Angelo Poletti

Several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), are characterized by the presence of misfolded proteins, thought to trigger neurotoxicity. Some familial forms of ALS (fALS), clinically indistinguishable from sporadic ALS (sALS), are linked to superoxide dismutase 1 (SOD1) gene mutations. It has been shown that the mutant SOD1 misfolds, forms insoluble aggregates and impairs the proteasome. Using transgenic G93A-SOD1 mice, we found that spinal cord motor neurons, accumulating mutant SOD1 also over-express the small heat shock protein HspB8. Using motor neuronal fALS models, we demonstrated that HspB8 decreases aggregation and increases mutant SOD1 solubility and clearance, without affecting wild-type SOD1 turnover. Notably, HspB8 acts on mutant SOD1 even when the proteasome activity is specifically blocked. The pharmacological blockage of autophagy resulted in a dramatic increase of mutant SOD1 aggregates. Immunoprecipitation studies, performed during autophagic flux blockage, demonstrated that mutant SOD1 interacts with the HspB8/Bag3/Hsc70/CHIP multiheteromeric complex, known to selectively activate autophagic removal of misfolded proteins. Thus, HspB8 increases mutant SOD1 clearance via autophagy. Autophagy activation was also observed in lumbar spinal cord of transgenic G93A-SOD1 mice since several autophago-lysosomal structures were present in affected surviving motor neurons. Finally, we extended our observation to a different ALS model and demonstrated that HspB8 exerts similar effects on a truncated version of TDP-43, another protein involved both in fALS and in sALS. Overall, these results indicate that the pharmacological modulation of HspB8 expression in motor neurons may have important implications to unravel the molecular mechanisms involved both in fALS and in sALS.

Macroautophagy/autophagy, a defense mechanism against aberrant stresses, in neurons counteracts aggregate-prone misfolded protein toxicity. Autophagy induction might be beneficial in neurodegenerative diseases (NDs). The natural compound trehalose promotes autophagy via TFEB (transcription factor EB), ameliorating disease phenotype in multiple ND models, but its mechanism is still obscure. We demonstrated that trehalose regulates autophagy by inducing rapid and transient lysosomal enlargement and membrane permeabilization (LMP). This effect correlated with the calcium-dependent phosphatase PPP3/calcineurin activation, TFEB dephosphorylation and nuclear translocation. Trehalose upregulated genes for the TFEB target and regulator Ppargc1a, lysosomal hydrolases and membrane proteins (Ctsb, Gla, Lamp2a, Mcoln1, Tpp1) and several autophagy-related components (Becn1, Atg10, Atg12, Sqstm1/p62, Map1lc3b, Hspb8 and Bag3) mostly in a PPP3- and TFEB-dependent manner. TFEB silencing counteracted the trehalose pro-degradative activity on misfolded protein causative of motoneuron diseases. Similar effects were exerted by trehalase-resistant trehalose analogs, melibiose and lactulose. Thus, limited lysosomal damage might induce autophagy, perhaps as a compensatory mechanism, a process that is beneficial to counteract neurodegeneration.Abbreviations: ALS: amyotrophic lateral sclerosis; AR: androgen receptor; ATG: autophagy related; AV: autophagic vacuole; BAG3: BCL2-associated athanogene 3; BECN1: beclin 1, autophagy related; CASA: chaperone-assisted selective autophagy; CTSB: cathepsin b; DAPI: 4ʹ,6-diamidino-2-phenylindole; DMEM: Dulbecco’s modified Eagle’s medium; EGFP: enhanced green fluorescent protein; fALS, familial amyotrophic lateral sclerosis; FRA: filter retardation assay; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GLA: galactosidase, alpha; HD: Huntington disease; hIPSCs: human induced pluripotent stem cells; HSPA8: heat shock protein A8; HSPB8: heat shock protein B8; IF: immunofluorescence analysis; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LGALS3: lectin, galactose binding, soluble 3; LLOMe: L-leucyl-L-leucine methyl ester; LMP: lysosomal membrane permeabilization; Lys: lysosomes; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MCOLN1: mucolipin 1; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; NDs: neurodegenerative diseases; NSC34: neuroblastoma x spinal cord 34; PBS: phosphate-buffered saline; PD: Parkinson disease; polyQ: polyglutamine; PPARGC1A: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; PPP3CB: protein phosphatase 3, catalytic subunit, beta isoform; RT-qPCR: real-time quantitative polymerase chain reaction; SBMA: spinal and bulbar muscular atrophy; SCAs: spinocerebellar ataxias; siRNA: small interfering RNA; SLC2A8: solute carrier family 2, (facilitated glucose transporter), member 8; smNPCs: small molecules neural progenitors cells; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STED: stimulated emission depletion; STUB1: STIP1 homology and U-box containing protein 1; TARDBP/TDP-43: TAR DNA binding protein; TFEB: transcription factor EB; TPP1: tripeptidyl peptidase I; TREH: trehalase (brush-border membrane glycoprotein); WB: western blotting; ZKSCAN3: zinc finger with KRAB and SCAN domains 3

Inflammatory activation of microglia is a hallmark of several disorders of the central nervous system. In addition to protecting the brain against inflammatory insults, microglia are neuroprotective and play a significant role in maintaining neuronal connectivity, but the prolongation of an inflammatory status may limit the beneficial functions of these immune cells. The finding that estrogen receptors are present in monocyte-derived cells and that estrogens prevent and control the inflammatory response raise the question of the role that this sex steroid plays in the manifestation and progression of pathologies that have a clear sex difference in prevalence, such as multiple sclerosis, Parkinson's disease, and Alzheimer's disease. The present review aims to provide a critical review of the current literature on the actions of estrogen in microglia and on the involvement of estrogen receptors in the manifestation of selected neurological disorders. This current understanding highlights a research area that should be expanded to identify appropriate replacement therapies to slow the progression of such diseases.

Stress granules (SGs) are ribonucleoprotein complexes induced by stress. They sequester mRNAs and disassemble when the stress subsides, allowing translation restoration. In amyotrophic lateral sclerosis (ALS), aberrant SGs cannot disassemble and therefore accumulate and are degraded by autophagy. However, the molecular events causing aberrant SG formation and the molecular players regulating this transition are largely unknown. We report that defective ribosomal products (DRiPs) accumulate in SGs and promote a transition into an aberrant state that renders SGs resistant to RNase. We show that only a minor fraction of aberrant SGs is targeted by autophagy, whereas the majority disassembles in a process that requires assistance by the HSPB8-BAG3-HSP70 chaperone complex. We further demonstrate that HSPB8-BAG3-HSP70 ensures the functionality of SGs and restores proteostasis by targeting DRiPs for degradation. We propose a system of chaperone-mediated SG surveillance, or granulostasis, which regulates SG composition and dynamics and thus may play an important role in ALS.

Small heat shock proteins (sHSPs) are present in all kingdoms of life and play fundamental roles in cell biology. sHSPs are key components of the cellular protein quality control system, acting as the first line of defense against conditions that affect protein homeostasis and proteome stability, from bacteria to plants to humans. sHSPs have the ability to bind to a large subset of substrates and to maintain them in a state competent for refolding or clearance with the assistance of the HSP70 machinery. sHSPs participate in a number of biological processes, from the cell cycle, to cell differentiation, from adaptation to stressful conditions, to apoptosis, and, even, to the transformation of a cell into a malignant state. As a consequence, sHSP malfunction has been implicated in abnormal placental development and preterm deliveries, in the prognosis of several types of cancer, and in the development of neurological diseases. Moreover, mutations in the genes encoding several mammalian sHSPs result in neurological, muscular, or cardiac age-related diseases in humans. Loss of protein homeostasis due to protein aggregation is typical of many age-related neurodegenerative and neuromuscular diseases. In light of the role of sHSPs in the clearance of un/misfolded aggregation-prone substrates, pharmacological modulation of sHSP expression or function and rescue of defective sHSPs represent possible routes to alleviate or cure protein conformation diseases. Here, we report the latest news and views on sHSPs discussed by many of the world’s experts in the sHSP field during a dedicated workshop organized in Italy (Bertinoro, CEUB, October 12–15, 2016).

Abstract Neurodegenerative diseases (NDs) are a wide class of disorders of the central nervous system (CNS) with unknown etiology. Several factors were hypothesized to be involved in the pathogenesis of these diseases, including genetic and environmental factors. Many of these diseases show a sex prevalence and sex steroids were shown to have a role in the progression of specific forms of neurodegeneration. Estrogens were reported to be neuroprotective through their action on cognate nuclear and membrane receptors, while adverse effects of male hormones have been described on neuronal cells, although some data also suggest neuroprotective activities. The response of the CNS to sex steroids is a complex and integrated process that depends on (i) the type and amount of the cognate steroid receptor and (ii) the target cell type—either neurons, glia, or microglia. Moreover, the levels of sex steroids in the CNS fluctuate due to gonadal activities and to local metabolism and synthesis. Importantly, biochemical processes involved in the pathogenesis of NDs are increasingly being recognized as different between the two sexes and as influenced by sex steroids. The aim of this review is to present current state-of-the-art understanding on the potential role of sex steroids and their receptors on the onset and progression of major neurodegenerative disorders, namely, Alzheimer’s disease, Parkinson’s diseases, amyotrophic lateral sclerosis, and the peculiar motoneuron disease spinal and bulbar muscular atrophy, in which hormonal therapy is potentially useful as disease modifier.

Each protein must be synthesized with the correct amino acid sequence, folded into its native structure, and transported to a relevant subcellular location and protein complex. If any of these steps fail, the cell has the capacity to break down aberrant proteins to maintain protein homeostasis (also called proteostasis). All cells possess a set of well-characterized protein quality control systems to minimize protein misfolding and the damage it might cause. Autophagy, a conserved pathway for the degradation of long-lived proteins, aggregates, and damaged organelles, was initially characterized as a bulk degradation pathway. However, it is now clear that autophagy also contributes to intracellular homeostasis by selectively degrading cargo material. One of the pathways involved in the selective removal of damaged and misfolded proteins is chaperone-assisted selective autophagy (CASA). The CASA complex is composed of three main proteins (HSPA, HSPB8 and BAG3), essential to maintain protein homeostasis in muscle and neuronal cells. A failure in the CASA complex, caused by mutations in the respective coding genes, can lead to (cardio)myopathies and neurodegenerative diseases. Here, we summarize our current understanding of the CASA complex and its dynamics. We also briefly discuss how CASA complex proteins are involved in disease and may represent an interesting therapeutic target.Abbreviation ALP: autophagy lysosomal pathway; ALS: amyotrophic lateral sclerosis; AMOTL1: angiomotin like 1; ARP2/3: actin related protein 2/3; BAG: BAG cochaperone; BAG3: BAG cochaperone 3; CASA: chaperone-assisted selective autophagy; CMA: chaperone-mediated autophagy; DNAJ/HSP40: DnaJ heat shock protein family (Hsp40); DRiPs: defective ribosomal products; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK1/HRI: eukaryotic translation initiation factor 2 alpha kinase 1; GABARAP: GABA type A receptor-associated protein; HDAC6: histone deacetylase 6; HSP: heat shock protein; HSPA/HSP70: heat shock protein family A (Hsp70); HSP90: heat shock protein 90; HSPB8: heat shock protein family B (small) member 8; IPV: isoleucine-proline-valine; ISR: integrated stress response; KEAP1: kelch like ECH associated protein 1; LAMP2A: lysosomal associated membrane protein 2A; LATS1: large tumor suppressor kinase 1; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOC: microtubule organizing center; MTOR: mechanistic target of rapamycin kinase; NFKB/NF-κB: nuclear factor kappa B; NFE2L2: NFE2 like bZIP transcription factor 2; PLCG/PLCγ: phospholipase C gamma; polyQ: polyglutamine; PQC: protein quality control; PxxP: proline-rich; RAN translation: repeat-associated non-AUG translation; SG: stress granule; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STUB1/CHIP: STIP1 homology and U-box containing protein 1; STK: serine/threonine kinase; SYNPO: synaptopodin; TBP: TATA-box binding protein; TARDBP/TDP-43: TAR DNA binding protein; TFEB: transcription factor EB; TPR: tetratricopeptide repeats; TSC1: TSC complex subunit 1; UBA: ubiquitin associated; UPS: ubiquitin-proteasome system; WW: tryptophan-tryptophan; WWTR1: WW domain containing transcription regulator 1; YAP1: Yes1 associated transcriptional regulator

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by motoneuron loss. Some familial cases (fALS) are linked to mutations of superoxide dismutase type-1 (SOD1), an antioxidant enzyme whose activity is preserved in most mutant forms. Owing to the similarities in sporadic and fALS forms, mutant SOD1 animal and cellular models are a useful tool to study the disease. In transgenic mice expressing either wild-type (wt) human SOD1 or mutant G93A-SOD1, we found that wtSOD1 was present in cytoplasm and in nuclei of motoneurons, whereas mutant SOD1 was mainly cytoplasmic. Similar results were obtained in immortalized motoneurons (NSC34 cells) expressing either wtSOD1 or G93A-SOD1. Analyzing the proteasome activity, responsible for misfolded protein clearance, in the two subcellular compartments, we found proteasome impairment only in the cytoplasm. The effect of G93A-SOD1 exclusion from nuclei was then analyzed after oxidative stress. Cells expressing G93A-SOD1 showed a higher DNA damage compared with those expressing wtSOD1, possibly because of a loss of nuclear protection. The toxicity of mutant SOD1 might, therefore, arise from an initial misfolding (gain of function) reducing nuclear protection from the active enzyme (loss of function in the nuclei), a process that may be involved in ALS pathogenesis.

Context: An increasing body of evidence suggests that testosterone may exert beneficial effects on the development of atherosclerosis. It was suggested that testosterone may act after conversion into estradiol and activation of the estrogen receptors; however, a direct role of androgens on the vascular wall has been proposed. Objective: We investigated the effects of dihydrotestosterone on the proinflammatory response observed in human endothelial cells. Design: Human endothelial cells isolated from umbilical cords were incubated with lipopolysaccharide or TNFα in the presence or absence of dihydrotestosterone (DHT). mRNA and cellular proteins were processed for gene expression studies, and transient transfection experiments were performed to investigate molecular mechanisms involved in the effects observed. Setting: These studies took place at the Department of Pharmacological Sciences, University of Milan, Milan, Italy. Results: Lipopolysaccharide and TNFα induced VCAM-1 and ICAM-1 mRNA and protein expression, as detected by real-time quantitative PCR, fluorescence-activated cell sorting, and confocal microscopy, but this effect was inhibited when cells were incubated with DHT. In addition, DHT inhibited mRNA expression of IL-6, MCP-1, CD40, TLR4, PAI-1, and Cox-2 and the release of cytokines and chemokines such as GRO, granulocyte-macrophage colony-stimulating factor, and TNF. The DHT effect was counteracted by bicalutamide, an antagonist of the androgen receptor. Furthermore, when cells were cotransfected with a Cox-2 promoter or a 3X-NF-κB luciferase reporter vector and a plasmid expressing the human androgen receptor, DHT treatment inhibited the increase of the luciferase activity observed with TNFα. Conclusion: DHT could positively regulate endothelial function through the control of the inflammatory response mediated by nuclear factor-κB in endothelial cells.

Stress granules (SGs) are mRNA-protein aggregates induced during stress, which accumulate in many neurodegenerative diseases. Previously, the autophagy-lysosome pathway and valosin-containing protein (VCP), key players of the protein quality control (PQC), were shown to regulate SG degradation. This is consistent with the idea that PQC may survey and/or assist SG dynamics. However, despite these observations, it is currently unknown whether the PQC actively participates in SG assembly. Here, we describe that inhibition of autophagy, lysosomes and VCP causes defective SG formation after induction. Silencing the VCP co-factors UFD1L and PLAA, which degrade defective ribosomal products (DRIPs) and 60S ribosomes, also impaired SG assembly. Intriguingly, DRIPs and 60S, which are released from disassembling polysomes and are normally excluded from SGs, were significantly retained within SGs in cells with impaired autophagy, lysosome or VCP function. Our results suggest that deregulated autophagy, lysosomal or VCP activities, which occur in several neurodegenerative (VCP-associated) diseases, may alter SG morphology and composition.

The ubiquitin-proteasome system (UPS) is the major intracellular proteolytic mechanism controlling the degradation of misfolded/abnormal proteins. A common hallmark in amyotrophic lateral sclerosis (ALS) and in other neurodegenerative disorders is the accumulation of misfolded/abnormal proteins into the damaged neurons, leading to the formation of cellular inclusions that are mostly ubiquitin-positive. Although proteolysis is a complex mechanism requiring the participation of different pathways, the abundant accumulation of ubiquitinated proteins strongly suggests an important contribution of UPS to these neuropathological features. The use of cellular and animal models of ALS, particularly those expressing mutant SOD1, the gene mutation most represented in familiar ALS, has provided significant evidence for a role of UPS in protein inclusions formation and motor neuron death. This review will specifically discuss this piece of evidence and provide suggestions of potential strategies for therapeutic intervention. We will also discuss the finding that, unlike the constitutive proteasome subunits, the inducible subunits are overexpressed early during disease progression in SOD1 mice models of ALS. These subunits form the immunoproteasome and generate peptides for the major histocompatibility complex class I molecules, suggesting a role of this system in the immune responses associated with the pathological features of ALS. Since recent discoveries indicate that innate and adaptive immunity may influence the disease process, in this review we will also provide evidence of a possible connection between immune-inflammatory reactions and UPS function, in the attempt to better understand the etiopathology of ALS and to identify appropriate targets for novel treatment strategies of this devastating disease.

Eukaryotic cells use autophagy and the ubiquitin–proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested to be involved in this switch. However, at present it is still unknown whether and to what extent BAG3 can indeed reroute proteasomal clients to the autophagosomal pathway. Here, we show that BAG3 induces the sequestration of ubiquitinated clients into cytoplasmic puncta colabeled with canonical autophagy linkers and markers. Following proteasome inhibition, BAG3 upregulation significantly contributes to the compensatory activation of autophagy and to the degradation of the (poly)ubiquitinated proteins. BAG3 binding to the ubiquitinated clients occurs through the BAG domain, in competition with BAG1, another BAG family member, that normally directs ubiquitinated clients to the proteasome. Therefore, we propose that following proteasome impairment, increasing the BAG3/BAG1 ratio ensures the “BAG-instructed proteasomal to autophagosomal switch and sorting” (BIPASS).

Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by motor neuron degeneration and associated with aggregation of nuclear RNA-binding proteins (RBPs), including FUS. How FUS aggregation and neurodegeneration are prevented in healthy motor neurons remain critically unanswered questions. Here, we use a combination of ALS patient autopsy tissue and induced pluripotent stem cell-derived neurons to study the effects of FUS mutations on RBP homeostasis. We show that FUS' tendency to aggregate is normally buffered by interacting RBPs, but this buffering is lost when FUS mislocalizes to the cytoplasm due to ALS mutations. The presence of aggregation-prone FUS in the cytoplasm causes imbalances in RBP homeostasis that exacerbate neurodegeneration. However, enhancing autophagy using small molecules reduces cytoplasmic FUS, restores RBP homeostasis and rescues motor function in vivo. We conclude that disruption of RBP homeostasis plays a critical role in FUS-ALS and can be treated by stimulating autophagy.

Amyotrophic lateral sclerosis (ALS) is a progressive adult-onset neurodegenerative disease, that affects cortical, bulbar and spinal motor neurons, and it is considered a proteinopathy, in which pathological proteins (SOD1, TDP-43, and FUS) may accumulate and interfere with neuronal functions eventually leading to cell death. These proteins can be released from cells and transported in the body fluids by extracellular vesicles (EVs). EVs are spherical vesicles, which are classified mainly in microvesicles (MVs) and exosomes (EXOs) based on their biogenesis, size and surface markers. In this study we characterized MVs and EXOs isolated from plasma of sporadic ALS patients and healthy controls and determined their number, size and SOD1, TDP-43, and FUS protein composition. No variation was found in the number of EVs between ALS patients and controls. However, the mean size both for MVs and for EXOs resulted increased in ALS patients compared to controls. MVs derived from ALS patients were enriched in SOD1, TDP-43, phospho-TDP-43, and FUS proteins compared to CTRLs. SOD1 was generally more concentrated in EXOs than in MVs, while TDP-43 and FUS protein levels were slightly higher in MVs than in EXOs. We demonstrated that MVs and EXOs size were increased in ALS patients compared to controls and that MVs of ALS patients were enriched with toxic proteins compared to CTRLs. EXOs did not show any protein changes. These data may suggest that MVs can transport toxic proteins and might play a role in prion-like propagation of ALS disease.

Perturbations in stress granule (SG) dynamics may be at the core of amyotrophic lateral sclerosis (ALS). Since SGs are membraneless compartments, modeling their dynamics in human motor neurons has been challenging, thus hindering the identification of effective therapeutics. Here, we report the generation of isogenic induced pluripotent stem cells carrying wild-type and P525L FUS-eGFP. We demonstrate that FUS-eGFP is recruited into SGs and that P525L profoundly alters their dynamics. With a screening campaign, we demonstrate that PI3K/AKT/mTOR pathway inhibition increases autophagy and ameliorates SG phenotypes linked to P525L FUS by reducing FUS-eGFP recruitment into SGs. Using a Drosophila model of FUS-ALS, we corroborate that induction of autophagy significantly increases survival. Finally, by screening clinically approved drugs for their ability to ameliorate FUS SG phenotypes, we identify a number of brain-penetrant anti-depressants and anti-psychotics that also induce autophagy. These drugs could be repurposed as potential ALS treatments.

Abstract In autophagy long‐lived proteins, protein aggregates or damaged organelles are engulfed by vesicles called autophagosomes prior to lysosomal degradation. Autophagy dysfunction is a hallmark of several neurodegenerative diseases in which misfolded proteins or dysfunctional mitochondria accumulate. Excessive autophagy can also exacerbate brain injury under certain conditions. In this review, we provide specific examples to illustrate the critical role played by autophagy in pathological conditions affecting the brain and discuss potential therapeutic implications. We show how a singular type of autophagy‐dependent cell death termed autosis has attracted attention as a promising target for improving outcomes in perinatal asphyxia and hypoxic‐ischaemic injury to the immature brain. We provide evidence that autophagy inhibition may be protective against radiotherapy‐induced damage to the young brain. We describe a specialized form of macroautophagy of therapeutic relevance for motoneuron and neuromuscular diseases, known as chaperone‐assisted selective autophagy, in which heat shock protein B8 is used to deliver aberrant proteins to autophagosomes. We summarize studies pinpointing mitophagy mediated by the serine/threonine kinase PINK1 and the ubiquitin–protein ligase Parkin as a mechanism potentially relevant to Parkinson's disease, despite debate over the physiological conditions in which it is activated in organisms. Finally, with the example of the autophagy‐inducing agent rilmenidine and its discrepant effects in cell culture and mouse models of motor neuron disorders, we illustrate the importance of considering aspects such a disease stage and aggressiveness, type of insult and load of damaged or toxic cellular components, when choosing the appropriate drug, timepoint and duration of treatment. image

Distal hereditary motor neuropathies (dHMNs) are clinically and genetically heterogeneous neurological conditions characterized by degeneration of the lower motor neurons. So far, 18 dHMN genes have been identified, however, about 80% of dHMN cases remain without a molecular diagnosis. By a combination of autozygosity mapping, identity-by-descent segment detection and whole-exome sequencing approaches, we identified two novel homozygous mutations in the SIGMAR1 gene (p.E138Q and p.E150K) in two distinct Italian families affected by an autosomal recessive form of HMN. Functional analyses in several neuronal cell lines strongly support the pathogenicity of the mutations and provide insights into the underlying pathomechanisms involving the regulation of ER-mitochondria tethering, Ca2+ homeostasis and autophagy. Indeed, in vitro, both mutations reduce cell viability, the formation of abnormal protein aggregates preventing the correct targeting of sigma-1R protein to the mitochondria-associated ER membrane (MAM) and thus impinging on the global Ca2+ signalling. Our data definitively demonstrate the involvement of SIGMAR1 in motor neuron maintenance and survival by correlating, for the first time in the Caucasian population, mutations in this gene to distal motor dysfunction and highlight the chaperone activity of sigma-1R at the MAM as a critical aspect in dHMN pathology.

Abstract Prostate cancer (PC) is one of the leading causes of death in males. Available treatments often lead to the appearance of chemoresistant foci and metastases, with mechanisms still partially unknown. Within tumour mass, autophagy may promote cell survival by enhancing cancer cells tolerability to different cell stresses, like hypoxia, starvation or those triggered by chemotherapic agents. Because of its connection with the apoptotic pathways, autophagy has been differentially implicated, either as prodeath or prosurvival factor, in the appearance of more aggressive tumours. Here, in three PC cells (LNCaP, PC3, and DU145), we tested how different autophagy inducers modulate docetaxel-induced apoptosis. We selected the mTOR-independent disaccharide trehalose and the mTOR-dependent macrolide lactone rapamycin autophagy inducers. In castration-resistant PC (CRPC) PC3 cells, trehalose specifically prevented intrinsic apoptosis in docetaxel-treated cells. Trehalose reduced the release of cytochrome c triggered by docetaxel and the formation of aberrant mitochondria, possibly by enhancing the turnover of damaged mitochondria via autophagy (mitophagy). In fact, trehalose increased LC3 and p62 expression, LC3-II and p62 (p62 bodies) accumulation and the induction of LC3 puncta. In docetaxel-treated cells, trehalose, but not rapamycin, determined a perinuclear mitochondrial aggregation (mito-aggresomes), and mitochondria specifically colocalized with LC3 and p62-positive autophagosomes. In PC3 cells, rapamycin retained its ability to activate autophagy without evidences of mitophagy even in presence of docetaxel. Interestingly, these results were replicated in LNCaP cells, whereas trehalose and rapamycin did not modify the response to docetaxel in the ATG5- deficient (autophagy resistant) DU145 cells. Therefore, autophagy is involved to alter the response to chemotherapy in combination therapies and the response may be influenced by the different autophagic pathways utilized and by the type of cancer cells.

Abstract Neurodegenerative diseases (NDs) are often associated with the presence of misfolded protein inclusions. The chaperone HSPB8 is upregulated in mice, the human brain and muscle structures affected during NDs progression. HSPB8 exerts a potent pro-degradative activity on several misfolded proteins responsible for familial NDs forms. Here, we demonstrated that HSPB8 also counteracts accumulation of aberrantly localized misfolded forms of TDP-43 and its 25 KDa fragment involved in most sporadic cases of Amyotrophic Lateral Sclerosis (sALS) and of Fronto Lateral Temporal Dementia (FLTD). HSPB8 acts with BAG3 and the HSP70/HSC70-CHIP complex enhancing the autophagic removal of misfolded proteins. We performed a high-through put screening (HTS) to find small molecules capable of inducing HSPB8 in neurons for therapeutic purposes. We identified two compounds, colchicine and doxorubicin, that robustly up-regulated HSPB8 expression. Both colchicine and doxorubicin increased the expression of the master regulator of autophagy TFEB, the autophagy linker p62/SQSTM1 and the autophagosome component LC3. In line, both drugs counteracted the accumulation of TDP-43 and TDP-25 misfolded species responsible for motoneuronal death in sALS. Thus, analogs of colchicine and doxorubicin able to induce HSPB8 and with better safety and tolerability may result beneficial in NDs models.

Aggregation of TAR-DNA-binding protein 43 (TDP-43) and of its fragments TDP-25 and TDP-35 occurs in amyotrophic lateral sclerosis (ALS). TDP-25 and TDP-35 act as seeds for TDP-43 aggregation, altering its function and exerting toxicity. Thus, inhibition of TDP-25 and TDP-35 aggregation and promotion of their degradation may protect against cellular damage. Upregulation of HSPB8 is one possible approach for this purpose, since this chaperone promotes the clearance of an ALS associated fragments of TDP-43 and is upregulated in the surviving motor neurones of transgenic ALS mice and human patients. We report that overexpression of HSPB8 in immortalized motor neurones decreased the accumulation of TDP-25 and TDP-35 and that protection against mislocalized/truncated TDP-43 was observed for HSPB8 in Drosophila melanogaster Overexpression of HSP67Bc, the functional ortholog of human HSPB8, suppressed the eye degeneration caused by the cytoplasmic accumulation of a TDP-43 variant with a mutation in the nuclear localization signal (TDP-43-NLS). TDP-43-NLS accumulation in retinal cells was counteracted by HSP67Bc overexpression. According with this finding, downregulation of HSP67Bc increased eye degeneration, an effect that is consistent with the accumulation of high molecular weight TDP-43 species and ubiquitinated proteins. Moreover, we report a novel Drosophila model expressing TDP-35, and show that while TDP-43 and TDP-25 expression in the fly eyes causes a mild degeneration, TDP-35 expression leads to severe neurodegeneration as revealed by pupae lethality; the latter effect could be rescued by HSP67Bc overexpression. Collectively, our data demonstrate that HSPB8 upregulation mitigates TDP-43 fragment mediated toxicity, in mammalian neuronal cells and flies.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two neurodegenerative diseases in which similar pathogenic mechanisms are involved. Both diseases associate to the high propensity of specific misfolded proteins, like TDP-43 or FUS, to mislocalize and aggregate. This is partly due to their intrinsic biophysical properties and partly as a consequence of failure of the neuronal protein quality control (PQC) system. Several familial ALS/FTD cases are linked to an expansion of a repeated G4C2 hexanucleotide sequence present in the C9ORF72 gene. The G4C2, which localizes in an untranslated region of the C9ORF72 transcript, drives an unconventional repeat-associated ATG-independent translation. This leads to the synthesis of five different dipeptide repeat proteins (DPRs), which are not “classical” misfolded proteins, but generate aberrant aggregation-prone unfolded conformations poorly removed by the PQC system. The DPRs accumulate into p62/SQSTM1 and ubiquitin positive inclusions. Here, we analyzed the biochemical behavior of the five DPRs in immortalized motoneurons. Our data suggest that while the DPRs are mainly processed via autophagy, this system is unable to fully clear their aggregated forms, and thus they tend to accumulate in basal conditions. Overexpression of the small heat shock protein B8 (HSPB8), which facilitates the autophagy-mediated disposal of a large variety of classical misfolded aggregation-prone proteins, significantly decreased the accumulation of most DPR insoluble species. Thus, the induction of HSPB8 might represent a valid approach to decrease DPR-mediated toxicity and maintain motoneuron viability.

Small Heat Shock Proteins (sHSPs) evolved early in the history of life; they are present in archaea, bacteria, and eukaryota. sHSPs belong to the superfamily of molecular chaperones: they are components of the cellular protein quality control machinery and are thought to act as the first line of defense against conditions that endanger the cellular proteome. In plants, sHSPs protect cells against abiotic stresses, providing innovative targets for sustainable agricultural production. In humans, sHSPs (also known as HSPBs) are associated with the development of several neurological diseases. Thus, manipulation of sHSP expression may represent an attractive therapeutic strategy for disease treatment. Experimental evidence demonstrates that enhancing the chaperone function of sHSPs protects against age-related protein conformation diseases, which are characterized by protein aggregation. Moreover, sHSPs can promote longevity and healthy aging in vivo. In addition, sHSPs have been implicated in the prognosis of several types of cancer. Here, sHSP upregulation, by enhancing cellular health, could promote cancer development; on the other hand, their downregulation, by sensitizing cells to external stressors and chemotherapeutics, may have beneficial outcomes. The complexity and diversity of sHSP function and properties and the need to identify their specific clients, as well as their implication in human disease, have been discussed by many of the world's experts in the sHSP field during a dedicated workshop in Québec City, Canada, on 26-29 August 2018.

The family of the human small Heat Shock Proteins (HSPBs) consists of ten members of chaperones (HSPB1-HSPB10), characterized by a low molecular weight and capable of dimerization and oligomerization forming large homo- or hetero-complexes. All HSPBs possess a highly conserved centrally located α-crystallin domain and poorly conserved N- and C-terminal domains. The main feature of HSPBs is to exert cytoprotective functions by preserving proteostasis, assuring the structural maintenance of the cytoskeleton and acting in response to cellular stresses and apoptosis. HSPBs take part in cell homeostasis by acting as holdases, which is the ability to interact with a substrate preventing its aggregation. In addition, HSPBs cooperate in substrates refolding driven by other chaperones or, alternatively, promote substrate routing to degradation. Notably, while some HSPBs are ubiquitously expressed, others show peculiar tissue-specific expression. Cardiac muscle, skeletal muscle and neurons show high expression levels for a wide variety of HSPBs. Indeed, most of the mutations identified in HSPBs are associated to cardiomyopathies, myopathies, and motor neuropathies. Instead, mutations in HSPB4 and HSPB5, which are also expressed in lens, have been associated with cataract. Mutations of HSPBs family members encompass base substitutions, insertions, and deletions, resulting in single amino acid substitutions or in the generation of truncated or elongated proteins. This review will provide an updated overview of disease-related mutations in HSPBs focusing on the structural and biochemical effects of mutations and their functional consequences.

BACKGROUND Atherosclerotic coronary artery disease, complicated by acute thrombosis, is the usual cause of sudden death in adults. This study addresses the pathology of coronary arteries in sudden death in the young (< or = 35 years old). METHODS AND RESULTS Among 200 consecutive cases of sudden death in youth in the Veneto region of Italy, 37 (33 men and 4 women, age 18 to 35 years; mean, 29.4 years) showed obstructive atherosclerotic coronary artery disease in the absence of other cardiac pathological conditions and causes of death. No patient had previous angina pectoris or myocardial infarction. Cardiac arrest occurred at rest in 30 subjects and was related to effort in 7. A histological study was carried out on the obstructive coronary plaques. Degree of lumen stenosis and extension of lipid core and intimal fibrocellular hyperplasia facing the lumen were calculated morphometrically. Immunohistochemistry and electron microscopy were used to further characterize the plaque cell population. Single-vessel disease was found in 33 patients and triple-vessel disease in 4, with an overall total of 45 obstructive plaques, 34 of which were located in the proximal left anterior descending coronary artery. At histological study, only 10 plaques from 10 patients showed acute thrombosis (occlusive in 5 and subocclusive in 5); the remaining 35 were uncomplicated. Thirty-one plaques were fibrous in nature, while the other 14 were atheromatous. Compared with the atheromatous lesions, the fibrous plaques were rarely complicated by thrombosis (3% versus 64%; P < .001) and distinctly exhibited a fairly well-preserved tunica media (81% versus 21%; P < .001) as well as a stratum of neointimal fibrocellular hyperplasia (68% versus 7%; P < .001), which on immunohistochemistry and electron microscopy appeared to be proliferating smooth muscle cells. CONCLUSIONS In our study population, sudden death was precipitated by acute coronary thrombosis in only 27% of patients with obstructive coronary atherosclerotic plaque. Most of the young victims of sudden death with obstructive coronary atherosclerosis showed single-vessel disease that affected the left anterior descending coronary artery and was due to fibrous plaques with neointimal smooth muscle cell hyperplasia and a preserved tunica media in the absence of acute thrombosis.

Abstract Prostate cancer growth depends, in its earlier stages, on androgens and is usually pharmacologically modulated with androgen blockade. However, androgen-ablation therapy may generate androgen-independent prostate cancer, often characterized by an increased invasiveness. We have found that the 5α-reduced testosterone derivative, dihydrotestosterone (the most potent natural androgen) inhibits cell migration with an androgen receptor–independent mechanism. We have shown that the dihydrotestosterone metabolite 5α-androstane-3β,17β-diol (3β-Adiol), a steroid which does not bind androgen receptors, but efficiently binds the estrogen receptor β (ERβ), exerts a potent inhibition of prostate cancer cell migration through the activation of the ERβ signaling. Very surprisingly, estradiol is not active, suggesting the existence of different pathways for ERβ activation in prostate cancer cells. Moreover, 3β-Adiol, through ERβ, induces the expression of E-cadherin, a protein known to be capable of blocking metastasis formation in breast and prostate cancer cells. The inhibitory effects of 3β-Adiol on prostate cancer cell migration is counteracted by short interfering RNA against E-cadherin. Altogether, the data showed that (a) circulating testosterone may act with estrogenic effects downstream in the catabolic process present in the prostate, and (b) that the estrogenic effect of testosterone derivatives (ERβ-dependent) results in the inhibition of cell migration, although it is apparently different from that linked to estradiol on the same receptor and may be protective against prostate cancer invasion and metastasis. These results also shed some light on clinical observations suggesting that alterations in genes coding for 3β-hydroxysteroid dehydrogenases (the enzymes responsible for 3β-Adiol formation) are strongly correlated with hereditary prostate cancer.

The present paper will summarize two important aspects of the interactions between steroids and the brain, which have recently been studied in the authors' laboratory. In particular the paper will consider data on: (1) the significance of the two isoforms of the 5α-R during brain ontogenesis and development, and (2) the cross-talk between glial and neuronal elements, particularly in relation to the metabolism of sex hormones. (1) The data obtained have shown that the 5α-R type 1 enzyme is constitutively expressed in the rat CNS at all stages of brain development. Moreover, the expression of the 5α-R type 1 is similar in males and in females, and does not appear to be controlled by androgens. The gene expression of the 5α-R type 2 is totally different. This isoform appears to be expressed in the rat brain almost exclusively in the late fetal/early post-natal life and is controlled by testosterone. (2) The present data show that two cell lines derived respectively from a rat glioma (C6 cell line) and from a human astrocytoma (1321N1 cell line) are able to convert testosterone and progesterone into their corresponding 5α-reduced metabolites dihydrotestosterone and dihydroprogesterone. The possibility that secretory products of normal and tumoral brain cells might be able to influence steroid metabolism occurring in the two glial cell lines previously mentioned has been considered.

Journal Article Right ventricular cardiomyopathy: Is there evidence of an inflammatory aetiology? Get access G. Thiene, G. Thiene Departments of Pathology and Cardiology, University of Padua Medical SchoolPadua, Italy Address for reprints. Gactano Thiene M.D. Istituto di Anatomia Patologica, Via A. Gabelli 61, 35121 Padova, Italy Search for other works by this author on: Oxford Academic PubMed Google Scholar D. Corrado, D. Corrado Departments of Pathology and Cardiology, University of Padua Medical SchoolPadua, Italy Search for other works by this author on: Oxford Academic PubMed Google Scholar A. Nava, A. Nava Departments of Pathology and Cardiology, University of Padua Medical SchoolPadua, Italy Search for other works by this author on: Oxford Academic PubMed Google Scholar L. Rossi, L. Rossi Departments of Pathology and Cardiology, University of Padua Medical SchoolPadua, Italy Search for other works by this author on: Oxford Academic PubMed Google Scholar A. Poletti, A. Poletti Departments of Pathology and Cardiology, University of Padua Medical SchoolPadua, Italy Search for other works by this author on: Oxford Academic PubMed Google Scholar G. M. Boffa, G. M. Boffa Departments of Pathology and Cardiology, University of Padua Medical SchoolPadua, Italy Search for other works by this author on: Oxford Academic PubMed Google Scholar L. Daliento, L. Daliento Departments of Pathology and Cardiology, University of Padua Medical SchoolPadua, Italy Search for other works by this author on: Oxford Academic PubMed Google Scholar N. Pennelli N. Pennelli Departments of Pathology and Cardiology, University of Padua Medical SchoolPadua, Italy Search for other works by this author on: Oxford Academic PubMed Google Scholar European Heart Journal, Volume 12, Issue suppl_D, August 1991, Pages 22–25, https://doi.org/10.1093/eurheartj/12.suppl_D.22 Published: 01 August 1991

The androgen receptor (AR) is a ligand-activated transcription factor which is responsible for the androgen responsiveness of target cells. Several types of mutations have been found in the AR and linked to endocrine dysfunctions. Surprisingly, the polymorphism involving the CAG triplet repeat expansion of the AR gene, coding for a polyglutamine (PolyGln) tract in the N-terminal transactivation domain of the AR protein, has been involved either in endocrine or neurological disorders. For example, among endocrine-related-diseases, the PolyGln size has been proposed to be associated to prostate cancer susceptibility, hirsutism, male infertility, cryptorchidism (in conjunction with polyglycine stretches polymorphism), etc.; the molecular mechanisms of these alterations are thought to involve a modulation of AR transcriptional competence, which inversely correlates with the PolyGln length. Among neurological alterations, a decreased AR function seems to be also involved in depression. Moreover, when the polymorphic PolyGln becomes longer than 35-40 contiguous glutamines (ARPolyGln), the ARPolyGln acquires neurotoxicity, because of an unknown gain-of-function. This mutation has been linked to a rare inherited X-linked motor neuronal disorder, the Spinal and Bulbar Muscular Atrophy, or Kennedy's disease. The disorder is characterized by death of motor neurons expressing high levels of AR. The degenerating motor neurons are mainly located in the anterior horns of the spinal cord and in the bulbar region; some neurons of the dorsal root ganglia may also be involved. Interestingly, the same type of PolyGln elongation has been found in other totally unrelated proteins responsible for different neurodegenerative diseases. A common feature of all these disorders is the formation of intracellular aggregates containing the mutated proteins; at present, but their role in the disease is largely debated. This review will discuss how the PolyGln neurotoxicity of SBMA AR may be either mediated or decreased by aggregates, and will present data on the dual role played by testosterone on motor neuronal functions and dysfunctions.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons. As with other age-dependent neurodegenerative disorders, ALS is linked to the presence of misfolded proteins that may perturb several intracellular mechanisms and trigger neurotoxicity. Misfolded proteins aggregate intracellularly generating insoluble inclusions that are a key neuropathological hallmark of ALS. Proteins involved in the intracellular degradative systems, signaling pathways and the human TAR DNA-binding protein TDP-43 are major components of these inclusions. While their role and cytotoxicity are still largely debated, aggregates represent a powerful marker to follow protein misfolding in the neurodegenerative processes. Using in vitro and in vivo models of mutant SOD1 associated familial ALS (fALS), we and other groups demonstrated that protein misfolding perturbs one of the major intracellular degradative pathways, the ubiquitin proteasome system, giving rise to a vicious cycle that leads to the further deposit of insoluble proteins and finally to the formation of inclusions. The aberrant response to mutated SOD1 thus leads to the activation of the cascade of events ultimately responsible for cell death. Hence, our idea is that, by assisting protein folding, we might reduce protein aggregation, restore a fully functional proteasome activity and/or other cascades of events triggered by the mutant proteins responsible for motor neuron death in ALS. This could be obtained by stimulating mutant protein turnover, using an alternative degradative pathway that could clear mutant SOD1, namely autophagy.

The family of the mammalian small heat-shock proteins consists of 10 members (sHSPs/HSPBs: HSPB1–HSPB10) that all share a highly conserved C-terminal alpha-crystallin domain, important for the modulation of both their structural and functional properties. HSPB proteins are biochemically classified as molecular chaperones and participate in protein quality control, preventing the aggregation of unfolded or misfolded proteins and/or assisting in their degradation. Thus, several members of the HSPB family have been suggested to be protective in a number of neurodegenerative and neuromuscular diseases that are characterized by protein misfolding. However, the pro-refolding, anti-aggregation or pro-degradative properties of the various members of the HSPB family differ largely, thereby influencing their efficacy and protective functions. Such diversity depends on several factors, including biochemical and physical properties of the unfolded/misfolded client, the expression levels and the subcellular localization of both the chaperone and the client proteins. Furthermore, although some HSPB members are inefficient at inhibiting protein aggregation, they can still exert neuroprotective effects by other, as yet unidentified, manners; e.g. by maintaining the proper cellular redox state or/and by preventing the activation of the apoptotic cascade. Here, we will focus our attention on how the differences in the activities of the HSPB proteins can influence neurodegenerative and neuromuscular disorders characterized by accumulation of aggregate-prone proteins. Understanding their mechanism of action may allow us to target a specific member in a specific cell type/disease for therapeutic purposes.

Motor neuron diseases (MNDs) are neurodegenerative disorders that specifically affect the survival and function of upper and/or lower motor neurons. Since motor neurons are responsible for the control of voluntary muscular movement, MNDs are characterized by muscle spasticity, weakness and atrophy. Different susceptibility genes associated with an increased risk to develop MNDs have been reported and several mutated genes have been linked to hereditary forms of MNDs. However, most cases of MNDs occur in sporadic forms and very little is known on their causes. Interestingly, several molecular mechanisms seem to participate in the progression of both the inherited and sporadic forms of MNDs. These include cytoskeleton organization, mitochondrial functions, DNA repair and RNA synthesis/processing, vesicle trafficking, endolysosomal trafficking and fusion, as well as protein folding and protein degradation. In particular, accumulation of aggregate-prone proteins is a hallmark of MNDs, suggesting that the protein quality control system (molecular chaperones and the degradative systems: ubiquitin–proteasome-system and autophagy) are saturated or not sufficient to allow the clearance of these altered proteins. In this review we mainly focus on the MNDs associated with disturbances in protein folding and protein degradation and on the potential implication of a specific class of molecular chaperones, the small heat shock proteins (sHSPs/HSPBs), in motor neuron function and survival. How boosting of specific HSPBs may be a potential useful therapeutic approach in MNDs and how mutations in specific HSPBs can directly cause motor neuron degeneration is discussed.

Motoneuron diseases, like spinal bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS), are associated with proteins that because of gene mutation or peculiar structures, acquire aberrant (misfolded) conformations toxic to cells. To prevent misfolded protein toxicity, cells activate a protein quality control (PQC) system composed of chaperones and degradative pathways (proteasome and autophagy). Inefficient activation of the PQC system results in misfolded protein accumulation that ultimately leads to neuronal cell death, while efficient macroautophagy/autophagy-mediated degradation of aggregating proteins is beneficial. The latter relies on an active retrograde transport, mediated by dynein and specific chaperones, such as the HSPB8-BAG3-HSPA8 complex. Here, using cellular models expressing aggregate-prone proteins involved in SBMA and ALS, we demonstrate that inhibition of dynein-mediated retrograde transport, which impairs the targeting to autophagy of misfolded species, does not increase their aggregation. Rather, dynein inhibition correlates with a reduced accumulation and an increased clearance of mutant ARpolyQ, SOD1, truncated TARDBP/TDP-43 and expanded polyGP C9ORF72 products. The enhanced misfolded protein clearance is mediated by the proteasome, rather than by autophagy and correlates with the upregulation of the HSPA8 cochaperone BAG1. In line, overexpression of BAG1 increases the proteasome-mediated clearance of these misfolded proteins. Our data suggest that when the misfolded proteins cannot be efficiently transported toward the perinuclear region of the cells, where they are either degraded by autophagy or stored into the aggresome, the cells activate a compensatory mechanism that relies on the induction of BAG1 to target the HSPA8-bound cargo to the proteasome in a dynein-independent manner.

Amyotrophic lateral sclerosis (ALS) is a disease of variable severity in terms of speed of progression of the disease course. We found a similar variability in disease onset and progression of 2 familial ALS mouse strains, despite the fact that they carry the same transgene copy number and express the same amount of mutant SOD1G93A messenger RNA and protein in the central nervous system. Comparative analysis of 2 SOD1G93A mouse strains highlights differences associated with the disease severity that are unrelated to the degree of motor neuron loss but that appear to promote early dysfunction of these cells linked to protein aggregation. Features of fast progressing phenotype are (1) abundant protein aggregates containing mutant SOD1 and multiple chaperones; (2) low basal expression of the chaperone alpha-B-crystallin (CRYAB) and β5 subunits of proteasome; and (3) downregulation of proteasome subunit expression at disease onset. In contrast, high levels of functional chaperones such as cyclophillin-A and CRYAB, combined with delayed alteration of expression of proteasome subunits and the sequestration of TDP43 into aggregates, are features associated with a more slowly progressing pathology. These data support the hypothesis that impairment of protein homeostasis caused by low-soluble chaperone levels, together with malfunction of the proteasome degradation machinery, contributes to accelerate motor neuron dysfunction and progression of disease symptoms. Therefore, modulating the activity of these systems could represent a rational therapeutic strategy for slowing down disease progression in SOD1-related ALS.

Amyotrophic lateral sclerosis (ALS) and spinal and bulbar muscular atrophy (SBMA) are two motoneuron diseases (MNDs) characterized by aberrant protein behavior in affected cells. In familial ALS (fALS) and in SBMA specific gene mutations lead to the production of neurotoxic proteins or peptides prone to misfold, which then accumulate in form of aggregates. Notably, some of these proteins accumulate into aggregates also in sporadic ALS (sALS) even if not mutated. To prevent proteotoxic stresses detrimental to cells, misfolded and/or aggregated proteins must be rapidly removed by the protein quality control (PQC) system. The small heat shock protein B8 (HSPB8) is a chaperone induced by harmful events, like proteasome inhibition. HSPB8 is expressed both in motoneuron and muscle cells, which are both targets of misfolded protein toxicity in MNDs. In ALS mice models, in presence of the mutant proteins, HSPB8 is upregulated both in spinal cord and muscle. HSPB8 interacts with the HSP70 co-chaperone BAG3 and enhances the degradation of misfolded proteins linked to sALS, or causative of fALS and of SBMA. HSPB8 acts by facilitating autophagy, thereby preventing misfolded protein accumulation in affected cells. BAG3 and BAG1 compete for HSP70-bound clients and target them for disposal to the autophagy or proteasome, respectively. Enhancing the selective targeting of misfolded proteins by HSPB8-BAG3-HSP70 to autophagy may also decrease their delivery to the proteasome by the BAG1-HSP70 complex, thereby limiting possible proteasome overwhelming. Thus, approaches aimed at potentiating HSPB8-BAG3 may contribute to the maintenance of proteostasis and may delay MNDs progression.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that primarily affects motoneurons, while non-neuronal cells may contribute to disease onset and progression. Most ALS cases are characterized by the mislocalization and aggregation of the TAR DNA binding protein 43 (TDP-43) in affected cells. TDP-43 aggregates contain C-terminal TDP-43 fragments of 35 kDa (TDP-35) and 25 kDa (TDP-25) and have been mainly studied in motoneurons, while little is currently known about their rate of accumulation and clearance in myoblasts. Here, we performed a comparative study in immortalized motoneuronal like (NSC34; i-motoneurons) cells and stabilized myoblasts (C2C12; s-myoblasts) to evaluate if these two cell types differentially accumulate and clear TDP forms. The most aggregating specie in i-motoneurons is the TDP-25 fragment, mainly constituted by the "prion-like" domain of TDP-43. To a lower extent, TDP-25 also aggregates in s-myoblasts. In both cell types, all TDP species are cleared by proteasome, but TDP-25 impairs autophagy. Interestingly, the routing of TDP-25 fragment to proteasome, by overexpressing BAG1, or to autophagy, by overexpressing HSPB8 or BAG3 decreased its accumulation in both cell types. These results demonstrate that promoting the chaperone-assisted clearance of ALS-linked proteins is beneficial not only in motoneurons but also in myoblasts.

Introduction Disruptions of proteasome and autophagy systems are central events in amyotrophic lateral sclerosis (ALS) and support the urgent need to find therapeutic compounds targeting these processes. The heat shock protein B8 (HSPB8) recognises and promotes the autophagy-mediated removal of misfolded mutant SOD1 and TDP-43 fragments from ALS motor neurons (MNs), as well as aggregating species of dipeptides produced in C9ORF72-related diseases. In ALS-SOD1 mice and in human ALS autopsy specimens, HSPB8 is highly expressed in spinal cord MNs that survive at the end stage of disease. Moreover, the HSPB8–BAG3–HSP70 complex maintains granulostasis, which avoids conversion of dynamic stress granules (SGs) into aggregation-prone assemblies. We will perform a randomised clinical trial (RCT) with colchicine, which enhances the expression of HSPB8 and of several autophagy players, blocking TDP-43 accumulation and exerting crucial activities for MNs function. Methods and analysis Colchicine in amyotrophic lateral sclerosis (Co-ALS) is a double-blind, placebo-controlled, multicentre, phase II RCT. ALS patients will be enrolled in three groups (placebo, colchicine 0.01 mg/day and colchicine 0.005 mg/day) of 18 subjects treated with riluzole; treatment will last 30 weeks, and follow-up will last 24 weeks. The primary aim is to assess whether colchicine decreases disease progression as measured by ALS Functional Rating Scale - Revised (ALSFRS-R) at baseline and at treatment end. Secondary aims include assessment of (1) safety and tolerability of Colchicine in patiets with ALS; (2) changes in cellular activity (autophagy, protein aggregation, and SG and exosome secretion) and in biomarkers of disease progression (neurofilaments); (3) survival and respiratory function and (4) quality of life. Preclinical studies with a full assessment of autophagy and neuroinflammation biomarkers in fibroblasts, peripheral blood mononuclear cells and lymphoblasts will be conducted in parallel with clinic assessment to optimise time and resources. Ethics and dissemination The study protocol was approved by the Ethics Committee of Area Vasta Emilia Nord and by Agenzia Italiana del Farmaco (EUDRACT N.2017-004459-21) based on the Declaration of Helsinki. This research protocol was written without patient involvement. Patients’ association will be involved in disseminating the study design and results. Results will be presented during scientific symposia or published in scientific journals. Trial registration number EUDRACT 2017-004459-21 ; NCT03693781 ; Pre-results.

Valosin containing protein (VCP) has emerged as a central protein in the regulation of the protein quality control (PQC) system. VCP mutations are causative of multisystem proteinopathies, which include neurodegenerative diseases (NDs), and share various signs of altered proteostasis, mainly associated with autophagy malfunctioning. Autophagy is a complex multistep degradative system essential for the maintenance of cell viability, especially in post-mitotic cells as neurons and differentiated skeletal muscle cells. Interestingly, many studies concerning NDs have focused on autophagy impairment as a pathological mechanism or autophagy activity boosting to rescue the pathological phenotype. The role of VCP in autophagy has been widely debated, but recent findings have defined new mechanisms associated with VCP activity in the regulation of autophagy, showing that VCP is involved in different steps of this pathway. Here we will discuss the multiple activity of VCP in the autophagic pathway underlying its leading role either in physiological or pathological conditions. A better understanding of VCP complexes and mechanisms in regulating autophagy could define the altered mechanisms by which VCP directly or indirectly causes or modulates different human diseases and revealing possible new therapeutic approaches for NDs.

Several steroid molecules, including androgens, estrogens, progestagens, and corticostereroids, are able to modulate the brain development and functions. These compounds are not always active in their own natural molecular configuration but they often need to be transformed at the level of their target cells into ‘active metabolites’. The two major metabolic pathways that transform steroids in the brain are: the 5alpha-reductase-3alpha-hydroxy-steroid dehydrogenase and the aromatase pathways. Both are present in the brain and probably exert specific roles in the mechanism of action of hormonal steroids. In this article we briefly review some important findings achieved in our own and in other laboratories concerning the cellular and subcellular brain distribution, development, regulation, cloning, and molecular characterization of the involved enzymes. In particular, the recent identification of two isoforms of the 5alpha-reductase, the type 1 and type 2, possessing different structural, biochemical, and distribution characteristics has attracted a considerable attention. The few data available on their brain distribution have been carefully considered. Finally, we have tried to focus on the role of the steroid metabolites in the brain, both when they interact with genomic and with membrane receptors. In particular, some unpublished observations on the effects of two 5alpha-reductase inhibitors on progesterone-induced anesthesia, a phenomenon mediated through the GABAA receptor, are presented.

Abstract In the brain, the spinal cord motor neurones express the highest levels of the androgen receptor (AR). Experimental data have suggested that neurite outgrowth in these neurones may be regulated by testosterone or its derivative 5α‐dihydrotestosterone (DHT), formed by the 5α‐reductase type 2 enzyme. In this study we have produced and characterized a model of immortalized motor neuronal cells expressing the mouse AR (mAR) [neuroblastoma‐spinal cord (NSC) 34/mAR] and analysed the role of androgens in motor neurones. Androgens either activated or repressed several genes; one has been identified as the mouse neuritin, a protein responsible for neurite elongation. Real‐time PCR analysis has shown that the neuritin gene is expressed in the basal condition in immortalized motor neurones and is selectively up‐regulated by androgens in NSC34/mAR cells; the DHT effect is counteracted by the anti‐androgen Casodex. Moreover, DHT induced neurite outgrowth in NSC34/mAR, while testosterone was less effective and its action was counteracted by the 5α‐reductase type 2 enzyme inhibitor finasteride. Finally, the androgenic effect on neurite outgrowth was abolished by silencing neuritin with siRNA. Therefore, the trophic effects of androgens in motor neurones may be explained by the androgenic regulation of neuritin, a protein linked to neurone development, elongation and regeneration.

The enzyme 5 alpha-reductase (5 alpha-R) activates several delta 4-3keto steroids to more potent derivatives which may also acquire new biological actions. Testosterone gives rise to the most potent natural androgen dihydrotestosterone (DHT), and progesterone to dihydroprogesterone (DHP), a precursor of the endogenous anxiolytic/anesthetic steroid tetrahydroprogesterone (THP). Two isoforms of 5 alpha-R, with a limited degree of homology, different biochemical properties and distinct tissue distribution have been cloned: 5 alpha-R type 1 and type 2. In androgen-dependent structures DHT is almost exclusively formed by 5 alpha-R type 2; 5 alpha-R type 1 is widely distributed in the body, with the highest levels in the liver, and may be involved in steroid catabolism. In the brain, the roles of the two isozymes are still largely unknown. This brief review will summarize recent experimental data from our laboratory which try to assign possible functional roles to the process of 5 alpha-reduction, and to the two 5 alpha-R isoforms in the CNS.

Lone atrial fibrillation (LAF) is defined by the presence of atrial fibrillation unassociated with other evidence of organic heart disease. There are conflicting data concerning the prognostic importance, rate of embolic complications, and survival in subjects affected by this arrhythmia.One hundred forty-five patients younger than 50 years at the time of the first diagnosis were identified; 96 had paroxysmal and 49 had chronic LAF. They were followed up with clinical and echocardiographic controls, and we recorded every thromboembolic complication and death. During the follow-up (10 +/- 8 years) among patients with paroxysmal LAF, 1 (1%) had an ischemic stroke, 2 a transient ischemic attack, and 1 a myocardial infarction. In the group with chronic LAF, 1 patient had moderate heart failure, 2 myocardial infarction, and 1 transient ischemic attack. In this group, 8 embolic complications in 7 (16.3%) patients were observed. One patient with intestinal embolism died during surgery; 2 (6.1%) patients died suddenly.The prognosis of young patients with paroxysmal LAF appears to be excellent, whereas patients with chronic LAF are at increased risk of embolic complications and higher mortality rates. Our results suggest that LAF is not always a benign disorder, as suggested by previous studies. Subgroups with substantially increased risk for thromboembolic events caused by LAF should be better identified.

This review summarizes the mechanisms of neurotoxicity associated to androgen receptor containing an elongated polyglutamine tract responsible for motor neuronal cell death.

Polyglutamine diseases are a family of nine neurodegenerative disorders caused by expansion in different genes of a CAG triplet repeat stretch, which encodes an elongated polyglutamine tract. This polyglutamine tract is thought to confer a toxic gain of function to the bearing proteins, which leads to late onset and progressive loss of neurons in specific regions of the central nervous system. The mechanisms underlying specificity for neuronal vulnerability remain enigmatic. One explanation is that the polyglutamine tract is not the only determinant of neurodegeneration and that protein context and post-translational events may also be crucial for pathogenesis. Here, we review how post-translational modifications of the polyglutamine proteins contribute to modulate neurotoxicity.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease caused by an abnormal expansion of a tandem CAG repeat in exon 1 of the androgen receptor (AR) gene that results in an abnormally long polyglutamine tract (polyQ) in the AR protein. As a result, the mutant AR (ARpolyQ) misfolds, forming cytoplasmic and nuclear aggregates in the affected neurons. Neurotoxicity only appears to be associated with the formation of nuclear aggregates. Thus, improved ARpolyQ cytoplasmic clearance, which indirectly decreases ARpolyQ nuclear accumulation, has beneficial effects on affected motoneurons. In addition, increased ARpolyQ clearance contributes to maintenance of motoneuron proteostasis and viability, preventing the blockage of the proteasome and autophagy pathways that might play a role in the neuropathy in SBMA. The expression of heat shock protein B8 (HspB8), a member of the small heat shock protein family, is highly induced in surviving motoneurons of patients affected by motoneuron diseases, where it seems to participate in the stress response aimed at cell protection. We report here that HspB8 facilitates the autophagic removal of misfolded aggregating species of ARpolyQ. In addition, though HspB8 does not influence p62 and LC3 (two key autophagic molecules) expression, it does prevent p62 bodies formation, and restores the normal autophagic flux in these cells. Interestingly, trehalose, a well-known autophagy stimulator, induces HspB8 expression, suggesting that HspB8 might act as one of the molecular mediators of the proautophagic activity of trehalose. Collectively, these data support the hypothesis that treatments aimed at restoring a normal autophagic flux that result in the more efficient clearance of mutant ARpolyQ might produce beneficial effects in SBMA patients.

Amyotrophic lateral sclerosis (ALS) is a fatal motoneuronal disease which occurs in sporadic or familial forms, clinically indistinguishable. About 15% of familial ALS cases are linked to mutations of the superoxide dismutase 1 (SOD1) gene that may induce misfolding in the coded protein, exerting neurotoxicity to motoneurons. However, other cell types might be target of SOD1 toxicity, because muscle-restricted expression of mutant SOD1 correlates with muscle atrophy and motoneurons death. We analysed the molecular behaviour of mutant SOD1 in motoneuronal NSC34 and muscle C2C12 cells. We found that misfolded mutant SOD1 clearance is much more efficient in muscle C2C12 than in motoneuronal NSC34 cells. Mutant SOD1 forms aggregates and impairs the proteasome only in motoneuronal NSC34 cells. Interestingly, NSC34 cells expressing mutant SOD1 are more sensitive to a superoxide-induced oxidative stress. Moreover, in muscle C2C12 cells mutant SOD1 remains soluble even when proteasome is inhibited with MG132. The higher mutant SOD1 clearance in muscle cells correlates with a more efficient proteasome activity, combined with a robust autophagy activation. Therefore, muscle cells seem to better manage misfolded SOD1 species, not because of an intrinsic property of the mutant protein, but in function of the cell environment, indicating also that the SOD1 toxicity at muscle level may not directly depend on its aggregation rate.

Several types of motorneuron diseases are linked to neurotoxic mutant proteins. These acquire aberrant conformations (misfolding) that trigger deleterious downstream events responsible for neuronal dysfunction and degeneration. The pharmacological removal of misfolded proteins might thus be useful in these diseases. We utilized a peculiar motorneuronal disease model, spinobulbar muscular atrophy (SBMA), in which the neurotoxicity of the protein involved, the mutant androgen receptor (ARpolyQ), can be modulated by its ligand testosterone (T). 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) has already been proven to exert beneficial action in SBMA. Here we demonstrated that 17-AAG exerts its pro-degradative activity on mutant ARpolyQ without impacting on proteasome functions. 17-AAG removes ARpolyQ misfolded species and aggregates by activating the autophagic system. We next analyzed the 17-AAG effects on two proteins (SOD1 and TDP-43) involved in related motorneuronal diseases, such as amyotrophic lateral sclerosis (ALS). In these models 17-AAG was unable to counteract protein aggregation.

The exposure to environmental endocrine disrupting compounds (EDC), as polychlorinated biphenyls (PCBs), widely diffused in the environment may produce epigenetic changes that affect the endocrine system. We found that PCBs activate AR transcriptional activity and that this effect is potentiated by the demethylase Jarid1b, a histone demethylase that catalyzes the removal of trimethylation of lysine 4 on histone H3 (H3K4me3), induced by PCB. The aim of the present study was to investigate the effect of the treatment of cultured cells (HEK293) with a mixture of the most diffused environmental PCBs and, also with dihydrotestosterone (DHT), on the functional interaction between AR and Jarid1b. Although the effect induced by DHT on the AR transactivation was considerably higher, the PCB mixture produced an AR-mediated transactivation in a dose-dependent manner. Cotransfection with plasmids expressing Jarid1b and various AR isoforms containing polyglutamine tracts (polyQ tracts) of different lengths showed that Jarid1b potentiates the AR transcriptional activity induced by PCBs but only with the shortest AR isoform. The potentiating effect of Jarid1b on the AR is mediated by a direct interaction of the enzyme with the AR promoter. In fact, utilizing constructs containing AR promoters with a different length and a luciferase reporter gene, we showed that the effect of PCBs, but not of DHT, needs the presence of Jarid1b and of at least two DNA binding sites for Jarid1b.

Amyotrophic lateral sclerosis (ALS) is a motoneuron disease characterized by misfolded proteins aggregation in affected motoneurons. In mutant SOD1 (mutSOD1) ALS models, aggregation correlates to impaired functions of proteasome and/or autophagy, both essential for the intracellular chaperone-mediated protein quality control (PQC), and to a reduced mutSOD1 clearance from motoneurons. Skeletal muscle cells are also sensitive to mutSOD1 toxicity, but no mutSOD1 aggregates are formed in these cells, that might better manage mutSOD1 than motoneurons. Thus, we analyzed in spinal cord and in muscle of transgenic (tg) G93A-SOD1 mice at presymptomatic (PS, 8 weeks) and symptomatic (S, 16 weeks) stages, and in age-matched control mice, whether mutSOD1 differentially modulates relevant PQC players, such as HSPB8, BAG3, and BAG1. Possible sex differences were also considered. No changes of HSPB8, BAG3, and BAG1 at PS stage (8 weeks) were seen in all tissues examined in tg G93A-SOD1 and control mice. At S stage (16 weeks), HSPB8 dramatically increased in skeletal muscle of tg G93A-SOD1 mice, while a minor increase occurred in spinal cord of male, but not female tg G93A-SOD1 mice. BAG3 expression increased both in muscle and spinal cord of tg G93A-SOD1 mice at S stage, BAG1 expression increased only in muscle of the same mice. Since, HSPB8-BAG3 complex assists mutSOD1 autophagic removal, we analyzed two well-known autophagic markers, LC3 and p62. Both LC3 and p62 mRNAs were significantly up-regulated in skeletal muscle of tg G93A-SOD1 mice at S stage (16 weeks). This suggests that mutSOD1 expression induces a robust autophagic response specifically in muscle. Together these results demonstrate that, in muscle mutSOD1-induced autophagic response is much higher than in spinal cord. In addition, if mutSOD1 exerts toxicity in muscle, this may not be mediated by misfolded proteins accumulation. It remains unclear whether in muscle mutSOD1 toxicity is related to aberrant autophagy activation.

Spinal and bulbar muscular atrophy (SBMA) is characterized by loss of motoneurons and sensory neurons, accompanied by atrophy of muscle cells. SBMA is due to an androgen receptor containing a polyglutamine tract (ARpolyQ) that misfolds and aggregates, thereby perturbing the protein quality control (PQC) system. Using SBMA AR113Q mice we analyzed proteotoxic stress-induced alterations of HSPB8-mediated PQC machinery promoting clearance of misfolded proteins by autophagy. In muscle of symptomatic AR113Q male mice, we found expression upregulation of Pax-7, myogenin, E2-ubiquitin ligase UBE2Q1 and acetylcholine receptor (AchR), but not of MyoD, and of two E3-ligases (MuRF-1 and Cullin3). TGFβ1 and PGC-1α were also robustly upregulated. We also found a dramatic perturbation of the autophagic response, with upregulation of most autophagic markers (Beclin-1, ATG10, p62/SQSTM1, LC3) and of the HSPB8-mediated PQC response. Both HSPB8 and its co-chaperone BAG3 were robustly upregulated together with other specific HSPB8 interactors (HSPB2 and HSPB3). Notably, the BAG3:BAG1 ratio increased in muscle suggesting preferential misfolded proteins routing to autophagy rather than to proteasome. Thus, mutant ARpolyQ induces a potent autophagic response in muscle cells. Alteration in HSPB8-based PQC machinery may represent muscle-specific biomarkers useful to assess SBMA progression in mice and patients in response to pharmacological treatments.

Breast cancer (BC) is one of the major causes of cancer death in women and is closely related to hormonal dysregulation.Estrogen receptor (ER)-positive BCs are generally treated with anti hormone therapy using antiestrogens or aromatase inhibitors.However, BC cells may become resistant to endocrine therapy, a process facilitated by autophagy, which may either promote or suppress tumor expansion.The autophagy facilitator HSPB8 has been found overexpressed in some BC.Here we found that HSPB8 is highly expressed and differentially modulated by natural or synthetic selective ER modulators (SERMs), in the triple-positive hormone-sensitive BC (MCF-7) cells, but not in triple-negative MDA-MB-231 BC cells.Specific SERMs induced MCF-7 cells proliferation in a HSPB8 dependent manner whereas, did not modify MDA-MB-231 cell growth.ER expression was unaffected in HSPB8-depleted MCF-7 cells.HSPB8 over-expression did not alter the distribution of MCF-7 cells in the various phases of the cell cycle.Conversely and intriguingly, HSPB8 downregulation resulted in an increased number of cells resting in the G0/G1 phase, thus possibly reducing the ability of the cells to pass through the restriction point.In addition, HSPB8 downregulation reduced the migratory ability of MCF-7 cells.None of these modifications were observed, when another small HSP (HSPB1), also expressed in MCF-7 cells, was downregulated.In conclusion, our data suggest that HSPB8 is involved in the mechanisms that regulate cell cycle and cell migration in MCF-7 cells.

Transforming growth factor beta (TGFB) is a pleiotropic cytokine known to be dysregulated in many neurodegenerative disorders and particularly in amyotrophic lateral sclerosis (ALS). This motor neuronal disease is non-cell autonomous, as it affects not only motor neurons but also the surrounding glial cells, and the target skeletal muscle fibers. Here, we analyze the multiple roles of TGFB in these cell types, and how TGFB signaling is altered in ALS tissues. Data reported support a crucial involvement of TGFB in the etiology and progression of ALS, leading us to hypothesize that an imbalance of TGFB signaling, diminished at the pre-symptomatic stage and then increased with time, could be linked to ALS progression. A reduced stimulation of the TGFB pathway at the beginning of disease blocks its neuroprotective effects and promotes glutamate excitotoxicity. At later disease stages, the persistent activation of the TGFB pathway promotes an excessive microglial activation and strengthens muscular dysfunction. The therapeutic potential of TGFB is discussed, in order to foster new approaches to treat ALS.

The cellular response to cancer-induced stress is one of the major aspects regulating cancer development and progression. The Heat Shock Protein B8 (HSPB8) is a small chaperone involved in chaperone-assisted selective autophagy (CASA). CASA promotes the selective degradation of proteins to counteract cell stress such as tumor-induced stress. HSPB8 is also involved in (i) the cell division machinery regulating chromosome segregation and cell cycle arrest in the G0/G1 phase and (ii) inflammation regulating dendritic cell maturation and cytokine production. HSPB8 expression and role are tumor-specific, showing a dual and opposite role. Interestingly, HSPB8 may be involved in the acquisition of chemoresistance to drugs. Despite the fact the mechanisms of HSPB8-mediated CASA activation in tumors need further studies, HSPB8 could represent an important factor in cancer induction and progression and it may be a potential target for anticancer treatment in specific types of cancer. In this review, we will discuss the molecular mechanism underlying HSPB8 roles in normal and cancer conditions. The basic mechanisms involved in anti- and pro-tumoral activities of HSPB8 are deeply discussed together with the pathways that modulate HSPB8 expression, in order to outline molecules with a beneficial effect for cancer cell growth, migration, and death.

Chaperone-assisted selective autophagy (CASA) is a highly selective pathway for the disposal of misfolding and aggregating proteins. In muscle, CASA assures muscle integrity by favoring the turnover of structural components damaged by mechanical strain. In neurons, CASA promotes the removal of aggregating substrates. A crucial player of CASA is HSPB8 (heat shock protein family B (small) member 8), which acts in a complex with HSPA, their cochaperone BAG3, and the E3 ubiquitin ligase STUB1. Recently, four novel HSPB8 frameshift (fs) gene mutations have been linked to neuromyopathies, and encode carboxy-terminally mutated HSPB8, sharing a common C-terminal extension. Here, we analyzed the biochemical and functional alterations associated with the HSPB8_fs mutant proteins. We demonstrated that HSPB8_fs mutants are highly insoluble and tend to form proteinaceous aggregates in the cytoplasm. Notably, all HSPB8 frameshift mutants retain their ability to interact with CASA members but sequester them into the HSPB8-positive aggregates together with two autophagy receptors SQSTM1/p62 and TAX1BP1. This copartitioning process negatively affects the CASA capability to remove its clients and causes a general failure in proteostasis response. Further analyses revealed that the aggregation of the HSPB8_fs mutants occurs independently of the other CASA members or from the autophagy receptors interaction, but it is an intrinsic feature of the mutated amino acid sequence. HSPB8_fs mutants aggregation alters the differentiation capacity of muscle cells and impairs sarcomere organization. Collectively, these results shed light on a potential pathogenic mechanism shared by the HSPB8_fs mutants described in neuromuscular diseases.Abbreviations : ACD: α-crystallin domain; ACTN: actinin alpha; BAG3: BAG cochaperone 3; C: carboxy; CASA: chaperone-assisted selective autophagy; CE: carboxy-terminal extension; CLEM: correlative light and electron microscopy; CMT2L: Charcot-Marie-Tooth type 2L; CTR: carboxy-terminal region; dHMNII: distal hereditary motor neuropathy type II; EV: empty vector; FRA: filter retardation assay; fs: frameshift; HSPA/HSP70: heat shock protein family A (Hsp70); HSPB1/Hsp27: heat shock protein family B (small) member 1; HSPB8/Hsp22: heat shock protein family B (small) member 8; HTT: huntingtin; KO: knockout; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MD: molecular dynamics; MTOC: microtubule organizing center; MYH: myosin heavy chain; MYOG: myogenin; NBR1: NBR1 autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; NSC34: Neuroblastoma X Spinal Cord 34; OPTN: optineurin; polyQ: polyglutamine; SQSTM1/p62: sequestosome 1; STUB1/CHIP: STIP1 homology and U-box containing protein 1; TARDBP/TDP-43: TAR DNA binding protein; TAX1BP1: Tax1 binding protein 1; TUBA: tubulin alpha; WT: wild-type.

Dilated cardiomyopathy is a heart muscle disease of unknown aetiology, characterized by left ventricular dilatation and impaired systolic function. Data on the incidence and prevalence of the disease is ambiguous, due to geographic variations, patient selection and the diagnostic criteria adopted.All the post-mortem and clinical cases observed in a consecutive series of 5252 patients resident in Trieste during the period November 1987-November 1989 were studied.Incidence of the disease discovered at autopsy was estimated at 4.5/100,000/year (24 cases), while clinical incidence in the same period was 2.45/100,000/year (13 cases). This is a total incidence of 6.95/100,000 new cases a year. A possible family history of heart muscle disease was found in three patients (12.5%). In 15 patients (62.5%) deaths were due to cardiological complications. Endocardial thickening (P = 0.03), fatty infiltration (P = 0.01) and arterial involvement (P = 0.04) were found more frequently in older patients (> 65 years).The study confirms that dilated cardiomyopathy in Europe has a higher incidence than previously suggested and emphasizes the need for greater diagnostic sensitivity, particularly since pharmacological treatment is now so effective.

It is now clearly established that the central and peripheral nervous systems have the ability to synthesize de novo steroids referred to as neurosteroids. The major evidence for biosynthesis of neuroactive steroids by nervous tissues is based on the expression of enzymes implicated in the formation of steroids in neural cells. The aim of the present review is to summarize the current knowledge regarding the presence of steroidogenic enzymes in the brain of vertebrates and to highlight the very considerable contribution of Professor Kazuyoshi Tsutsui in this domain. The data indicate that expression of steroid-producing enzymes in the brain appeared early during vertebrate evolution and has been preserved from fish to mammals.

Prostate trophism depends on DHT formed from T by the enzyme 5alpha-R. Two 5alpha-R isoforms with different biochemical characteristics have been cloned. Also estrogens might contribute to the prostate growth; however, their intraglandular formation by the enzyme aromatase is still debated. The aim of the present study was to verify whether (a) only one or both isoforms of the 5alpha-Rs are expressed in the prostate cancer cell line LNCaP and in BPH, or (b) the aromatase is present in these samples.The profile of the pH optimum of the 5alpha-Rs was evaluated "in vitro" in LNCaP cells by the production of labeled 5alpha-reduced metabolites either from [14C]-T or [14C]-D4 at pH 3.5-8. The gene expression of the two 5alpha-Rs and of the aromatase in LNCaP cells and in BPH specimens was analyzed by RT-PCR combined to Southern blot analysis, using specific sets of oligonucleotides. The tissue localization of 5alpha-R1 was analyzed by immunohistochemistry using an anti-5alpha-R1 polyclonal antibody.(a) In LNCaP cells, the formation of 5alpha-reduced metabolites from the respective precursors increases progressively as a function of pH, being the highest at neutral pH values; (b) the 5alpha-R1 isoform is expressed in both LNCaP cells and in BPH, while the 5alpha-R2 mRNA is present only in BPH, but not in LNCaP cells; and (c) no aromatase transcripts were observed either in BPH or in LNCaP cells.A careful examination of the possible differential expression of T-activating enzymes, particularly in prostate cancer, would be of help to choose the appropriate treatment.

The distribution of the 5 alpha-reductase, the enzyme which converts testosterone into its 'active' metabolite dihydrotestosterone (DHT), has been studied in neurons, astrocytes and oligodendrocytes isolated from the brain of male rats by density gradient ultracentrifugation and in neurons and glial cells grown in cultures. Purity of cellular preparations was examined by electron and light microscopy. Purified neurons, astrocytes and oligodendrocytes, obtained from the brain of adult male rats, are all able to form DHT from testosterone and consequently possess a 5 alpha-reductase activity. Among the 3 cell types studied, neurons appear to be more active than oligodendrocytes and astrocytes. Moreover, between the two population of glial cells, the oligodendrocytes seem to possess a slightly higher enzymatic activity than that present in the astrocytes. Neurons appeared more active in metabolizing testosterone than glial cells also in cell culture experiments. It is presently believed that the 5 alpha-reduction of testosterone to DHT provides one of the mechanisms through which the hormone becomes effective in the CNS. This is supported by the present findings, which indicate that neurons are the cell population in which the 5 alpha-reductase is more concentrated. However, the presence of a considerable 5 alpha-reductase activity in glial cells indicates that also non-neuronal cells might participate in androgen-mediated events occurring in the brain.

Previous results obtained in this laboratory indicate that in the rat brain the 5 alpha-reductase, the enzymatic activity involved in metabolizing testosterone into 5 alpha-androstan-17 beta-ol-3-one (dihydrotestosterone), is particularly concentrated in the white matter. In the present experiments, this enzymatic activity was studied in the following white matter structures, which were microdissected using the punch technique of Palkovits: anterior commissure (CA), fornix (FX), habenulo-interpeduncular tract (HP), corpus callosum (CC), stria medullaris (SM), optic chiasm (CO), fimbria of the hippocampus (FI), cerebral peduncle (PC), pontine fibers (FP), cerebellar medulla (CMD) and corticospinal tract (TCS). Moreover brain myelin was isolated and purified by sucrose density gradient ultracentrifugation. The results obtained confirm that, in the rat brain, the enzymes involved in testosterone 5 alpha-reduction are preferentially localized in the white matter. However, clearcut differences in the metabolic activity exist between the different structures examined so far. DHT formation increases rostro-caudally, so that the highest activity has been recorded in the white matter structures punched at the level of pons (FP), medulla oblungata (TCS) and cerebellum (CMD). The high metabolic activity associated with the white matter structures appears to be linked to the presence of myelin, since the specific activity of the enzyme is particularly elevated in purified preparations of myelin sheaths.

Aggregates, a hallmark of most neurodegenerative diseases, may have different properties, and possibly different roles in neurodegeneration. We analysed ubiquitin-proteasome pathway functions during cytoplasmic aggregation in polyglutamine (polyQ) diseases, using a unique model of motor neuron disease, the SpinoBulbar Muscular Atrophy. The disease, which is linked to a polyQ tract elongation in the androgen receptor (ARpolyQ), has the interesting feature that ARpolyQ aggregation is triggered by the AR ligand, testosterone. Using immortalized motor neurons expressing ARpolyQ, we found that a proteasome reporter, YFPu, accumulated in absence of aggregates; testosterone treatment, which induced ARpolyQ aggregation, allowed the normal clearance of YFPu, suggesting that aggregation contributed to proteasome de-saturation, an effect not related to AR nuclear translocation. Using AR antagonists to modulate the kinetic of ARpolyQ aggregation, we demonstrated that aggregation, by removing the neurotoxic protein from the soluble compartment, protected the proteasome from an excess of misfolded protein to be processed.

Following crush injury to the facial nerve in Syrian hamsters, treatment with androgens enhances axonal regeneration rates and decreases time to recovery. It has been demonstrated in vitro that the ability of androgen to enhance neurite outgrowth in motoneurons is dependent on neuritin-a protein that is involved in the re-establisment of neuronal connectivity following traumatic damage to the central nervous system and that is under the control of several neurotrophic and neuroregenerative factors--and we have hypothesized that neuritin is a mediator of the ability of androgen to increase peripheral nerve regeneration rates in vivo. Testosterone treatment of facial nerve-axotomized hamsters resulted in an approximately 300% increase in neuritin mRNA levels 2 days post-injury. Simultaneous treatment with flutamide, an androgen receptor blocker that is known to prevent androgen enhancement of nerve regeneration, abolished the ability of testosterone to upregulate neuritin mRNA levels. In a corroborative in vitro experiment, the androgen dihydrotestosterone induced an approximately 100% increase in neuritin mRNA levels in motoneuron-neuroblastoma cells transfected with androgen receptors, but not in cells without androgen receptors. These data confirm that neuritin is under the control of androgens, and suggest that neuritin is an important effector of androgen in enhancing peripheral nerve regeneration following injury. Given that neuritin has now been shown to be involved in responses to both central and peripheral injuries, and appears to be a common effector molecule for several neurotrophic and neurotherapeutic agents, understanding the neuritin pathway is an important goal for the clinical management of traumatic nervous system injuries.

Androgens act on the CNS to affect motor function through interaction with a widespread distribution of intracellular androgen receptors (AR). This review highlights our work on androgens and process outgrowth in motoneurons, both in vitro and in vivo. The actions of androgens on motoneurons involve the generation of novel neuronal interactions that are mediated by the induction of androgen-dependent neurite or axonal outgrowth. Here, we summarize the experimental evidence for the androgenic regulation of the extension and regeneration of motoneuron neurites in vitro using cultured immortalized motoneurons, and axons in vivo using the hamster facial nerve crush paradigm. We place particular emphasis on the relevance of these effects to SBMA and peripheral nerve injuries.

Prostate cancer (PC) develops in response to an abnormal activation of androgen receptor induced by circulating androgens and, in its initial stages, is pharmacologically controlled by androgen blockade. However, androgen ablation therapy often allows androgen-independent PC development, generally characterized by increased invasiveness. We previously reported that 5α-androstane-3β,17β-diol (3β-Adiol) inhibits the migration of PC cell lines via the estrogen receptor β (ERβ) activation. Here, by combining in vitro assays and in vivo imaging approaches, we analyzed the effects of 3β-Adiol on PC proliferation, migration, invasiveness, and metastasis in cultured cells and in xenografts using luciferase-labeled PC3 (PC3-Luc) cells. We found that 3β-Adiol not only inhibits PC3-Luc cell migratory properties, but also induces a broader anti-tumor phenotype by decreasing the proliferation rate, increasing cell adhesion, and reducing invasive capabilities in vitro . All these 3β-Adiol activities are mediated by ERβ and cannot be reproduced by the physiological estrogen, 17β-estradiol, suggesting the existence of different pathways activated by the two ERβ ligands in PC3-Luc cells. In vivo , continuous administration of 3β-Adiol reduces growth of established tumors and counteracts metastasis formation when PC3-Luc cells are engrafted s.c. in nude mice or are orthotopically injected into the prostate. Since 3β-Adiol has no androgenic activity, and cannot be converted to androgenic compounds, the effects here described entail a novel potential application of this agent against human PC.

Spinal and bulbar muscular atrophy (SBMA or Kennedy's disease) is a fatal neurodegenerative disease characterized by the selective loss of motor neurons in the bulbar region of the brain and in the anterior horns of the spinal cord. The disease has been associated to an expansion of a CAG triplet repeat present in the first coding exon of the androgen receptor (AR) gene. SBMA was the first identified member of a large class of neurodegenerative diseases now known as CAG-related diseases, which includes Huntington's disease (HD), several types of spinocerebellar ataxia (SCAs), and dentatorubral and pallidoluysian atrophy (DRPLA). The expanded CAG tract is translated to an aberrantly long polyglutamine tract (ARpolyQ) in the N-terminal region of the AR protein. The elongated polyQ tract seems to confer a neurotoxic gain-of-function to the mutant AR, possibly via the generation of aberrant conformations (misfolding). Protein misfolding is thought to be a trigger of neurotoxicity, since it perturbs a wide variety of motor neuronal functions. The first event is the accumulation of the ARpolyQ into ubiquitinated aggregates in a ligand (testosterone) dependent manner. The mutant ARpolyQ also impairs proteasome functions. The autophagic pathway may be activated to compensate these aberrant events by clearing the mutant ARpolyQ from motor neuronal cells. This review illustrates the mechanisms at the basis of ARpolyQ degradation via the proteasomal and autophagic systems.

In the initial stages, human prostate cancer (PC) is an androgen-sensitive disease, which can be pharmacologically controlled by androgen blockade. This therapy often induces selection of androgen-independent PC cells with increased invasiveness. We recently demonstrated, both in cells and mice, that a testosterone metabolite locally synthetized in prostate, the 5α-androstane-3β, 17β-diol (3β-Adiol), inhibits PC cell proliferation, migration and invasion, acting as an anti-proliferative/anti-metastatic agent. 3β-Adiol is unable to bind androgen receptor (AR), but exerts its protection against PC by specifically interacting with estrogen receptor beta (ERβ). Because of its potential retro-conversion to androgenic steroids, 3β-Adiol cannot be used “in vivo”, thus, the aims of this study were to investigate the capability of four ligands of ERβ (raloxifen, tamoxifen, genistein and curcumin) to counteract PC progression by mimicking the 3β-Adiol activity. Our results demonstrated that raloxifen, tamoxifen, genistein and curcumin decreased DU145 and PC3 cell proliferation in a dose-dependent manner; in addition, all four compounds significantly decreased the detachment of cells seeded on laminin or fibronectin. Moreover, raloxifen, tamoxifen, genistein and curcumin-treated DU145 and PC3 cells showed a significant decrease in cell migration. Notably, all these effects were reversed by the anti-estrogen, ICI 182,780, suggesting that their actions are mediated by the estrogenic pathway, via the ERβ, the only isoform present in these PCs. In conclusion, these data demonstrate that by selectively activating the ERβ, raloxifen, tamoxifen, genistein and curcumin inhibit human PC cells proliferation and migration favoring cell adesion. These synthetic and natural modulators of ER action may exert a potent protective activity against the progression of PC even in its androgen-independent status.

Human MutationVolume 39, Issue 12 p. 2060-2071 RESEARCH ARTICLE Concurrent AFG3L2 and SPG7 mutations associated with syndromic parkinsonism and optic atrophy with aberrant OPA1 processing and mitochondrial network fragmentation Stefania Magri, Stefania Magri Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, ItalySearch for more papers by this authorValentina Fracasso, Valentina Fracasso Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, ItalySearch for more papers by this authorMassimo Plumari, Massimo Plumari Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy Present address: Genomic and Post-genomic Center, IRCCS Foundation "C. Mondino" National Neurological Institute, Pavia, Italy.Search for more papers by this authorEnrico Alfei, Enrico Alfei Unit of Developmental Neurology, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy Present address: Pediatric Neurology Unit, Department of Pediatrics, Ospedale dei Bambini "Vittore Buzzi," ASST Fatebenefratelli-Sacco, Milan, Italy.Search for more papers by this authorDaniele Ghezzi, Daniele Ghezzi Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università degli Studi di Milano, Milan, ItalySearch for more papers by this authorCinzia Gellera, Cinzia Gellera Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, ItalySearch for more papers by this authorPaola Rusmini, Paola Rusmini Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Centro di Eccellenza sulle Malattie Neurodegenerative, Università degli Studi di Milano, Milan, ItalySearch for more papers by this authorAngelo Poletti, Angelo Poletti Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Centro di Eccellenza sulle Malattie Neurodegenerative, Università degli Studi di Milano, Milan, ItalySearch for more papers by this authorDaniela Di Bella, Daniela Di Bella Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, ItalySearch for more papers by this authorAntonio E. Elia, Antonio E. Elia Unit of Neurology 1, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, ItalySearch for more papers by this authorChiara Pantaleoni, Chiara Pantaleoni Unit of Developmental Neurology, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, ItalySearch for more papers by this authorFranco Taroni, Corresponding Author Franco Taroni franco.taroni@istituto-besta.it orcid.org/0000-0002-2420-5233 Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy Correspondence Franco Taroni, MD, Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, via Celoria 11, 20133 Milan, Italy. Email: franco.taroni@istituto-besta.itSearch for more papers by this author Stefania Magri, Stefania Magri Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, ItalySearch for more papers by this authorValentina Fracasso, Valentina Fracasso Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, ItalySearch for more papers by this authorMassimo Plumari, Massimo Plumari Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy Present address: Genomic and Post-genomic Center, IRCCS Foundation "C. Mondino" National Neurological Institute, Pavia, Italy.Search for more papers by this authorEnrico Alfei, Enrico Alfei Unit of Developmental Neurology, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy Present address: Pediatric Neurology Unit, Department of Pediatrics, Ospedale dei Bambini "Vittore Buzzi," ASST Fatebenefratelli-Sacco, Milan, Italy.Search for more papers by this authorDaniele Ghezzi, Daniele Ghezzi Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università degli Studi di Milano, Milan, ItalySearch for more papers by this authorCinzia Gellera, Cinzia Gellera Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, ItalySearch for more papers by this authorPaola Rusmini, Paola Rusmini Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Centro di Eccellenza sulle Malattie Neurodegenerative, Università degli Studi di Milano, Milan, ItalySearch for more papers by this authorAngelo Poletti, Angelo Poletti Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Centro di Eccellenza sulle Malattie Neurodegenerative, Università degli Studi di Milano, Milan, ItalySearch for more papers by this authorDaniela Di Bella, Daniela Di Bella Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, ItalySearch for more papers by this authorAntonio E. Elia, Antonio E. Elia Unit of Neurology 1, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, ItalySearch for more papers by this authorChiara Pantaleoni, Chiara Pantaleoni Unit of Developmental Neurology, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, ItalySearch for more papers by this authorFranco Taroni, Corresponding Author Franco Taroni franco.taroni@istituto-besta.it orcid.org/0000-0002-2420-5233 Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy Correspondence Franco Taroni, MD, Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, via Celoria 11, 20133 Milan, Italy. Email: franco.taroni@istituto-besta.itSearch for more papers by this author First published: 25 September 2018 https://doi.org/10.1002/humu.23658Citations: 23 Funding Information: This work was supported by grants from the Ministero della Salute (RF-2011-02351165), Istituto Superiore di Sanità (E-Rare-2 JTC 2011 EuroSCAR), and Fondazione Telethon (GGP09301) to F.T. Communicated by Christine Van Broeckhoven Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Mitochondrial dynamics and quality control are crucial for neuronal survival and their perturbation is a major cause of neurodegeneration. m-AAA complex is an ATP-dependent metalloprotease located in the inner mitochondrial membrane and involved in protein quality control. Mutations in the m-AAA subunits AFG3L2 and paraplegin are associated with autosomal dominant spinocerebellar ataxia (SCA28) and autosomal recessive hereditary spastic paraplegia (SPG7), respectively. We report a novel m-AAA-associated phenotype characterized by early-onset optic atrophy with spastic ataxia and L-dopa-responsive parkinsonism. The proband carried a de novo AFG3L2 heterozygous mutation (p.R468C) along with a heterozygous maternally inherited intragenic deletion of SPG7. Functional analysis in yeast demonstrated the pathogenic role of AFG3L2 p.R468C mutation shedding light on its pathogenic mechanism. Analysis of patient's fibroblasts showed an abnormal processing pattern of OPA1, a dynamin-related protein essential for mitochondrial fusion and responsible for most cases of hereditary optic atrophy. Consistently, assessment of mitochondrial morphology revealed a severe fragmentation of the mitochondrial network, not observed in SCA28 and SPG7 patients' cells. This case suggests that coincidental mutations in both components of the mitochondrial m-AAA protease may result in a complex phenotype and reveals a crucial role for OPA1 processing in the pathogenesis of neurodegenerative disease caused by m-AAA defects. CONFLICTS OF INTEREST The authors declare no conflict of interest. Citing Literature Supporting Information Filename Description humu23658-sup-0001-SuppMat.pdf5 MB Concurrent AFG3L2 and SPG7 mutations associated with syndromic parkinsonism and optic atrophy with aberrant OPA1 processing and mitochondrial network fragmentation Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Volume39, Issue12December 2018Pages 2060-2071 RelatedInformation

We already demonstrated that in peripheral blood mononuclear cells (PBMCs) of sporadic amyotrophic lateral sclerosis (sALS) patients, superoxide dismutase 1 (SOD1) was present in an aggregated form in the cytoplasmic compartment. Here, we investigated the possible effect of soluble SOD1 decrease and its consequent aggregation. We found an increase in DNA damage in patients PBMCs characterized by a high level of aggregated SOD1, while we found no DNA damage in PBMCs with normal soluble SOD1. We found an activation of ataxia-telangiectasia-mutated (ATM)/Chk2 and ATM and Rad3-related (ATR)/Chk1 DNA damage response pathways, which lead to phosphorylation of SOD1. Moreover, data showed that phosphorylation allows SOD1 to shift from the cytoplasm to the nucleus, protecting DNA from oxidative damage. Such pathway was finally confirmed in our cellular model. Our data lead us to suppose that in a sub-group of patients this physiologic pathway is non-functional, leading to an accumulation of DNA damage that causes the death of particularly susceptible cells, like motor neurons. In conclusion, during oxidative stress SOD1 is phosphorylated by Chk2 leading to its translocation in the nuclear compartment, in which SOD1 protects DNA from oxidative damage. This pathway, inefficient in sALS patients, could represent an innovative therapeutic target.

Extracellular vesicles (EVs) play a central role in neurodegenerative diseases (NDs) since they may either spread the pathology or contribute to the intracellular protein quality control (PQC) system for the cellular clearance of NDs-associated proteins. Here, we investigated the crosstalk between large (LVs) and small (SVs) EVs and PQC in the disposal of TDP-43 and its FTLD and ALS-associated C-terminal fragments (TDP-35 and TDP-25). By taking advantage of neuronal cells (NSC-34 cells), we demonstrated that both EVs types, but particularly LVs, contained TDP-43, TDP-35 and TDP-25. When the PQC system was inhibited, as it occurs in NDs, we found that TDP-35 and TDP-25 secretion via EVs increased. In line with this observation, we specifically detected TDP-35 in EVs derived from plasma of FTLD patients. Moreover, we demonstrated that both neuronal and plasma-derived EVs transported components of the chaperone-assisted selective autophagy (CASA) complex (HSP70, BAG3 and HSPB8). Neuronal EVs also contained the autophagy-related MAP1LC3B-II protein. Notably, we found that, under PQC inhibition, HSPB8, BAG3 and MAP1LC3B-II secretion paralleled that of TDP-43 species. Taken together, our data highlight the role of EVs, particularly of LVs, in the disposal of disease-associated TDP-43 species, and suggest a possible new role for the CASA complex in NDs.

Spinal and bulbar muscular atrophy (SBMA) is characterized by motor neuron (MN) degeneration that leads to slowly progressive muscle weakness. It is considered a neuromuscular disease since muscle has a primary role in disease onset and progression. SBMA is caused by a CAG triplet repeat expansion in the androgen receptor (AR) gene. The translated poly-glutamine (polyQ) tract confers a toxic gain of function to the mutant AR altering its folding, causing its aggregation into intracellular inclusions, and impairing the autophagic flux. In an in vitro SBMA neuronal model, we previously showed that the antiandrogen bicalutamide and trehalose, a natural disaccharide stimulating autophagy, block ARpolyQ activation, reduce its nuclear translocation and toxicity and facilitate the autophagic degradation of cytoplasmic AR aggregates. Here, in a knock-in SBMA mouse model (KI AR113Q), we show that bicalutamide and trehalose ameliorated SBMA pathology. Bicalutamide reversed the formation of the AR insoluble forms in KI AR113Q muscle, preventing autophagic flux blockage. We demonstrated that apoptosis is activated in KI AR113Q muscle, and that both compounds prevented its activation. We detected a decrease of mtDNA and an increase of OXPHOS enzymes, already at early symptomatic stages; these alterations were reverted by trehalose. Overall, bicalutamide and/or trehalose led to a partial recovery of muscle morphology and function, and improved SBMA mouse motor behavior, inducing an extension of their survival. Thus, bicalutamide and trehalose, by counteracting ARpolyQ toxicity in skeletal muscle, are valuable candidates for future clinical trials in SBMA patients.

Sixty subjects with uncomplicated essential hypertension and 60 matched normal subjects were submitted to neuropsychological tests in order to establish whether some impairment of cognitive functions can be evidenced even in those hypertensive subjects that are in this respect asymptomatic on standard examination and interview. The hypertensive subjects obtained significantly poorer results than normotensive subjects on memory, visuo-motor and performance tests. In the control group, the classic negative correlation pattern between age and scores was observed, while in the patient group this correlation could be confirmed only in a few tests. Subgrouping of patients according to hypertension duration and treatment showed that the impairment of cognitive functions manifested itself very early and did not tend to progress within 6-10 years of hypertension duration.

An increasing body of evidence suggests that testosterone may exert beneficial effects against the development of atherosclerosis. These effects are thought to be the consequence of its conversion into estradiol and the activation of the estrogen receptors; however a direct role of androgens, such as dihydrotestosterone, has also been proposed. More recently, it has been shown that the transformation of the dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) and 5alpha-androstane-3beta,17beta-diol (3beta-Adiol), generates two molecules unable to bind the androgen receptor, but with a high affinity for the estrogen receptors (ERs) in particular the beta isoform. As the actions of testosterone may result from the balance between androgenic and estrogenic molecules originating from its catabolism, we investigated the effects of the 3beta-Adiol on inflammatory responses in vitro in human endothelial cells and ex vivo in mice aortas.3beta-Adiol reverts the pro-inflammatory gene expression pattern induced by TNF-alpha in HUVECs as determined by a cDNA microrray approach. Q-real-time PCR and protein array approaches confirmed that TNF-alpha-induced ICAM-1, VCAM-1 and ELAM-1 as well as MCP-1 and IL-6 induction was affected upon 3beta-Adiol pre-incubation. ICI 182780, an estrogen receptor antagonist and R,R-THC, an estrogen receptor beta antagonist, counteracted the effect of 3beta-Adiol while bicalutamide, an androgen receptor antagonist, had minor effects. 3beta-Adiol exerted a similar action on macrophages. Finally in castrated male mice, 3beta-Adiol significantly counteracted the LPS mediated mRNA induction of IL-6, ELAM-1and PECAM-1 in the aortas.3beta-Adiol reverts in vitro the TNF-alpha and LPS induced pro-inflammatory activation of endothelial cells and macrophages. 3beta-Adiol in vivo modulates the inflammatory response induced by LPS in the arterial vascular wall.

Anabolic/androgenic steroids (AAS) are drugs that enhance muscle mass, and are often illegally utilized in athletes to improve their performances. Recent data suggest that the increased risk for amyotrophic lateral sclerosis (ALS) in male soccer and football players could be linked to AAS abuse. ALS is a motor neuron disease mainly occurring in sporadic (sALS) forms, but some familial forms (fALS) exist and have been linked to mutations in different genes. Some of these, in their wild type (wt) form, have been proposed as risk factors for sALS, i.e. superoxide dismutase 1 (SOD1) gene, whose mutations are causative of about 20% of fALS. Notably, SOD1 toxicity might occur both in motor neurons and in muscle cells. Using gastrocnemius muscles of mice overexpressing human mutant SOD1 (mutSOD1) at different disease stages, we found that the expression of a selected set of genes associated to muscle atrophy, MyoD, myogenin, atrogin-1, and transforming growth factor (TGF)β1, is up-regulated already at the presymptomatic stage. Atrogin-1 gene expression was increased also in mice overexpressing human wtSOD1. Similar alterations were found in axotomized mouse muscles and in cultured ALS myoblast models. In these ALS models, we then evaluated the pharmacological effects of the synthetic AAS nandrolone on the expression of the genes modified in ALS muscle. Nandrolone administration had no effects on MyoD, myogenin, and atrogin-1 expression, but it significantly increased TGFβ1 expression at disease onset. Altogether, these data suggest that, in fALS, muscle gene expression is altered at early stages, and AAS may exacerbate some of the alterations induced by SOD1 possibly acting as a contributing factor also in sALS.

Spinal and bulbar muscular atrophy (SBMA) or Kennedy's disease is an X-linked disease associated with the expansion of the CAG triplet repeat present in exon 1 of the androgen receptor (AR) gene. This results in the production of a mutant AR containing an elongated polyglutamine tract (polyQ) in its N-terminus. Interestingly, the ARpolyQ becomes toxic only after its activation by the natural androgenic ligands, possibly because of aberrant androgen-induced conformational changes of the ARpolyQ, which generate misfolded species. These misfolded ARpolyQ species must be cleared from motoneurons and muscle cells, and this process is mediated by the protein quality control (PQC) system. Experimental evidence suggested that failure of the PQC pathways occurs in disease, leading to ARpolyQ accumulation and toxicity in the target cells. In this review, we summarized the overall impact of mutant and misfolded ARpolyQ on the PQC system and described how molecular chaperones and the degradative pathways (ubiquitin-proteasome system (UPS), the autophagy-lysosome pathway (ALP), and the unfolded protein response (UPR), which activates the endoplasmic reticulum-associated degradation (ERAD)) are differentially affected in SBMA. We also extensively and critically reviewed several molecular and pharmacological approaches proposed to restore a global intracellular activity of the PQC system. Collectively, these data suggest that the fine and delicate equilibrium existing among the different players of the PQC system could be restored in a therapeutic perspective by the synergic/additive activities of compounds designed to tackle sequential or alternative steps of the intracellular defense mechanisms triggered against proteotoxic misfolded species.

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions are neurodegenerative disorders that share the cytosolic deposition of TDP-43 (TAR DNA-binding protein 43) in the CNS. TDP-43 is well known as being actively degraded by both the proteasome and macroautophagy. The well-documented decrease in the efficiency of these clearance systems in aging and neurodegeneration, as well as the genetic evidence that many of the familial forms of TDP-43 proteinopathies involve genes that are associated with them, suggest that a failure of these protein degradation systems is a major factor that contributes to the onset of TDP-43-associated disorders. Here, we inserted preformed human TDP-43 aggregates in the cytosol of murine NSC34 and N2a cells in diffuse form and observed their degradation under conditions in which exogenous TDP-43 is not expressed and endogenous nuclear TDP-43 is not recruited, thereby allowing a time zero to be established in TDP-43 degradation and to observe its disposal kinetically and analytically. TDP-43 degradation was observed in the absence and presence of selective inhibitors and small interfering RNAs against the proteasome and autophagy. We found that cytosolic diffuse aggregates of TDP-43 can be distinguished in 3 different classes on the basis of their vulnerability to degradation, which contributed to the definition-with previous reports-of a total of 6 distinct classes of misfolded TDP-43 species that range from soluble monomer to undegradable macroaggregates. We also found that the proteasome and macroautophagy-degradable pools of TDP-43 are fully distinguishable, rather than in equilibrium between them on the time scale required for degradation, and that a significant crosstalk exists between the 2 degradation processes.-Cascella, R., Fani, G., Capitini, C., Rusmini, P., Poletti, A., Cecchi, C., Chiti, F. Quantitative assessment of the degradation of aggregated TDP-43 mediated by the ubiquitin proteasome system and macroautophagy.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of upper and lower motor neurons and skeletal muscle atrophy. Epidemiologic and experimental evidence suggest the involvement of androgens in ALS pathogenesis, but the mechanism through which androgens modify the ALS phenotype is unknown. Here, we show that androgen ablation by surgical castration extends survival and disease duration of a transgenic mouse model of ALS expressing mutant human SOD1 (hSOD1-G93A). Furthermore, long-term treatment of orchiectomized hSOD1-G93A mice with nandrolone decanoate (ND), an anabolic androgenic steroid, worsened disease manifestations. ND treatment induced muscle fiber hypertrophy but caused motor neuron death. ND negatively affected survival, thereby dissociating skeletal muscle pathology from life span in this ALS mouse model. Interestingly, orchiectomy decreased androgen receptor levels in the spinal cord and muscle, whereas ND treatment had the opposite effect. Notably, stimulation with ND promoted the recruitment of endogenous androgen receptor into biochemical complexes that were insoluble in sodium dodecyl sulfate, a finding consistent with protein aggregation. Overall, our results shed light on the role of androgens as modifiers of ALS pathogenesis via dysregulation of androgen receptor homeostasis.

Aim: The demonstration that chaperone-mediated autophagy (CMA) contributes to the degradation of TDP-43, the main constituent of cytoplasmic inclusions typically found in motor neurons of patients with sporadic amyotrophic lateral sclerosis (sALS), has pointed out a possible involvement of CMA in aggregate formation. To explore this possibility, in this study, we verified the presence of a possible systemic CMA alteration in sALS patients and its effect on TDP-43 expression. Materials and methods: Gene and protein expression of the cytosolic chaperone HSC70 and the lysosome receptor LAMP2A, the two pivotal mediators of CMA, was assessed in peripheral blood mononuclear cells (PBMCs) derived from 30 sALS patients and 30 healthy controls. The expression of TDP-43 and co-chaperones BAG1 and BAG3 was also analyzed. Results: We found reduced HSC70 expression in patient cells, with no change in LAMP2A, and increased insoluble TDP-43 protein levels, with an aberrant intracellular localization. We also observed an unbalanced expression of co-chaperones BAG1 and BAG3. HSC70 down-regulation was confirmed in immortalized lymphoblastoid cell lines derived from sporadic and TARDBP mutant ALS patients. Lastly, we demonstrated that HSC70 silencing directly increases TDP-43 protein levels in human neuroblastoma cells. Discussion: Our results do not support the existence of a systemic CMA alteration in sALS patients but indicate a direct involvement of HSC70 alterations in ALS pathogenesis.

Article18 November 2021Open Access Source DataTransparent process C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation Nausicaa V Licata Nausicaa V Licata orcid.org/0000-0003-0750-0692 Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy These authors contributed equally to this work Search for more papers by this author Riccardo Cristofani Riccardo Cristofani orcid.org/0000-0003-2719-846X Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy These authors contributed equally to this work Search for more papers by this author Sally Salomonsson Sally Salomonsson orcid.org/0000-0001-6717-3369 Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK UK Dementia Research Institute at UCL, UCL Queen Square Institute of Neurology, London, UK Search for more papers by this author Katherine M Wilson Katherine M Wilson Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK UK Dementia Research Institute at UCL, UCL Queen Square Institute of Neurology, London, UK Search for more papers by this author Liam Kempthorne Liam Kempthorne orcid.org/0000-0002-9790-8968 Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK UK Dementia Research Institute at UCL, UCL Queen Square Institute of Neurology, London, UK Search for more papers by this author Deniz Vaizoglu Deniz Vaizoglu orcid.org/0000-0001-6572-1544 Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK UK Dementia Research Institute at UCL, UCL Queen Square Institute of Neurology, London, UK Search for more papers by this author Vito G D'Agostino Vito G D'Agostino orcid.org/0000-0003-3379-2254 Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy Search for more papers by this author Daniele Pollini Daniele Pollini orcid.org/0000-0001-7782-7960 Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy Search for more papers by this author Rosa Loffredo Rosa Loffredo orcid.org/0000-0001-7981-9227 Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy Search for more papers by this author Michael Pancher Michael Pancher orcid.org/0000-0002-3783-6069 HTS Core Facility, Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy Search for more papers by this author Valentina Adami Valentina Adami orcid.org/0000-0002-0617-9393 HTS Core Facility, Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy Search for more papers by this author Paola Bellosta Paola Bellosta orcid.org/0000-0003-1913-5661 Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy Department of Medicine, NYU at Grossman School of Medicine, NY, USA Search for more papers by this author Antonia Ratti Antonia Ratti orcid.org/0000-0002-4264-6614 Department of Neurology, Stroke Unit and Laboratory of Neuroscience, Istituto Auxologico Italiano, IRCCS, Milan, Italy Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy Search for more papers by this author Gabriella Viero Gabriella Viero orcid.org/0000-0002-6755-285X Institute of Biophysics, CNR Unit at Trento, Trento, Italy Search for more papers by this author Alessandro Quattrone Alessandro Quattrone orcid.org/0000-0003-3333-7630 Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy Search for more papers by this author Adrian M Isaacs Adrian M Isaacs orcid.org/0000-0002-6820-5534 Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK UK Dementia Research Institute at UCL, UCL Queen Square Institute of Neurology, London, UK Search for more papers by this author Angelo Poletti Corresponding Author Angelo Poletti [email protected] orcid.org/0000-0002-8883-0468 Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy Search for more papers by this author Alessandro Provenzani Corresponding Author Alessandro Provenzani [email protected] orcid.org/0000-0003-1652-3415 Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy Search for more papers by this author Nausicaa V Licata Nausicaa V Licata orcid.org/0000-0003-0750-0692 Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy These authors contributed equally to this work Search for more papers by this author Riccardo Cristofani Riccardo Cristofani orcid.org/0000-0003-2719-846X Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy These authors contributed equally to this work Search for more papers by this author Sally Salomonsson Sally Salomonsson orcid.org/0000-0001-6717-3369 Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK UK Dementia Research Institute at UCL, UCL Queen Square Institute of Neurology, London, UK Search for more papers by this author Katherine M Wilson Katherine M Wilson Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK UK Dementia Research Institute at UCL, UCL Queen Square Institute of Neurology, London, UK Search for more papers by this author Liam Kempthorne Liam Kempthorne orcid.org/0000-0002-9790-8968 Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK UK Dementia Research Institute at UCL, UCL Queen Square Institute of Neurology, London, UK Search for more papers by this author Deniz Vaizoglu Deniz Vaizoglu orcid.org/0000-0001-6572-1544 Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK UK Dementia Research Institute at UCL, UCL Queen Square Institute of Neurology, London, UK Search for more papers by this author Vito G D'Agostino Vito G D'Agostino orcid.org/0000-0003-3379-2254 Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy Search for more papers by this author Daniele Pollini Daniele Pollini orcid.org/0000-0001-7782-7960 Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy Search for more papers by this author Rosa Loffredo Rosa Loffredo orcid.org/0000-0001-7981-9227 Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy Search for more papers by this author Michael Pancher Michael Pancher orcid.org/0000-0002-3783-6069 HTS Core Facility, Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy Search for more papers by this author Valentina Adami Valentina Adami orcid.org/0000-0002-0617-9393 HTS Core Facility, Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy Search for more papers by this author Paola Bellosta Paola Bellosta orcid.org/0000-0003-1913-5661 Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy Department of Medicine, NYU at Grossman School of Medicine, NY, USA Search for more papers by this author Antonia Ratti Antonia Ratti orcid.org/0000-0002-4264-6614 Department of Neurology, Stroke Unit and Laboratory of Neuroscience, Istituto Auxologico Italiano, IRCCS, Milan, Italy Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy Search for more papers by this author Gabriella Viero Gabriella Viero orcid.org/0000-0002-6755-285X Institute of Biophysics, CNR Unit at Trento, Trento, Italy Search for more papers by this author Alessandro Quattrone Alessandro Quattrone orcid.org/0000-0003-3333-7630 Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy Search for more papers by this author Adrian M Isaacs Adrian M Isaacs orcid.org/0000-0002-6820-5534 Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK UK Dementia Research Institute at UCL, UCL Queen Square Institute of Neurology, London, UK Search for more papers by this author Angelo Poletti Corresponding Author Angelo Poletti [email protected] orcid.org/0000-0002-8883-0468 Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy Search for more papers by this author Alessandro Provenzani Corresponding Author Alessandro Provenzani [email protected] orcid.org/0000-0003-1652-3415 Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy Search for more papers by this author Author Information Nausicaa V Licata1, Riccardo Cristofani2, Sally Salomonsson3,4, Katherine M Wilson3,4, Liam Kempthorne3,4, Deniz Vaizoglu3,4, Vito G D'Agostino1, Daniele Pollini1, Rosa Loffredo1, Michael Pancher5, Valentina Adami5, Paola Bellosta1,6, Antonia Ratti7,8, Gabriella Viero9, Alessandro Quattrone1, Adrian M Isaacs3,4, Angelo Poletti *,2 and Alessandro Provenzani *,1 1Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy 2Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy 3Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK 4UK Dementia Research Institute at UCL, UCL Queen Square Institute of Neurology, London, UK 5HTS Core Facility, Department of Cellular, Computational and Integrative Biology, University of Trento, Trento, Italy 6Department of Medicine, NYU at Grossman School of Medicine, NY, USA 7Department of Neurology, Stroke Unit and Laboratory of Neuroscience, Istituto Auxologico Italiano, IRCCS, Milan, Italy 8Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milan, Italy 9Institute of Biophysics, CNR Unit at Trento, Trento, Italy *Corresponding author. Tel: +39 02 50318215; E-mail: [email protected] *Corresponding author. Tel: +39 0461 283176; E-mail: [email protected] The EMBO Journal (2022)41:e105026https://doi.org/10.15252/embj.2020105026 PDFDownload PDF of article text and main figures. 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 Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD. Synopsis GGGGCC repeat expansion in the C9orf72 gene causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in part via neurotoxic dipeptide-repeat proteins (DPRs) produced by repeat-associated non-AUG translation. Here, a high-throughput drug screen identifies novel positive and negative small-molecule modulators of DPR levels. cAMP-elevating compounds increase DPR levels by enhancing protein kinase A (PKA) activity. PKA inhibition reduces DPR levels in cells and in C9ALS/FTD patient-derived iPSC motor neurons. Inhibition or knockdown of PKA ameliorates motility and survival in a Drosophila model of C9orf72 ALS/FTD. Knockdown of PKA catalytic subunits correlates with reduced DPR translation efficiency. HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduce DPR levels by promoting protein degradation. Introduction Repeat-associated non-AUG (RAN) translation is an unconventional translation mechanism associated with several nucleotide-repeat expansion disorders. The hexanucleotide repeat expansion GGGGCCn, also known as (G4C2)n, is localized in the first intron of the C9orf72 gene (DeJesus-Hernandez et al, 2011; Renton et al, 2011) and it is the most common genetic cause of familial forms of ALS and FTD (hereafter C9ALS/FTD) (Gijselinck et al, 2012). The pathogenic mechanisms proposed for C9ALS/FTD suggest that sense (G4C2)n- and antisense (C4G2)n-containing transcripts cause two different mechanisms of toxicity. The first is mediated by the formation of RNA foci that bind and sequester RNA-binding proteins, thereby leading to impairment of RNA metabolism (Donnelly et al, 2013; Lee et al, 2013; Mori et al, 2013b; Xu et al, 2013; Cooper-Knock et al, 2014; Haeusler et al, 2014; Wen et al, 2014; Zhang et al, 2015; Conlon et al, 2016; Swinnen et al, 2018); and the second mediated by their unconventional RAN translation into five different dipeptide-repeat proteins (DPRs: poly-GA, poly-GP, poly-GR, poly-PA, poly-PR) (Ash et al, 2013; Mori et al, 2013a, 2013c). In addition, pathological expansions of (G4C2)n reduce C9orf72 transcription and translation with decreased C9orf72 protein levels (DeJesus-Hernandez et al, 2011; Renton et al, 2011); this latter event can also be associated with endosomal trafficking, autophagy dysfunction, which synergizes with repeat-associated toxicity (Shi et al, 2018; Boivin et al, 2020; Zhu et al, 2020). DPR-induced toxicity has been shown in several cell lines, in iPSC-derived neurons (May et al, 2014; Su et al, 2014; Yamakawa et al, 2015; Yang et al, 2015a; Westergard et al, 2019), in Drosophila (Mizielinska et al, 2014; Wen et al, 2014; Chew et al, 2015; Freibaum et al, 2015; Yang et al, 2015a; Boeynaems et al, 2016) and in mouse models (Zhang et al, 2016, Zhang et al, 2018, Zhang et al, 2019; Schludi et al, 2017; Choi et al, 2019; Hao et al, 2019). Multiple studies demonstrated proteasome dysfunction due to the sequestration of proteasomal proteins by poly-GA in both in vitro (May et al, 2014; Yamakawa et al, 2015) and in vivo models (Zhang et al, 2016). RAN translation of (G4C2)n-RNAs has been recently shown to require a near-cognate start codon upstream of the repeat in the +1 frame (Green et al, 2017; Tabet et al, 2018) and to be triggered by stress conditions in a cap-dependent (Kearse et al, 2016; Green et al, 2017; Tab et et al, 2018) or cap-independent way (Cheng et al, 2018; Sonobe et al, 2018). However, the mechanisms regulating RAN translation have not yet been completely elucidated. Antisense oligonucleotides (ASOs) (Jiang et al, 2016; Gendron et al, 2017) and small molecules targeting the (G4C2)n (Su et al, 2014; Simone et al, 2018; Wang et al, 2019) and/or r(CGG)n RNAs (Yang et al, 2015b, 2016; Green et al, 2019) have been proposed as possible therapeutic approaches, but no clinically approved drugs are known to selectively modulate RAN translation. The small molecules available at present alter RNA secondary structures providing a proof of principle as to how their binding to (G4C2)n can inhibit both RNA foci formation and RAN translation. A genetic screen recently identified the ribosomal protein RPS25 as a regulator of RAN translation of different repeat sequence expansions (Yamada et al, 2019). Other modifiers of DPR production were identified by two independent genome-wide CRISPR-Cas9 screens (Kramer et al, 2018; Cheng et al, 2019; Wilson et al, 2019). In addition, the RNA helicase DHX36 has been shown to favour (G4C2)n and FMR1-associated RAN translation (Tseng et al, 2021), whereas the RNA helicase DDX3X inhibits (G4C2)n RAN translation (Cheng et al, 2019) but it promotes FMR1-associated RAN translation (Linsalata et al, 2019). Here, we used a chemical genomic approach to identify small molecules and relative molecular targets. These small molecules modulate DPR levels by either increasing protein clearance or inhibiting translation of (G4C2)n-containing RNAs. Among these small molecules, we found that Geldanamycin (GELD, an inhibitor of Heat Shock Protein 90, HSP90) increases proteasome activity and that Spironolactone (SPL, an aldosterone antagonist) modulates DPR autophagy degradation. Moreover, we found for the first time that cAMP-elevating compounds increase DPR levels by boosting protein kinase A (PKA) activity, while PKA silencing, or inhibition reverted these effects. This suggests a novel mechanism in which PKA is involved in pathways that aberrantly enhance the translation of C9orf72 (G4C2)n mRNA to neurotoxic DPRs. Results Development of a HTS assay for identifying modulators of C9-DPR levels We set up a cell-based high-throughput screen (HTS) (full HTS protocol in Appendix Materials and Methods), to find small molecules capable of modulating DPR levels. In the HTS we used an artificial reporter containing 58 G4C2 repeats outside of the native C9orf72 sequence, and with the GFP sequence in the GP frame (Freibaum et al, 2015) (hereafter polyGP-GFP) (Appendix Tables S1 and S2). We obtained a consistent signal of polyGP-GFP across experimental repeats using a reverse transfection approach in HEK293T cells (Fig EV1A and B). In the HTS we also co-transfected an AUG-RFP plasmid (Fig 1A) to report on AUG-mediated translation. In the screen, Cycloheximide (CHX) was used to model general translation inhibition. We used the variation in the number of GFP- or RFP-positive cells as the read-out of the assay. Due to the lack of a positive control, small molecule RAN translation inhibitor, the variability and the robustness of the assay were optimized to perform a HTS based on the effect of CHX on RFP-expressing cells (Z′-factor = 0.5) (Zhang et al, 1999) (Fig EV1C). We observed that CHX did not decrease the fluorescent intensity or the number of cells expressing polyGP-GFP (Fig EV1D), consistent with a recent report showing non-AUG translation to be resistant to elongation inhibitors (Kearse et al, 2019). Click here to expand this figure. Figure EV1. Set up of a High-Throughput Screen (HTS) for identifying modulators of DPR levels SH-SY5Y and HEK293T cells were co-transfected with AUG-RFP and polyGP-GFP plasmids by standard (SH-SY5Y and HEK293Ta) or reverse (HEK293Tb) transfection method. Images were acquired 24 h after using Operetta High-Content Imaging System and transfection efficiency were calculated on the ratio of cells expressing RFP/tot and cells expressing GFP/tot. Data are mean ± SD from three biological replicates. Images in HEK293Tb are from DMSO in Fig 2A. Scale bars 200 μm. Immunoblot for testing the level of polyGP-GFP and AUG-RFP in lysates from HEK293T cells co-transfected with both plasmids or with polyGP-GFP or AUG-RFP and mock. Distribution of cells expressing AUG-RFP (upper inset) or AUG-RFP fluorescent intensity (lower inset) in negative (DMSO) and positive (CHX, 5 μM) controls. Data used to calculate Z′-factor. Data are mean ± SD from 45 (upper inset) and 35 (lower inset) technical replicates. Distribution of cells expressing polyGP-GFP (upper inset) and polyGP-GFP fluorescent intensity (lower inset) in negative (DMSO) and positive (CHX, 5 μM) controls. Data are mean ± SD from 45 (upper inset) and 35 (lower inset) technical replicates. HTS. HEK293T cells were co-transfected with the constructs shown in Fig 1A. Scatter plot shows the distribution of negative (DMSO, orange dots) and positive (CHX 5 μM, purple dots) controls added 3 h after reverse co-transfection. On the Y-axis reported the Z-score values of cells expressing AUG-RFP and on the X-axis the Z-score values of cells expressing polyGP-GFP. Grid lines represent the thresholds arbitrarily set up around the DMSO distribution (polyGP-GFP ± 1.5 on the X-axis and ± 1.5 for AUG-RFP on the Y-axis). Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Primary and confirmatory screening for identifying modulators of C9orf72-derived DPR levels A. Schematic representation of the constructs utilized in (B) and (C). The first construct contains 58 (G4C2) repeats outside of the native C9orf72 sequence and GFP in the GP frame (polyGP-GFP). The start codon of GFP was removed. The second construct AUG-RFP is used as a positive control of canonical translation. B. HTS. HEK293T cells were co-transfected with the constructs in (A) and, negative (DMSO, orange dots) and positive (CHX 5 µM, purple dots) controls added 3 h after reverse co-transfection with compound-libraries (5 µM). Images and data acquisition collected after about 30 h of treatment. Scatter plot shows the distribution of compounds. On the Y-axis reported the Z-score values of cells expressing AUG-RFP and on the X-axis the Z-score values of cells expressing polyGP-GFP. Grid lines represent the thresholds arbitrarily set up around DMSO distribution (polyGP-GFP ± 1.5 on the X-axis and ± 1.5 for AUG-RFP on the Y-axis) to select compounds for the confirmatory screening and eliminate the ones without effect (orange square). C. Confirmatory screening performed as described above. Schematic distribution of compounds based on Z-score values of cells expressing polyGP-GFP and AUG-RFP (above) and on Z-score values of the fluorescent intensity of the two reporters per each compound (below). Data are from four technical replicates, boxes are the Z-score mean value and whiskers represent ± SD. Baseline of Z-score = 0 indicates that Z-score is identical to the mean score. Asterisk (*) represents the selected compounds: 1 Forskolin (FSK), 2 Erysolin (ERY), 3 Geldanamycin (GELD), 4 Helenin (HLN) and 5 Spironolactone (SPL). Number sign (#) represents cellular stress inducers: #1 Thapsigargin and #2 Tunicamycin. D, E. Dose-response analysis of ERY, HLN, SPL, FSK and GELD. Cells were co-transfected with AUG-RFP and polyGP-GFP plasmids and treated with two concentration ranges 0.5, 1, 5 and 10 µM (D) and 20, 40 and 60 µM (E) for 24 h. CHX used only low dosages. Data are mean ± SD from three biological replicates. One-way ANOVA followed by Dunnett's multiple comparison tests: *P < 0.5; **P < 0.01; ***P < 0.001. Download figure Download PowerPoint We screened approximately 2,500 compounds with biological activity from different chemical libraries (see Materials and Methods). The compounds were added 3 h after plasmid reverse co-transfection. GFP and RFP reporter signals were measured approximately 30 h later. Plotting the Z-score of the number of cells expressing AUG-products (RFP, Y-axis) versus the Z-score of the number of cells expressing DPR-products (polyGP-GFP, X-axis), we obtained a graphical representation of the simultaneous effect of the small molecules on both AUG and RAN translation-dependent products (Fig 1B). The majority of tested compounds did not modify the levels of the reporters. Their signals overlapped with the distribution of negative controls (Figs 1B and EV1E), indirectly proving the assay quality. We selected effective compounds using an arbitrary threshold of ± 1.5 Z-score for cells expressing polyGP-GFP and ± 1.5 for AUG-derived positive cells. These thresholds gave a significant separation of DMSO treated from CHX-treated samples (see Appendix for full details on thresholding calculations). We excluded highly toxic compounds using a threshold Z-score nuclei ≤ −2, indicating that < 50% of cells survived. A confirmatory screen was next performed as described above but increased the number of replicates from one to four. As expected, while only a few compounds were able to reduce levels of the RAN products, many others had the opposite effect (Fig 1C). This comes as no surprise because many RAN-increasing compounds were cellular stressors (Thapsigargin, (Green et al, 2017; Westergard et al, 2019) and Tunicamycin (Green et al, 2017; Westergard et al, 2019)) present in the chemical library (Fig 1C). We selected three small molecules according to their capability to specifically reduce or increase the number of cells expressing polyGP-GFP and/or the fluorescent intensity of GFP (Fig 1C, Table 1 and Dataset EV1). GELD and SPL reduced RAN products, whilst cAMP-elevating compounds, with Forskolin (FSK) being the most potent one, increased them. Interestingly, FSK, which activates adenylyl cyclase (AC) and enhances intracellular cAMP levels, triggers a multitude of PKA-dependent and/or -independent pathways resulting in pleiotropic effects on cells. These events include the activation of many intracellular signalling cascades and of the cAMP Response Elements Binding (CREB) family of transcription factors that, upon phosphorylation, regulate the expression of genes containing cAMP Response Elements (CREs) in their promoters (Seamon et al, 1981; Sapio et al, 2014; Kanne et al, 2015). We also identified two phytochemicals with undefined mechanisms of action, Erysolin (ERY) and Helenin (HLN) that effectively reduced RAN products. These results were then validated in the confirmatory screen (Dataset EV1). Table 1. List of the small molecules selected from the confirmatory screening. Small molecules Cells expressing polyGP-GFP Cells expressing AUG-RFP polyGP-GFP intensity AUG-RFP intensity Number of cells DMSO −0.06 −0.2 0.3 −0.12 −0.2 Erysolin (ERY) −3.5 −0.2 −1.34 −0.7 −1.1 Forskolin (FSK) 20.3 −2.4 27 −0.8 0.05 Geldanamycin (GELD) −5 1.6 0.002 3 −2.6 Helenin (HLN) −1.05 −0.7 −2.8 −1.1 −1.3 Spironolactone (SPL) −0.5 0.7 −2.3 0.6 −0.9 Mean of compounds from four biological replicates. All the compounds were added after 3h of reverse co-transfection and used at the final concentration of 5 µM for approximately 30 h. Mean of DMSO from 64 biological replicates (32 replicates per each 384-well plate). Data are reported as Z-score values. The entire list of small molecules used is reported in Dataset EV1. To obtain a confidence interval of safe utilization of the selected compounds, we then performed dose–response experiments by treating cells with two concentration ranges for each compound (Fig 1D and E), simultaneously checking their toxicity and the effects on DPR levels. All the compounds confirmed their activity in modulating the number of polyGP-GFP-positive cells, although to various extents. The most potent compound was FSK that selectively increased polyGP-GFP-positive cells compared to AUG-RFP-positive cells. HLN decreased both products, whereas ERY, GELD and SPL decreased more efficiently the polyGP-GFP products than the AUG-RFP. All these compounds were moderately toxic at concentrations higher than 40 μM, with HLN the most toxic. Therefore, we excluded HLN due to its toxicity and proceeded with the other four molecules to gain information about their molecular mechanism of action. GELD, SPL and FSK modulate DPR levels independently of the near-cognate CUG start codon To understand whether the four selected DPR modulators (Fig 2A) affect general transcription and translation, we used the incorporation of the modified nucleoside 5‑ethynyl uridine (EU) to evaluate general RNA transcription. In parallel, we took advantage of O-propargyl-puromycin (OPP) incorporation assay to evaluate de novo protein synthesis. GELD marginally induced general transcription (Fig 2B). None of the compounds modulated translation (Fig 2C). The molecular mechanism of RAN translation initiation (Kearse et al, 2016) is still a matter of debate and a near-cognate CUG start codon within C9orf72 first intron 1A has been suggested to play a key role in (G4C2)n RAN translation (Green et al, 2017; Tab et et al, 2018). The polyGP-GFP reporter used in the HTS did not contain the native sequence upstream of the repeat. Therefore, to evaluate whether the effect of these compounds was CUG independent, we used the (G4C2)x66 construct (hereafter 66R) that contains repeats within the native C9orf72 sequence and a specific C-term tag for each frame (Gendron et al, 2013). To prove the efficacy of GELD and SPL in motor neuron-like cells, we used NSC34 cells (Appendix Fig S1A). To ensure the proper evaluation of the total amounts of DPRs produced in cells, we quantified both the soluble DPR levels and the PBS-insoluble DPR aggregate fraction by immunoblot analysis and filter retardation assay, respectively. GELD and SPL significantly reduced the accumulation of DPRs, while FSK significantly increased poly-GA in HEK293T (Fig 2D), poly-GP and its PBS-insoluble fraction in NSC34 cells (Fig 2E and F), despite the presence of the upstream CUG codon. In contrast, ERY did not show any effect in modulating DPR levels in either cell line (Fig 2D–F). We further challenged the selected compounds using C9RAN NLuc reporters (in the GA frame) with the native (CUG) or mutated (CCC) start codon (Green et al, 2017). C

In the rat brain, several steroids can be converted by specific enzymes to either more potent compounds or to derivatives showing new biological effects. One of the most studied enzyme is the 5alpha-reductase (5alpha-R), which acts on 3keto-delta4 steroids. In males, testosterone is the main substrate and gives rise to the most potent natural androgen dihydrotestosterone. In females, progesterone is reduced to dihydroprogesterone, a precursor of allopregnanolone, a natural anxiolytic/anesthetic steroid. Other substrates are some gluco- and minero-corticoids. Two isoforms of the 5alpha-R, with limited degree of homology, have been cloned: 5alpha-R type 1 and type 2. The 5alpha-R type 1 possesses low affinity for the various substrates and is widely distributed in the body, with the highest levels in the liver; in the brain, this isoform is expressed throughout life and does not appear to be controlled by androgens. 5Alpha-R type 1 in the rat brain is mainly concentrated in myelin membranes, where it might be involved in the catabolism of potentially neurotoxic steroids. The 5alpha-R type 2 shows high affinity for the various substrates, a peculiar pH optimum at acidic values and is localized in androgen-dependent structures. In the rat brain, the type 2 isoform is expressed at high levels only in the perinatal period and is controlled by androgens, at least in males. In adulthood, the type 2 gene appears to be specifically expressed in localised brain regions, like the hypothalamus and the hippocampus. The 5alpha-R type 2 is present in the GT1 cells, a model of LHRH-secreting neurons. These cells also contain the androgen receptor, which is probably involved in the central negative feedback effect exerted by androgens on the hypothalamic-pituitary-gonadal axis. The physiological significance of these and additional data will be discussed.

The formation of the 5alpha-reduced metabolites of testosterone (T) and of progesterone (P) is a very active process in the brain, since the enzyme 5alpha-reductase (5alpha-R) is present in almost any central nervous system (CNS) structure. A particularly elevated 5alpha-R activity has been shown in myelin sheaths. Two isoforms of the enzyme have been cloned, with different localisation as well as different biochemical properties. The present study was performed to determine whether both isoforms of the 5alpha-R, or only one of them, are/is responsible for the enzymatic activity observed in myelin. Kinetic analyses have been performed on purified myelin membranes prepared from the male or female rat brain, using both T and P as substrates. The 5alpha-R present appears to possess a pH optimum at basic values. The Vmax values obtained in the Lineweaver-Burk analysis were comparable in male and female preparations independently on whether T or P were used as the substrates, suggesting that a single enzymatic form is present in all samples examined; the Km obtained using [14C]T (Km: male 1.14 microM; female 1.46 microM) or [14C]P (Km: male 0.5 microM; female 0.64 microM) as substrates, were in good agreement with those obtained for the recombinant type 1 isoform. These data suggest that the type 1 isoform is the most relevant 5alpha-R present in myelin. To confirm this, a new polyclonal antibody was raised against the type 1 5alpha-R enzymatic protein, and used in immunohistochemical studies. The experiments were performed on the optic nerve, a myelinated structure very rich in 5alpha-R activity and the results clearly indicated the presence of a specific type 1 enzyme immunoreactivity in the myelin sheaths of axons.

LHRH synthesis and release are modulated in vivo by gonadal steroids. Although immunocytochemical and autoradiographic studies failed to detect appreciable amounts of estrogen or androgen receptor in LHRH-producing neurons, the recent finding that the promoter region of the LHRH gene contains several steroid hormone-responsive elements indicates a possible direct effect of sex steroids on these specialized neurons. The immortalized LHRH-producing neuronal cell line, GT1, which became recently available, may allow the study of LHRH dynamics. The presence of specific binding sites for estrogen and androgens as well as the presence of the two major enzymatic pathways involved in modulation of androgen action (the 5 alpha-reductase/3 alpha-hydroxysteroid dehydrogenase and the aromatase) have been studied in the GT1-1 clone. High affinity, low capacity binding sites for [3H]estradiol (Kd, 0.11 nM; binding capacity, 6.2 fmol/mg protein) and for a ligand of the androgen receptor, [3H]R1881 (Kd, 0.054 nM; binding capacity, 9.58 fmol/mg protein), have been identified in this cell line. A 2-fold induction of androgen-binding sites has been observed after 3 days of treatment of GT1-1 cells with estradiol (1 microM), indicating that the estradiol binding is probably linked to a functional estrogen receptor. Aromatase and 5 alpha-reductase/3 alpha-hydroxysteroid dehydrogenase activities have been also tested in GT1-1 cells. Under the culture conditions adopted, no detectable aromatization of [1 beta 3H]delta 4-androstenedione to estrone was observed using the tritiated water method. On the other hand, GT1-1 cells efficiently converted testosterone into dihydrotestosterone and subsequently into 5 alpha-androstan-3 alpha,17 beta-diol. In conclusion, GT1-1 cells possess several elements of the machinery through which sex steroids may influence LHRH dynamics.

The central nervous system (CNS) is considered a target structure for the action of all the classes of hormonal steroids produced by the organism. Well-characterized genomic and less well-understood membrane mechanisms of action are probably involved in the steroid modulation of brain activities. Moreover, some classes of steroids need to be converted into "active" metabolites before interacting with their effector systems. In particular, testosterone (T) exerts many of its effects after conversion to 5 alpha-dihydrotestosterone (DHT) and estrogens. The CNS possesses both the 5 alpha-reductase, the enzyme which produces DHT and the aromatase which transforms T into estrogens; however, the relative role and distribution of these enzymes in the various structural components of the CNS has not been clarified so far. The 5 alpha-reductase has been found to be present in high concentrations in brain white matter structures because these are particularly rich in myelin membranes, to which the enzymatic activity appears to be associated. This membrane localization might suggest a possible involvement of steroidal 5 alpha-reduced metabolites in membrane-mediated events in the CNS. Moreover, the distribution of 5 alpha-reductase was studied in neurons, astrocytes and oligodendrocytes isolated from the brain of male rats by density gradient ultracentrifugation, as well as in neurons and glial cells grown in culture. The aromatase activity was also evaluated in neurons and glial cells grown in culture and in isolated oligodendrocytes. Among the three cell types isolated, neurons appear to be more active than oligodendrocytes and astrocytes, respectively, in converting T into DHT. Also, in cell culture experiments, neurons are more active in forming DHT than glial cells. Only neurons possess aromatase activity, while glial cells are apparently unable to aromatize T.

In the central nervous system of the rat, the 5 alpha-reductase, the enzyme which converts testosterone into dihydrotestosterone, appears to be concentrated in the white matter and in particular to be associated with myelin. In order to verify whether a temporal correlation might exist between the formation of myelin membranes and the variations of the 5 alpha-reductase activity observed in the brain, the enzymatic activity was studied in the cerebral cortex and in the hypothalamus of male rat in the age range of 3-60 days, in myelin purified from animals of 15-60 days of life and in oligodendrocytes (i.e. in the cells responsible for the formation of the myelin) isolated from the brain of adult and very young rats (7th day of life, when the myelination process is not yet initiated). The results show that the formation of 5 alpha-androstane-17 beta-ol-3-one (DHT) in the cerebral cortex and in the hypothalamus has a peak activity in the first two weeks of life, before the beginning of the myelination process; purified myelin has an enzymatic activity always much higher than that present in the cerebral cortex and in the hypothalamus and shows a peak in the formation of DHT in the first period of myelinogenesis, on the third week of life. Finally the oligodendrocytes of young rats possess a much higher ability to convert testosterone into the 5 alpha-reduced metabolites than the oligodendrocytes of adult animals. A possible involvement of this enzyme in the myelin function may be hypothesized.

Maca is the edible root of the Peruvian plant Lepidum meyenii, traditionally employed for its purported aphrodisiac and fertility-enhancing properties. This study aimed at testing the hypothesis that Maca contains testosterone-like compounds, able to bind the human androgen receptor and promote transcription pathways regulated by steroid hormone signaling. Maca extracts (obtained with different solvents: methanol, ethanol, hexane and chloroform) are not able to regulate GRE (glucocorticoid response element) activation. Further experiments are needed to assess which compound, of the several Maca's components, is responsible of the observed in vivo effects.

Previous reports from this laboratory indicate that the 5 alpha-reductase, the enzyme which converts testosterone into its "active" metabolite 5 alpha-androstan-17 beta-ol-3-one (dihydrotestosterone, DHT) is highly concentrated in the white matter structures of the CNS, which are mainly composed of myelinated fibers. No studies have been performed up to now, in order to evaluate the possible presence of the 5 alpha-reductase activity in peripheral myelinated nerves. To this purpose the 5 alpha-reductase activity has been evaluated in the sciatic nerve of the rat and compared to that present in the cerebral cortex and in the subcortical white matter, a central structure mainly composed of myelinated fibers. The study has been performed in normal adult male rats (60-90-day-old) and in aged (20-month-old) animals. The data obtained in 60-90-day-old animals indicate the presence of an active metabolism of testosterone at the level of the sciatic nerve. In this structure, testosterone is actively transformed into DHT and 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol); in the sciatic nerve, the formation of DHT is equal to that found in the subcortical white matter and higher than that found in the cerebral cortex. Moreover, at variance with what happens in CNS structures, where 3 alpha-diol is produced only in small amounts, in the sciatic nerve this metabolite is produced in amounts similar to those of DHT. The study in aged rats has shown that in the sciatic nerve, the formation of DHT and particularly that of 3 alpha-diol are much lower than in younger animals. No age-related variations in the 5 alpha-reductase activity in the cerebral cortex and in the subcortical white matter have been observed.

The molecular mechanism of the presynaptic toxicity of secreted phospholipase A2 (sPLA2) neurotoxins, including that of ammodytoxin A (AtxA), has not been resolved. Here we report the action of AtxA on mouse motoneuron-like cells, on which it induced characteristic neurotoxic effects on synaptic vesicles and on the reorganization of F-actin. AtxA also released fatty acids from the plasmalemma. Its significantly less neurotoxic V31W mutant showed similar effects on cells but with a much higher rate of hydrolysis than the wild-type, indicating that high enzymatic activity alone is not sufficient for the observed effects. The neurotoxic action was observed by confocal microscopy of a fluorescently labelled AtxA and by electron microscopy of a nanogold-labelled toxin. The Atx-binding proteins were tagged by a photo-cross-linking reagent conjugated to the toxin. AtxA was taken up rapidly by the cells, where it interacted within minutes with calmodulin and 14-3-3 proteins in the cytosol. These data demonstrate, for the first time, the translocation of an sPLA2 from the extracellular space into the cytosol of a cell. Such an event may thus be important in explaining the action of a range of homologous endogenous sPLA2 enzymes in mammals whose roles in various cellular processes are not yet completely understood.

ALS (amyotrophic lateral sclerosis), a fatal motoneuron (motor neuron) disease, occurs in clinically indistinguishable sporadic (sALS) or familial (fALS) forms. Most fALS-related mutant proteins identified so far are prone to misfolding, and must be degraded in order to protect motoneurons from their toxicity. This process, mediated by molecular chaperones, requires proteasome or autophagic systems. Motoneurons are particularly sensitive to misfolded protein toxicity, but other cell types such as the muscle cells could also be affected. Muscle-restricted expression of the fALS protein mutSOD1 (mutant superoxide dismutase 1) induces muscle atrophy and motoneuron death. We found that several genes have an altered expression in muscles of transgenic ALS mice at different stages of disease. MyoD, myogenin, atrogin-1, TGFβ1 (transforming growth factor β1) and components of the cell response to proteotoxicity [HSPB8 (heat shock 22kDa protein 8), Bag3 (Bcl-2-associated athanogene 3) and p62] are all up-regulated by mutSOD1 in skeletal muscle. When we compared the potential mutSOD1 toxicity in motoneuron (NSC34) and muscle (C2C12) cells, we found that muscle ALS models possess much higher chymotryptic proteasome activity and autophagy power than motoneuron ALS models. As a result, mutSOD1 molecular behaviour was found to be very different. MutSOD1 clearance was found to be much higher in muscle than in motoneurons. MutSOD1 aggregated and impaired proteasomes only in motoneurons, which were particularly sensitive to superoxide-induced oxidative stress. Moreover, in muscle cells, mutSOD1 was found to be soluble even after proteasome inhibition. This effect could be associated with a higher mutSOD1 autophagic clearance. Therefore muscle cells seem to manage misfolded mutSOD1 more efficiently than motoneurons, thus mutSOD1 toxicity in muscle may not directly depend on aggregation.

MNDs (motorneuron diseases) are neurodegenerative disorders in which motorneurons located in the motor cortex, in the brainstem and in the spinal cord are affected. These diseases in their inherited or sporadic forms are mainly characterized by motor dysfunctions, occasionally associated with cognitive and behavioural alterations. Although these diseases show high variability in onset, progression and clinical symptoms, they share common pathological features, and motorneuronal loss invariably leads to muscle weakness and atrophy. One of the most relevant aspect of these disorders is the occurrence of defects in axonal transport, which have been postulated to be either a direct cause, or a consequence, of motorneuron degeneration. In fact, due to their peculiar morphology and high energetic metabolism, motorneurons deeply rely on efficient axonal transport processes. Dysfunction of axonal transport is known to adversely affect motorneuronal metabolism, inducing progressive degeneration and cell death. In this regard, the understanding of the fine mechanisms at the basis of the axonal transport process and of their possible alterations may help shed light on MND pathological processes. In the present review, we will summarize what is currently known about the alterations of axonal transport found to be either causative or a consequence of MNDs.

Amyotrophic Lateral Sclerosis (ALS) is the most common form of adult-onset motor neuron disease. It is now considered a multi-factorial and multi-systemic disorder in which alterations of the crosstalk between neuronal and non-neuronal cell types might influence the course of the disease. In this review, we will provide evidence that dysfunctions of affected muscle cells are not only a marginal consequence of denervation associated to motor neurons loss, but a direct consequence of cell muscle toxicity of mutant SOD1. In muscle, the misfolded state of mutant SOD1 protein, unlike in motor neurons, does not appear to have direct effects on protein aggregation and mitochondrial functionality. Muscle cells are, in fact, more capable than motor neurons to handle misfolded proteins, suggesting that mutant SOD1 toxicity in muscle is not mediated by classical mechanisms of intracellular misfolded proteins accumulation. Several recent works indicate that a higher activation of molecular chaperones and degradative systems is present in muscle cells, which for this reason are possibly able to better manage misfolded mutant SOD1. However, several alterations in gene expression and regenerative potential of skeletal muscles have also been reported as a consequence of the expression of mutant SOD1 in muscle. Whether these changes in muscle cells are causative of ALS or a consequence of motor neuron alterations is not yet clear, but their elucidation is very important, since the understanding of the mechanisms involved in mutant SOD1 toxicity in muscle may facilitate the design of treatments directed toward this specific tissue to treat ALS or at least to delay disease progression.

Abstract Several of the identified genetic factors in Amyotrophic Lateral Sclerosis (ALS) point to dysfunction in RNA processing as a major pathogenic mechanism. However, whether a precise RNA pathway is particularly affected remains unknown. Evidence suggests that FUS, that is mutated in familial ALS, and SMN, the causative factor in Spinal Muscular Atrophy (SMA), cooperate to the same molecular pathway, i.e. regulation of alternative splicing, and that disturbances in SMN-regulated functions, either caused by depletion of SMN protein (as in the case of SMA) or by pathogenic interactions between FUS and SMN (as in the case of ALS) might be a common theme in both diseases. In this work, we followed these leads and tested their pathogenic relevance in vivo . FUS-associated ALS recapitulates, in transgenic mice, crucial molecular features that characterise mouse models of SMA, including defects in snRNPs distribution and in the alternative splicing of genes important for motor neurons. Notably, altering SMN levels by haploinsufficiency or overexpression does not impact the phenotypes of mouse or Drosophila models of FUS-mediated toxicity. Overall, these findings suggest that FUS and SMN functionally interact and that FUS may act downstream of SMN-regulated snRNP assembly in the regulation of alternative splicing and gene expression.

Misfolding protein diseases are a wide class of disorders in which the aberrantly folded protein aggregate accumulating in affected cells. In the brain and in the skeletal muscle, misfolded protein accumulation induces a variety of cell dysfunctions that frequently lead to cell death. In motoneuron diseases (MNDs), misfolded proteins accumulate primarily in motoneurons, glial cells and/or skeletal muscle cells, altering motor function. The deleterious effects of misfolded proteins can be counteracted by the activity of the protein quality control (PQC) system, composed of chaperone proteins and degradative systems. Here, we focus on the PQC system components: heat shock protein family B (small) member 8 (HSPB8), a chaperone induced by harmful stressful events, including proteotoxicity. In motoneuron and muscle cells, misfolded proteins activate HSPB8 transcription and enhance HSPB8 levels, which contributes to prevent aggregate formation and their harmful effects. HSPB8 acts not only as chaperone, but also facilitates the autophagy process, to enable the efficient clearance of the misfolded proteins. HSPB8 acts as a dimer bound to the HSP70 co-chaperone BAG3, a scaffold protein that is also capable of binding to HSP70 (associated with the E3-ligase CHIP) and dynein. When this complex is formed, it is transported by dynein to the microtubule organization center (MTOC), where aggresomes are formed. Here, misfolded proteins are engulfed into nascent autophagosomes to be degraded via the chaperone-assisted selective autophagy (CASA). When CASA is insufficient or impaired, HSP70 and CHIP, associate with an alternative co-chaperone, BAG1, which routes misfolded proteins to the proteasome for degradation. The finely-tuned equilibrium between proteasome and CASA activity is thought to be crucial for maintaining the functional cell homeostasis during proteotoxic stresses, which in turn is essential for cell survival. This fine equilibrium seems to be altered in MNDs, like Amyotrophic lateral sclerosis (ALS) and spinal and bulbar muscular atrophy (SBMA), contributing to the onset and the progression of disease. Here, we will review how misfolded proteins may affect the PQC system, and how the proper activity of this system can be restored by boosting or regulating HSPB8 activity, with the aim to ameliorate disease progression in these two fatal MNDs.

Myofibrillar myopathies (MFMs) are genetically heterogeneous dystrophies characterized by the disintegration of Z-disks and myofibrils and are associated with mutations in genes encoding Z-disk or Z-disk-related proteins. The c.626 C > T (p.P209L) mutation in the BAG3 gene has been described as causative of a subtype of MFM. We report a sporadic case of a 26-year-old Italian woman, affected by MFM with axonal neuropathy, cardiomyopathy, rigid spine, who carries the c.626 C > T mutation in the BAG3 gene. The patient and her non-consanguineous healthy parents and brother were studied with whole exome sequencing (WES) to further investigate the genetic basis of this complex phenotype. In the patient, we found that the BAG3 mutation is associated with variants in the NRAP and FHL1 genes that encode muscle-specific, LIM domain containing proteins. Quantitative real time PCR, immunohistochemistry and Western blot analysis of the patient's muscular biopsy showed the absence of NRAP expression and FHL1 accumulation in aggregates in the affected skeletal muscle tissue. Molecular dynamic analysis of the mutated FHL1 domain showed a modification in its surface charge, which could affect its capability to bind its target proteins. To our knowledge this is the first study reporting, in a BAG3 MFM, the simultaneous presence of genetic variants in the BAG3 and FHL1 genes (previously described as independently associated with MFMs) and linking the NRAP gene to MFM for the first time.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease (MND) caused by a mutant androgen receptor (AR) containing an elongated polyglutamine (polyQ) tract. ARpolyQ toxicity is triggered by androgenic AR ligands, which induce aberrant conformations (misfolding) of the ARpolyQ protein that aggregates. Misfolded proteins perturb the protein quality control (PQC) system leading to cell dysfunction and death. Spinal cord motoneurons, dorsal root ganglia neurons and skeletal muscle cells are affected by ARpolyQ toxicity. Here, we found that, in stabilized skeletal myoblasts (s-myoblasts), ARpolyQ formed testosterone-inducible aggregates resistant to NP-40 solubilization; these aggregates did not affect s-myoblasts survival or viability. Both wild type AR and ARpolyQ were processed via proteasome, but ARpolyQ triggered (and it was also cleared via) autophagy. ARpolyQ reduced two pro-autophagic proteins expression (BAG3 and VCP), leading to decreased autophagic response in ARpolyQ s-myoblasts. Overexpression of two components of the chaperone assisted selective autophagy (CASA) complex (BAG3 and HSPB8), enhanced ARpolyQ clearance, while the treatment with the mTOR independent autophagy activator trehalose induced complete ARpolyQ degradation. Thus, trehalose has beneficial effects in SBMA skeletal muscle models even when autophagy is impaired, possibly by stimulating CASA to assist the removal of ARpolyQ misfolded species/aggregates.

Motor neuron diseases (MNDs) are neurodegenerative disorders characterized by upper and/or lower MN loss. MNDs include amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and spinal and bulbar muscular atrophy (SBMA). Despite variability in onset, progression, and genetics, they share a common skeletal muscle involvement, suggesting that it could be a primary site for MND pathogenesis. Due to the key role of muscle-specific microRNAs (myomiRs) in skeletal muscle development, by real-time PCR we investigated the expression of miR-206, miR-133a, miR-133b, and miR-1, and their target genes, in G93A-SOD1 ALS, Δ7SMA, and KI-SBMA mouse muscle during disease progression. Further, we analyzed their expression in serum of SOD1-mutated ALS, SMA, and SBMA patients, to demonstrate myomiR role as noninvasive biomarkers. Our data showed a dysregulation of myomiRs and their targets, in ALS, SMA, and SBMA mice, revealing a common pathogenic feature associated with muscle impairment. A similar myomiR signature was observed in patients’ sera. In particular, an up-regulation of miR-206 was identified in both mouse muscle and serum of human patients. Our overall findings highlight the role of myomiRs as promising biomarkers in ALS, SMA, and SBMA. Further investigations are needed to explore the potential of myomiRs as therapeutic targets for MND treatment.

Mitochondria alterations are present in tissues derived from patients and animal models, but no data are available for peripheral blood mononuclear cells (PBMCs) of ALS patients. This work aims to investigate mitophagy in PBMCs of sporadic (sALS) patients and how this pathway can be tuned by using small molecules. We found the presence of morphologically atypical mitochondria by TEM and morphological abnormalities by MitoTracker™. We found a decreased number of healthy mitochondria in sALS PBMCs and an impairment of mitophagy with western blot and immunofluorescence. After rapamycin treatment, we found a higher increase in the LC3 marker in sALS PBMCs, while after NH4Cl treatment, we found a lower increase in the LC3 marker. Finally, mTOR-independent autophagy induction with trehalose resulted in a significant decrease in the lysosomes level sALS PBMCs. Our data suggest that the presence of morphologically altered mitochondria and an inefficient turnover of damaged mitochondria in PBMCs of sALS patients rely on the impairment of the mitophagy pathway. We also found that the induction of the mTOR-independent autophagy pathway leads to a decrease in lysosomes level, suggesting a more sensitivity of sALS PBMCs to trehalose. Such evidence suggests that trehalose could represent an effective treatment for ALS patients.

Four phosphorylation sites have been identified in the chicken progesterone receptor. Two of these sites exhibit basal phosphorylation which is enhanced upon treatment with hormone and two of the sites are phosphorylated in response to hormone. Mutation of one of these hormone dependent sites, Ser530 to Ala530, causes a decrease in transcriptional activation at low concentrations of hormone, but the activity is unaffected at high concentrations. However, the hormone binding of the mutant is unaffected suggesting that phosphorylation of Ser530 plays a role in facilitating the response of the receptor to low concentrations of hormone. The chicken progesterone receptor can be activated by modulators of kinases in the absence of hormone. The finding that signals initiated by tyrosine phosphorylation (through treatment with EGF) or through the dopamine receptor suggests that there are multiple means of activating chicken progesterone receptor. In contrast, the human progesterone receptor does not exhibit ligand independent activation; however, its activity in the presence of the agonist R5020 is enhanced by treatment with 8-Br-cAMP, an activator of protein kinase A, and treatment with 8-Br-cAMP causes the antagonist, RU486, to act as an agonist.

Neuritin is a small, highly conserved GPI-anchored protein involved in neurite outgrowth. We have analyzed the involvement of neuritin in NGF-induced differentiation of PC12 cells by investigating the time-course of neuritin expression, the effects of its overexpression or silencing, and the possible mechanisms of its regulation and action. Real-time PCR analysis has shown that neuritin gene is upregulated by NGF in PC12 cells hours before neurite outgrowth becomes appreciable. PC12 cells transfected with a plasmid expressing neuritin display a significant increase in the response to NGF: 1) in the levels of SMI312 positive phosphorylated neurofilament proteins (markers for axonal processes) and tyrosine hydroxylase; 2) in the percentage of cells bearing neurites; as well as 3) in the average length of neurites when compared to control cells. On the contrary, neuritin silencing significantly reduces neurite outgrowth. These data suggest that neuritin is a modulator of NGF-induced neurite extension in PC12 cells. We also showed that neuritin potentiated the NGF-induced differentiation of PC12 cells without affecting TrkA or EGF receptor mRNAs expression. Moreover, the S-methylisothiourea (MIU), a potent inhibitor of inducible nitric oxide synthases, partially counteracts the NGF-mediated neuritin induction. These data suggest that NGF regulates neuritin expression in PC12 cells via the signaling pathway triggered by NO. This study reports the first evidence that neuritin plays a role in modulating neurite outgrowth during the progression of NGF-induced differentiation of PC12 cells. PC12 cells could be considered a valuable model to unravel the mechanism of action of neuritin on neurite outgrowth. (c) 2007 Wiley-Liss, Inc.

Motor neuron diseases (MNDs) are fatal diseases characterized by loss of motor neurons in the brain cortex, in the bulbar region, and/or in the anterior horns of the spinal cord. While generally sporadic, inherited forms linked to mutant genes encoding altered RNA/protein products have also been described. Several different mechanisms have been found altered or dysfunctional in MNDs, like the protein quality control (PQC) system. In this review, we will discuss how the PQC system is affected in two MNDs-spinal and bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS)-and how this affects the clearance of aberrantly folded proteins, which accumulate in motor neurons, inducing dysfunctions and their death. In addition, we will discuss how the PQC system can be targeted to restore proper cell function, enhancing the survival of affected cells in MNDs.

BACKGROUND The presence and possible role of androgen-metabolizing enzymes in androgen-independent prostate carcinoma (CaP) are still unclear. The aim of the present study was: 1) to evaluate the pattern of androgen metabolism (relative production of 5α-reduced vs. 17-keto androgens); and 2) to analyze whether one or both the two known 5α-reductase isoforms (5α-R1 and 5α-R2) and the aromatase (Aro) are expressed and active in this pathology. METHODS Two different cell lines (DU145 and PC3) were used as a model of androgen-independent human CaP. In these cells, the expression of the two 5α-Rs and of Aro were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot, using specific sets of oligoprimers and of [32P]-labeled oligoprobes; the enzymatic activities of 5α-R and of Aro were evaluated by radioenzymatic methods. The pH optimum for the activity of the two 5α-Rs was assessed in cell homogenates at different pH (from 3.5–8), using substrate concentrations similar either to 5α-R1 or to 5α-R2 Kms. RESULTS The two CaP cell lines DU145 and PC3, although unresponsive to androgens, possess the enzymatic machinery involved in the metabolism of this class of hormonal steroids: 5α-Rs, which allow their transformation into 5α-reduced steroids (5α-dihydrotestosterone, DHT, and 5α-androstandione, 5α-A), and 17β-hydroxysteroid-oxidoreductase (17β-HSD), which interconverts testosterone (T) and androstenedione (ADIONE); however, the two cell lines show differences in the rate of formation of these metabolites. Furthermore, two cell lines expressed the type 1 isoform of 5α-R, but only DU145 cells also possess 5α-R2. Aro is expressed and active in DU145 as well as in PC3 cells. CONCLUSIONS The present findings suggest that T might still be indirectly active in androgen-unresponsive CaP through its local conversion into estrogens by the action of Aro; the biological role played by the two 5α-Rs in androgen-independent CaP deserves further investigation. Prostate 41:224–232, 1999. © 1999 Wiley-Liss, Inc.

Abstract Gonadal steroids are potent modulators of gonadotropin releasing hormone (GnRH) secretion, and androgen binding sites and 5α‐reductase activity have been found in the immortalized GnRH secreting cell line GT1‐1, suggesting the existence of a direct androgenic control of GnRH dynamics. Two isoforms of the 5α‐reductase have been cloned with very different biochemical/functional properties: 5α‐reductase type 1 (widely distributed in the body) and 5α‐reductase type 2 (confined in androgen target structures). We have analysed whether, in GT1‐1, androgen binding sites are linked to ‘classical’ androgen receptor, and which 5α‐reductase isoform is active. Reverse transcriptase‐polymerase chain reaction analysis showed that the mRNAs coding for androgen receptor and for the two 5α‐reductase isoforms are all expressed in GT1‐1 cells. However, the 5α‐reductase enzymatic reaction showed a peak of activity at a narrow pH around 5.5, the optimum for the 5α‐reductase type 2. The affinity for testosterone, of the enzyme present in GT1‐1 cells, was very similar to that observed for the recombinant type 2 isozyme expressed in yeasts. The data indicate that GT1‐1 cells (i) express a ‘classical’ androgen receptor and (ii) contain the 5α‐reductase type 2 isoform, a specific marker of androgen‐responsiveness.