphatase activity. To see if the substitution of these two invariant residues in PTPα-D2 affected D2 structure, we modeled PTPα-D1 and -D2 (Fig. 1 B). Although the x-ray structure of PTPα-D1 has been reported (31Bilwes A.M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar), it is not complexed with substrate, so models were prepared based on the structure of PTP1B complexed with phosphopeptide (13Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (558) Google Scholar). Inspection of all three superimposed structures showed that the molecules were largely similar. One area of interest and notable difference among the structures was the WPD loop region. The carboxylate group of Asp-382 in PTPα-D1 sits close to the phosphate (approximately 4 Å), whereas the carboxylate group of Glu-671 in the WPE loop of PTPα-D2 sits about 7.45 Å from the phosphate, likely due to the larger glutamate side chain having steric hindrance as well as repulsive forces from adjacent negative charges within the active site pocket (Fig. 1 B, inset). The distances between these carboxylate groups and the phenolic oxygen are 3.7 and 6.94 Å, respectively (not shown). The aspartate thus fits within the active site, whereas the glutamate is forced to remain at a distance from active site residues. Additional models were made of mutants where the loop sequence of PTPα-D1 was changed to WPE and that of PTPα-D2 to WPD (data not shown). The resulting models had the aspartate and glutamate residues at the expected locations so that glutamate was outside the active site pocket and aspartate was nestled inside the pocket of D2. The conserved KNRY region containing the invariant tyrosine (recognition region) was also analyzed. The model reveals that, as in PTP1B, Tyr-243 of PTPα-D1 supplies a hydrophobic environment to the phosphotyrosine-binding pocket. The Val-536 in the analogous sequence position in PTPα-D2 could result in reduced tenacity of binding due to the absence of a complementary surface for phosphotyrosines. The roles of Val-536 and Glu-671 in PTPα-D2 catalysis were tested by mutating these residues, singly and in combination, to the corresponding residues in PTPα-D1. Likewise, the counterpart invariant residues Tyr-243 and Asp-382 of PTPα-D1 were mutated to those present in PTPα-D2. A schematic representation of these mutants is shown in Fig. 2 A. Recombinant wild-type and mutant forms of the PTPα catalytic domains were expressed as GST-fusion proteins and cleaved with thrombin. The integrity, purity, and amounts of the released PTPα proteins were evaluated by SDS-polyacrylamide gel electrophoresis (Fig. 2 B) and densitometric scanning prior to the assays described below. To test the theory that proton donation to the phenolic oxygen of phosphotyrosine by the more distant hydroxyl moiety of glutamate in wild-type PTPα-D2 would be less catalytically favorable than from a closer hydroxyl moiety of aspartate, Glu-671 was mutated to either Asp or Ala (E671D and E671A, respectively). The kinetic parameters of activity of these PTPα-D2 mutants were assayed toward pNPP and the RR-src phosphotyrosyl peptide. As predicted, the E671D substitution positively affected PTPα-D2 activity toward pNPP, resulting in an enzyme with a 10-fold increased turnover number (k cat) intermediate to those of wild-type PTPα-D1 and -D2 and with an overall 4-fold increase in catalytic efficiency ratio (k cat/K m) (Table I). In contrast, the E671D substitution had surprisingly little effect on the kinetics of RR-src dephosphorylation by PTPα-D2 (Table I), indicating that Glu-671 in wild-type D2 is not responsible for the very low activity toward this substrate. Consistent with a role for Glu-671 as a general acid in catalysis, the PTPα-D2 E671A mutant exhibited less favorableK m and V max values than wild-type D2, with a 60-fold reduction in the catalytic efficiency ratio of pNPP dephosphorylation and such low activity toward RR-src that it could not be reliably measured (Table I).Table IKinetic parameters of activity of WPD and KNRY mutants of PTPαpNPPRR-srcK mV maxk catk cat/K mK mV maxk catk cat/K mmmnmol/min/mgs−1m−1s−1(102)mmnmol/min/mgs−1m−1s−1(102)PTPα-D11-aValues as previously determined in Ref. 19.0.281887211.644160.03380004.931494PTPα-D1(D382E)1.9422761.407.220.049400.024.08(±0.25)(±74)(±0.014)(±12)PTPα-D1(D382A)1.471770.110.750.0391.509.25 × 10−40.24(±0.25)(±4)(±0.009)(±0.29)PTPα-D1(Y243V)2.2365464.0418.10.153210.010.65(±0.55)(±543)(±0.041)(±3)PTPα-D1(Y243V/D382E)3.124550.280.900.1380.160.99 × 10−40.01(±0.27)(±16)(±0.049)(±0.07)PTPα-D21-aValues as previously determined in Ref. 19.1.689240.513.040.1330.6673.67 × 10−40.03PTPα-D2(E671D)3.8688024.8412.50.1330.8334.58 × 10−40.03(±0.25)(±736)(±0.047)(±0.24)PTPα-D2(E671A)3.81350.020.05ND1-bND, not determined.ND1-bND, not determined.(±0.96)(±11)PTPα-D2(V536Y)0.4539662.1848.40.0932.3312.8 × 10−40.14(±0.02)(±142)(±0.028)(±0.72)PTPα-D2(V536Y/E671D)0.766299634.654560.112580.032.68(±0.10)(±5633)(±0.023)(±25)The K m and V max values represent the mean ± S.E. of experiments conducted with at least three independent preparations of purified proteins.1-a Values as previously determined in Ref. 19Lim K.L. Lai D.S.Y. Kalousek M.B. Wang Y. Pallen C.J. Eur. J. Biochem. 1997; 245: 693-700Crossref PubMed Scopus (35) Google Scholar.1-b ND, not determined. Open table in a new tab The K m and V max values represent the mean ± S.E. of experiments conducted with at least three independent preparations of purified proteins. The mutation of Asp-382 to Glu or Ala in PTPα-D1 (D382E or D382A, respectively) had pronounced effects on the kinetics of dephosphorylation of pNPP and RR-src peptide (Table I). Toward pNPP, the PTPα-D1 D382E mutant exhibited a 7-fold increase in K m and an 8-fold decrease in V max relative to wild-type PTPα-D1, resulting in a turnover number and a catalytic efficiency ratio only 2–3-fold higher than wild-type PTPα-D2. However, toward RR-src, the PTPα-D1 D382E mutant had an essentially unchanged K m value relative to wild-type PTPα-D1, whereas theV max decreased 200-fold. Despite this,k cat and the catalytic efficiency ratio of this mutant D1 were still 54- and 136-fold higher than those of wild-type D2. The D1 mutant containing alanine rather than an acidic residue in position 382 (D382A) exhibited K m values similar to those of the D382E mutant for both substrates but had a further reduced rate of activity (Table I). The D1(D382A) mutant and wild-type D1 showed no essential difference in K m values for RR-src, although the K m of the mutant for pNPP was about 5-fold higher than that of wild-type D1 (Table I). The latter contrasts with a report that this same mutation in PTPα-D1 has virtually no effect on the K m for pNPP (24Wu L. Buist A. den Hertog J. Zhang Z.-Y. J. Biol. Chem. 1997; 272: 6994-7002Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). We do not know the reason for this difference, but in our experiments the mutation of Glu-671 to Ala in D2 had a similar effect on theK m value for pNPP (Table I). The catalytic efficiency ratios of PTPα-D1 D382A were about 560- and 6200-fold lower than wild-type D1 for pNPP and RR-src, respectively, consistent with a role of Asp-382 as a general acid in catalysis. The above results suggest that in the case of pNPP, PTPα-D1 and -D2 can be induced to behave more, but not entirely, like the other catalytic domain (i.e., D1 like D2 and vice versa) by mutation of their different putative general acids to that present in the counterpart domain, but in the case of the peptide substrate RR-src, the D1 a

Disruption of the ubiquitin-proteasome system, which normally identifies and degrades unwanted intracellular proteins, is thought to underlie neurodegeneration. Supporting this, mutations of Parkin, a ubiquitin ligase, are associated with autosomal recessive parkinsonism. Remarkably, Parkin can protect neurons against a wide spectrum of stress, including those that promote proteasome dysfunction. Although the mechanism underlying the preservation of proteasome function by Parkin is hitherto unclear, we have previously proposed that Parkin-mediated K63-linked ubiquitination (which is usually uncoupled from the proteasome) may serve to mitigate proteasomal stress by diverting the substrate load away from the machinery. By means of linkage-specific antibodies, we demonstrated here that proteasome inhibition indeed promotes K63-linked ubiquitination of proteins especially in Parkin-expressing cells. Importantly, we further demonstrated that the recruitment of Ubc13 (an E2 that mediates K63-linked polyubiquitin chain formation exclusively) by Parkin is selectively enhanced under conditions of proteasomal stress, thus identifying a mechanism by which Parkin could promote K63-linked ubiquitin modification in cells undergoing proteolytic stress. This mode of ubiquitination appears to facilitate the subsequent clearance of Parkin substrates via autophagy. Consistent with the proposed protective role of K63-linked ubiquitination in times of proteolytic stress, we found that Ubc13-deficient cells are significantly more susceptible to cell death induced by proteasome inhibitors compared to their wild type counterparts. Taken together, our study suggests a role for Parkin-mediated K63 ubiquitination in maintaining cellular protein homeostasis, especially during periods when the proteasome is burdened or impaired.

Exposure to divalent metals such as iron and manganese is thought to increase the risk for Parkinson's disease (PD). Under normal circumstances, cellular iron and manganese uptake is regulated by the divalent metal transporter 1 (DMT1). Accordingly, alterations in DMT1 levels may underlie the abnormal accumulation of metal ions and thereby disease pathogenesis. Here, we have generated transgenic mice overexpressing DMT1 under the direction of a mouse prion promoter and demonstrated its robust expression in several regions of the brain. When fed with iron-supplemented diet, DMT1-expressing mice exhibit rather selective accumulation of iron in the substantia nigra, which is the principal region affected in human PD cases, but otherwise appear normal. Alongside this, the expression of Parkin is also enhanced, likely as a neuroprotective response, which may explain the lack of phenotype in these mice. When DMT1 is overexpressed against a Parkin null background, the double-mutant mice similarly resisted a disease phenotype even when fed with iron- or manganese-supplemented diet. However, these mice exhibit greater vulnerability toward 6-hydroxydopamine-induced neurotoxicity. Taken together, our results suggest that iron accumulation alone is not sufficient to cause neurodegeneration and that multiple hits are required to promote PD.

Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease, affecting about 7 to 10 million patients worldwide. The major pathological features of PD include loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) of the midbrain and the presence of α-synuclein-enriched Lewy bodies. Although the mechanism underlying PD pathogenesis remains to be elucidated, oxidative stress induced by the overproduction of reactive oxygen species (ROS) is widely accepted to be a key pathogenic factors. ROS cause oxidative damage to proteins, lipids, and DNA, which subsequently lead to neurodegeneration. Great efforts have been made to slow or stop the progress of PD. Unfortunately there is no effective cure for PD till now. Compounds with good antioxidant activity represent the promising candidates for therapeutics of PD. Some natural molecules from Chinese herbs are found to have good antioxidant activity. Both in vitro and in vivo studies demonstrate that these natural molecules could mitigate the oxidative stress and rescue the neuronal cell death in PD models. In present review, we summarized the reported natural molecules that displayed protective effects in PD. We also addressed the possible signal pathway through which natural molecules achieved their antioxidative effects and mitigate PD phenotypes. Hopefully it will pave the way to better recognize and utilize Chinese herbs for the treatment of PD.

Emerging studies implicate energy dysregulation as an underlying trigger for Parkinson's disease (PD), suggesting that a better understanding of the molecular pathways governing energy homeostasis could help elucidate therapeutic targets for the disease. A critical cellular energy regulator is AMP kinase (AMPK), which we have previously shown to be protective in PD models. However, precisely how AMPK function impacts on dopaminergic neuronal survival and disease pathogenesis remains elusive. Here, we showed that Drosophila deficient in AMPK function exhibits PD-like features, including dopaminergic neuronal loss and climbing impairment that progress with age. We also created a tissue-specific AMPK-knockout mouse model where the catalytic subunits of AMPK are ablated in nigral dopaminergic neurons. Using this model, we demonstrated that loss of AMPK function promotes dopaminergic neurodegeneration and associated locomotor aberrations. Accompanying this is an apparent reduction in the number of mitochondria in the surviving AMPK-deficient nigral dopaminergic neurons, suggesting that an impairment in mitochondrial biogenesis may underlie the observed PD-associated phenotypes. Importantly, the loss of AMPK function enhances the susceptibility of nigral dopaminergic neurons in these mice to 6-hydroxydopamine-induced toxicity. Notably, we also found that AMPK activation is reduced in post-mortem PD brain samples. Taken together, these findings highlight the importance of neuronal energy homeostasis by AMPK in PD and position AMPK pathway as an attractive target for future therapeutic exploitation.

Report18 October 2022 Transparent process Parkin coordinates mitochondrial lipid remodeling to execute mitophagy Chao-Chieh Lin Chao-Chieh Lin orcid.org/0000-0001-5890-9004 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA Contribution: Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, ​Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Jin Yan Jin Yan orcid.org/0000-0002-0116-7705 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Contribution: Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, ​Investigation, Methodology, Writing - original draft Search for more papers by this author Meghan D Kapur Meghan D Kapur Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Contribution: Conceptualization, Data curation, Formal analysis, Funding acquisition, ​Investigation, Writing - original draft Search for more papers by this author Kristi L Norris Kristi L Norris Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Methodology, Writing - original draft Search for more papers by this author Cheng-Wei Hsieh Cheng-Wei Hsieh orcid.org/0000-0003-2453-2071 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Contribution: Methodology Search for more papers by this author De Huang De Huang Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Contribution: Methodology Search for more papers by this author Nicolas Vitale Nicolas Vitale orcid.org/0000-0002-4752-4907 Institut des Neurosciences Cellulaires et IntégrativesUPR-3212 CNRS - Université de Strasbourg, Strasbourg, France Contribution: Methodology Search for more papers by this author Kah-Leong Lim Kah-Leong Lim orcid.org/0000-0002-5440-2588 Lee Kong Chian School of Medicine, Singapore City, Singapore Contribution: Conceptualization Search for more papers by this author Ziqiang Guan Ziqiang Guan Department of Biochemistry, Duke University Medical Center, Durham, NC, USA Contribution: Methodology Search for more papers by this author Xiao-Fan Wang Xiao-Fan Wang Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Contribution: Conceptualization, Funding acquisition Search for more papers by this author Jen-Tsan Chi Jen-Tsan Chi orcid.org/0000-0003-3433-903X Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA Contribution: Conceptualization, Funding acquisition Search for more papers by this author Wei-Yuan Yang Wei-Yuan Yang orcid.org/0000-0002-8939-651X Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Contribution: Conceptualization, Methodology Search for more papers by this author Tso-Pang Yao Corresponding Author Tso-Pang Yao [email protected] orcid.org/0000-0001-8914-3224 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Contribution: Conceptualization, Resources, Supervision, Funding acquisition, ​Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Chao-Chieh Lin Chao-Chieh Lin orcid.org/0000-0001-5890-9004 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA Contribution: Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, ​Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Jin Yan Jin Yan orcid.org/0000-0002-0116-7705 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Contribution: Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, ​Investigation, Methodology, Writing - original draft Search for more papers by this author Meghan D Kapur Meghan D Kapur Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Contribution: Conceptualization, Data curation, Formal analysis, Funding acquisition, ​Investigation, Writing - original draft Search for more papers by this author Kristi L Norris Kristi L Norris Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Methodology, Writing - original draft Search for more papers by this author Cheng-Wei Hsieh Cheng-Wei Hsieh orcid.org/0000-0003-2453-2071 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Contribution: Methodology Search for more papers by this author De Huang De Huang Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Contribution: Methodology Search for more papers by this author Nicolas Vitale Nicolas Vitale orcid.org/0000-0002-4752-4907 Institut des Neurosciences Cellulaires et IntégrativesUPR-3212 CNRS - Université de Strasbourg, Strasbourg, France Contribution: Methodology Search for more papers by this author Kah-Leong Lim Kah-Leong Lim orcid.org/0000-0002-5440-2588 Lee Kong Chian School of Medicine, Singapore City, Singapore Contribution: Conceptualization Search for more papers by this author Ziqiang Guan Ziqiang Guan Department of Biochemistry, Duke University Medical Center, Durham, NC, USA Contribution: Methodology Search for more papers by this author Xiao-Fan Wang Xiao-Fan Wang Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Contribution: Conceptualization, Funding acquisition Search for more papers by this author Jen-Tsan Chi Jen-Tsan Chi orcid.org/0000-0003-3433-903X Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA Contribution: Conceptualization, Funding acquisition Search for more papers by this author Wei-Yuan Yang Wei-Yuan Yang orcid.org/0000-0002-8939-651X Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Contribution: Conceptualization, Methodology Search for more papers by this author Tso-Pang Yao Corresponding Author Tso-Pang Yao [email protected] orcid.org/0000-0001-8914-3224 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Contribution: Conceptualization, Resources, Supervision, Funding acquisition, ​Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Author Information Chao-Chieh Lin1,2,†, Jin Yan1,†, Meghan D Kapur1,†, Kristi L Norris1,†, Cheng-Wei Hsieh3, De Huang1, Nicolas Vitale4, Kah-Leong Lim5, Ziqiang Guan6, Xiao-Fan Wang1, Jen-Tsan Chi2, Wei-Yuan Yang3 and Tso-Pang Yao *,1 1Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA 2Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA 3Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan 4Institut des Neurosciences Cellulaires et IntégrativesUPR-3212 CNRS - Université de Strasbourg, Strasbourg, France 5Lee Kong Chian School of Medicine, Singapore City, Singapore 6Department of Biochemistry, Duke University Medical Center, Durham, NC, USA † These authors contributed equally to this work *Corresponding author. Tel: +1 919 613 8654; E-mail: [email protected] EMBO Reports (2022)23:e55191https://doi.org/10.15252/embr.202255191 Full textView the full text of the articlePDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Autophagy has emerged as the prime machinery for implementing organelle quality control. In the context of mitophagy, the ubiquitin E3 ligase Parkin tags impaired mitochondria with ubiquitin to activate autophagic degradation. Although ubiquitination is essential for mitophagy, it is unclear how ubiquitinated mitochondria activate autophagosome assembly locally to ensure efficient destruction. Here, we report that Parkin activates lipid remodeling on mitochondria targeted for autophagic destruction. Mitochondrial Parkin induces the production of phosphatidic acid (PA) and its subsequent conversion to diacylglycerol (DAG) by recruiting phospholipase D2 and activating the PA phosphatase, Lipin-1. The production of DAG requires mitochondrial ubiquitination and ubiquitin-binding autophagy receptors, NDP52 and optineurin (OPTN). Autophagic receptors, via Golgi-derived vesicles, deliver an autophagic activator, EndoB1, to ubiquitinated mitochondria. Inhibition of Lipin-1, NDP52/OPTN, or EndoB1 results in a failure to produce mitochondrial DAG, autophagosomes, and mitochondrial clearance, while exogenous cell-permeable DAG can induce autophagosome production. Thus, mitochondrial DAG production acts downstream of Parkin to enable the local assembly of autophagosomes for the efficient disposal of ubiquitinated mitochondria. Synopsis Parkin orchestrates mitophagy by coordinating mitochondrial lipid remodeling and sequential production of mitochondrial phosphatic acid and diacylglycerol. Parkin engages phospholipase D2 (PLD2) to generate mitochondrial phosphatidic acid. Parkin-mediated mitochondrial ubiquitination recruits the ubiquitin-binding autophagic receptors, optineurin and NDP52, which promote mitochondrial diacylglycerol production via Lipin-1. Mitochondrial diacylglycerol production stimulates autophagosome assembly and mitophagy. Previous ArticleNext Article Read MoreAbout the coverClose modalView large imageVolume 23,Issue 12,06 December 2022This month's cover highlights the article Cryo–EM structure of the octameric pore of Clostridium perfringens β–toxin by Julia Bruggisser, Ioan Iacovache, Horst Posthaus, Benoît Zuber and colleagues. The cover shows two octameric pores of C. Perfringens β–toxin in a lipid bilayer. One pore is shown bound to its receptor CD31 while the second is shown as cut through at the edge of the membrane. Alternating protomers in the oligomer are shown in different shades of blue while for the receptor, which was model based on an AlphaFold prediction, alternating Ig domains are shown in different shades of yellow. (Scientific image by Ioan Iacovache and Julia Bruggisser. Illustration created in Blender (http://www.blender.org) using a phospholipid bilayer template by Dr. Joseph G. Manion (https://cgfigures.gumroad.com/l/eJzxj. Copyright (author or institute): Institute of Anatomy, University of Bern) Volume 23Issue 126 December 2022In this issue ReferencesRelatedDetailsLoading ...

Abstract Monoamine oxidases (MAOs) are the enzymes that catalyze the oxidation of monoamines, such as dopamine, norepinephrine, and serotonin, which serve as key neurotransmitters in the central nervous system (CNS). MAOs play important roles in maintaining the homeostasis of monoamines, and the aberrant expression or activation of MAOs underlies the pathogenesis of monoamine neurotransmitter disorders, including neuropsychiatric and neurodegenerative diseases. Clearly, detecting and inhibiting the activities of MAOs is of great value for the diagnosis and therapeutics of these diseases. Accordingly, many specific detection probes and inhibitors have been developed and substantially contributed to basic and clinical studies of these diseases. In this review, progress in the detecting and inhibiting of MAOs and their applications in mechanism exploration and treatment of neurotransmitter‐related disorders is summarized. Notably, how the detection probes and inhibitors of MAOs were developed has been specifically addressed. It is hoped that this review will benefit the design of more effective and sensitive probes and inhibitors for MAOs, and eventually the treatment of monoamine neurotransmitter disorders.

The authors designed and synthesized a mitochondria-targeted polydopamine nanoprobe for visualizing endogenous SO₃²⁻/HSO₃⁻ by the nucleophilic addition reaction. The nanoprobe was used for imaging SO₂ derivatives both in the mitochondria of cells, zebrafish, and hippocampus of a rat model of epilepsy.

MAO-A promotes the proliferation of human glioma cells. Herein, we report a series of MAO-A specific two-photon small molecular fluorescent probes (A1-5) based on an intramolecular charge transfer enhancing strategy. The activity of endogenous MAO-A can be selectively imaged using A3 as a representative probe in different biological samples including human glioma cells/tissues via two-photon fluorescence microscopy. The study provides new tools for the visual detection of glioma.

Retinal pigment epithelial (RPE) cell dysfunction and death are characteristics of age-related macular degeneration. A promising therapeutic option is RPE cell transplantation. Development of clinical grade stem-cell derived RPE requires efficient in vitro differentiation and purification methods. Enzymatic purification of RPE relies on the relative adherence of RPE and non-RPE cells to the culture plate. However, morphology and adherence of non-RPE cells differ for different stem cell sources. In cases whereby the non-RPE adhered as strongly as RPE cells to the culture plate, enzymatic method of purification is unsuitable. Thus, we hypothesized the need to customize purification strategies for RPE derived from different stem cell sources. We systematically compared five different RPE purification methods, including manual, enzymatic, flow cytometry-based sorting or combinations thereof for parameters including cell throughput, yield, purity and functionality. Flow cytometry-based approach was suitable for RPE isolation from heterogeneous cultures with highly adherent non-RPE cells, albeit with lower yield. Although all five purification methods generated pure and functional RPE, there were significant differences in yield and processing times. Based on the high purity of the resulting RPE and relatively short processing time, we conclude that a combination of enzymatic and manual purification is ideal for clinical applications.

Alzheimer's disease (AD) and Parkinson's disease (PD) are the most common neurodegenerative diseases (NDDs) threatening the lives of millions of people worldwide, including especially elderly people. Currently, due to the lack of a timely diagnosis and proper intervention strategy, AD and PD largely remain incurable. Innovative diagnosis and therapy are highly desired. Exosomes are small vesicles that are present in various bodily fluids, which contain proteins, nucleic acids, and active biomolecules, and which play a crucial role especially in intercellular communication. In recent years, the role of exosomes in the pathogenesis, early diagnosis, and treatment of diseases has attracted ascending attention. However, the exact role of exosomes in the pathogenesis and theragnostic of AD and PD has not been fully illustrated. In the present review, we first introduce the biogenesis, components, uptake, and function of exosomes. Then we elaborate on the involvement of exosomes in the pathogenesis of AD and PD. Moreover, the application of exosomes in the diagnosis and therapeutics of AD and PD is also summarized and discussed. Additionally, exosomes serving as drug carriers to deliver medications to the central nervous system are specifically addressed. The potential role of exosomes in AD and PD is explored, discussing their applications in diagnosis and treatment, as well as their current limitations. Given the limitation in the application of exosomes, we also propose future perspectives for better utilizing exosomes in NDDs. Hopefully, it would pave ways for expanding the biological applications of exosomes in fundamental research as well as theranostics of NDDs.

Abstract Background Lipodystrophy-associated metabolic disorders caused by Seipin deficiency lead to not only severe lipodystrophy but also neurological disorders. However, the underlying mechanism of Seipin deficiency-induced neuropathy is not well elucidated, and the possible restorative strategy needs to be explored. Methods In the present study, we used Seipin knockout (KO) mice, combined with transcriptome analysis, mass spectrometry imaging, neurobehavior test, and cellular and molecular assay to investigate the systemic lipid metabolic abnormalities in lipodystrophic mice model and their effects on adult neurogenesis in the subventricular zone (SVZ) and olfactory function. After subcutaneous adipose tissue (AT) transplantation, metabolic and neurological function was measured in Seipin KO mice to clarify whether restoring lipid metabolic homeostasis would improve neurobehavior. Results It was found that Seipin KO mice presented the ectopic accumulation of lipids in the lateral ventricle, accompanied by decreased neurogenesis in adult SVZ, diminished new neuron formation in the olfactory bulb, and impaired olfactory-related memory. Transcriptome analysis showed that the differentially expressed genes (DEGs) in SVZ of adult Seipin KO mice were significantly enriched in lipid metabolism. Mass spectrometry imaging showed that the levels of glycerophospholipid and diglyceride (DG) were significantly increased. Furthermore, we found that AT transplantation rescued the abnormality of peripheral metabolism in Seipin KO mice and ameliorated the ectopic lipid accumulation, concomitant with restoration of the SVZ neurogenesis and olfactory function. Mechanistically, PKCα expression was up-regulated in SVZ tissues of Seipin KO mice, which may be a potential mediator between lipid dysregulation and neurological disorder. DG analogue (Dic8) can up-regulate PKCα and inhibit the proliferation and differentiation of neural stem cells (NSCs) in vitro, while PKCα inhibitor can block this effect. Conclusion This study demonstrates that Seipin deficiency can lead to systemic lipid disorder with concomitant SVZ neurogenesis and impaired olfactory memory. However, AT restores lipid homeostasis and neurogenesis. PKCα is a key mediator mediating Seipin KO-induced abnormal lipid metabolism and impaired neurogenesis in the SVZ, and inhibition of PKCα can restore the impaired neurogenesis. This work reveals the underlying mechanism of Seipin deficiency-induced neurological dysfunction and provides new ideas for the treatment of neurological dysfunction caused by metabolic disorders.

Cholesterol is important for membrane integrity and cell signaling, and dysregulation of the distribution of cellular cholesterol is associated with numerous diseases, including neurodegenerative disorders. While regulated transport of a specific pool of cholesterol, known as "accessible cholesterol", contributes to the maintenance of cellular cholesterol distribution and homeostasis, tools to monitor accessible cholesterol in live cells remain limited. Here, we engineer a highly sensitive accessible cholesterol biosensor by taking advantage of the cholesterol-sensing element (the GRAM domain) of an evolutionarily conserved lipid transfer protein, GRAMD1b. Using this cholesterol biosensor, which we call GRAM-W, we successfully visualize in real time the distribution of accessible cholesterol in many different cell types, including human keratinocytes and iPSC-derived neurons, and show differential dependencies on cholesterol biosynthesis and uptake for maintaining levels of accessible cholesterol. Furthermore, we combine GRAM-W with a dimerization-dependent fluorescent protein (ddFP) and establish a strategy for the ultrasensitive detection of accessible plasma membrane cholesterol. These tools will allow us to obtain important insights into the molecular mechanisms by which the distribution of cellular cholesterol is regulated.

Aging results from the accumulation of molecular damage that impairs normal biochemical processes. We previously reported that age-linked damage to amino acid sequence NGR (Asn-Gly-Arg) results in "gain-of-function" conformational switching to isoDGR (isoAsp-Gly-Arg). This integrin-binding motif activates leukocytes and promotes chronic inflammation, which are characteristic features of age-linked cardiovascular disorders. We now report that anti-isoDGR immunotherapy mitigates lifespan reduction of Pcmt1-/- mouse. We observed extensive accumulation of isoDGR and inflammatory cytokine expression in multiple tissues from Pcmt1-/- and naturally aged WT animals, which could also be induced via injection of isoDGR-modified plasma proteins or synthetic peptides into young WT animals. However, weekly injection of anti-isoDGR mAb (1 mg/kg) was sufficient to significantly reduce isoDGR-protein levels in body tissues, decreased pro-inflammatory cytokine concentrations in blood plasma, improved cognition/coordination metrics, and extended the average lifespan of Pcmt1-/- mice. Mechanistically, isoDGR-mAb mediated immune clearance of damaged isoDGR-proteins via antibody-dependent cellular phagocytosis (ADCP). These results indicate that immunotherapy targeting age-linked protein damage may represent an effective intervention strategy in a range of human degenerative disorders.

Cholesterol plays a significant role in maintaining membrane integrity and cell signaling. Dysregulation of the distribution of cellular cholesterol is associated with numerous diseases, including cardiovascular and neurological diseases. While regulated transport of a specific pool of cholesterol, known as "accessible cholesterol" contributes to the maintenance of cellular cholesterol distribution and homeostasis, tools to visualize accessible cholesterol in live cells remain limited. Here, we engineered a highly sensitive accessible cholesterol biosensor by utilizing the cholesterol-sensing domain (the GRAM domain) of an evolutionarily conserved lipid transfer protein, GRAMD1b.

Abstract Although multiple cellular pathways have been implicated in a-Synuclein (a-syn)-associated Parkinson’s disease (PD), the role of lipid metabolism remains elusive. Using the Drosophila system as a genetic screening tool, we identified mino , which encodes the mitochondrial isoform of the lipid synthesis enzyme glycerol 3-phosphate acyltransferase (GPAT), as a potent modifier of a-syn. Silencing the expression of mino significantly suppresses a-syn-induced PD phenotypes in Drosophila , including dopaminergic neuronal loss and locomotion defects as well as circadian rhythm-related activities, whereas mino overexpression yields opposite effects. Mechanistically, we found that mino modulates the levels of mitochondrial reactive oxygen speciesand lipid peroxidation. Importantly, treatment of a-syn-expressing flies with FSG67, a GPAT inhibitor, reproduces the benefits of mino knockdown. FSG67 also inhibited a-syn aggregation and lipid peroxidation in mouse primary neurons transfected with a-syn preformed fibrils. Our study elucidates an important factor contributing to a-syn toxicity and offers a novel therapeutic direction for PD.

Abstract Parkinson’s disease (PD) is a prevalent neurodegenerative disorder affecting millions of elderly individuals worldwide. Clinically, PD is diagnosed based on the presentation of motoric symptoms. Other methods such as F-DOPA PET scan or α-Synuclein detection from the cerebral spinal fluid are either too expensive or invasive for routine use. Omics platforms such as transcriptomics, proteomics, and metabolomics may identify PD biomarkers from blood, which can reduce cost and increase efficiency. However, there are many biological moieties being measured and issues with false positives/negatives. It is also unknown which omics platform offers most useful information. Therefore, it is important to assess the reliability of these omics studies. Here, we shortlisted and analysed nearly 80 published reports across transcriptomics, proteomics and metabolomics in search of overlapping blood-based biomarkers for PD. The top biomarkers were reported across 29%, 42% and 12.5% of shortlisted papers in transcriptomics, proteomics and metabolomics respectively. These percentages increased to 42%, 60% and 50% accordingly when studies were grouped by specific blood subtypes for analysis, demonstrating the need for test kits to be blood-subtype specific. Following systematic analyses, we propose six novel PD biomarkers: two mRNAs (Whole blood, WB) – Arg1 and SNCA, two proteins (Plasma EV) – SNCA and APOA1, and two metabolites (WB) – 8-OHdG and uric acid for further validation. While these proposed biomarkers are useful, they are also snapshots, representing subsets of larger pathways of origin where the different omics levels corroborate. Indeed, identifying the interconnections across different biological layers can strengthen contextual reasoning, which in turn, would give rise to better quality biomarkers. Knowledge integration across the omics spectrum revealed consistent aberrations on the same neuroinflammation pathway, showcasing the value of integrative (i)-omics agreements for increasing confidence of biomarker selection. We believe that our findings could pave the way for identifying reproducible PD biomarkers, with potential for clinical deployment. Graphical Abstract Six Proposed blood-based biomarkers. Seventy-nine publications across transcriptomics, proteomics and metabolomics were shortlisted and analysed for reported biomarkers. The proposed biomarkers are SNCA, APOA1, Arg1, 8-OHdG and Uric acid.

Parkinson's disease (PD) is an age-related neurodegenerative disease characterized by histopathological hallmarks of Lewy bodies formed by accumulation of α-synuclein (αSyn) and progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain, with clinical symptoms of motor deficits. Toxic protein accumulation of αSyn in PD is associated with autolysosomal acidification dysfunction that contributes to defective autophagy-lysosomal degradation system. While lysosome-acidifying nanoparticles have been applied as therapeutics to ameliorate dopaminergic neurodegeneration in neurotoxin mediated or αSyn aggregates induced mouse model of sporadic PD, lysosome-targeted approach has not yet been applied in synucleinopathy models of familial PD. Here, we report the first application of the new poly(ethylene tetrafluorosuccinate-co-succinate) (PEFSU)-based acidic nanoparticles (AcNPs) in A30P αSyn overexpressing SH-SY5Y cells and Drosophila models of PD. In the cellular model, we showed that AcNPs restore lysosomal acidification, promote autophagic clearance of αSyn, improve mitochondrial turnover and function, and rescue A30P αSyn induced death in SH-SY5Y cells. In the Drosophila model, we demonstrated that AcNPs enhance clearance of αSyn and rescue dopaminergic neuronal loss in fly brains and improve their locomotor activity. Our results highlight AcNPs as a new class of lysosome-acidifying therapeutic for treatment of PD and other proteinopathies in general.