Darren J. Moore

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 ( PINK1 ) and PARK2/Parkin mutations cause autosomal recessive forms of Parkinson's disease. Upon a loss of mitochondrial membrane potential (ΔΨ m ) in human cells, cytosolic Parkin has been reported to be recruited to mitochondria, which is followed by a stimulation of mitochondrial autophagy. Here, we show that the relocation of Parkin to mitochondria induced by a collapse of ΔΨ m relies on PINK1 expression and that overexpression of WT but not of mutated PINK1 causes Parkin translocation to mitochondria, even in cells with normal ΔΨ m . We also show that once at the mitochondria, Parkin is in close proximity to PINK1, but we find no evidence that Parkin catalyzes PINK1 ubiquitination or that PINK1 phosphorylates Parkin. However, co-overexpression of Parkin and PINK1 collapses the normal tubular mitochondrial network into mitochondrial aggregates and/or large perinuclear clusters, many of which are surrounded by autophagic vacuoles. Our results suggest that Parkin, together with PINK1, modulates mitochondrial trafficking, especially to the perinuclear region, a subcellular area associated with autophagy. Thus by impairing this process, mutations in either Parkin or PINK1 may alter mitochondrial turnover which, in turn, may cause the accumulation of defective mitochondria and, ultimately, neurodegeneration in Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder that results primarily from the death of dopaminergic neurons in the substantia nigra. Although the etiology of PD is incompletely understood, the recent discovery of genes associated with rare monogenic forms of the disease, together with earlier studies and new experimental animal models, has provided important and novel insight into the molecular pathways involved in disease pathogenesis. Increasing evidence indicates that deficits in mitochondrial function, oxidative and nitrosative stress, the accumulation of aberrant or misfolded proteins, and ubiquitin-proteasome system dysfunction may represent the principal molecular pathways or events that commonly underlie the pathogenesis of sporadic and familial forms of PD .

Mutations in the leucine-rich repeat kinase 2 gene ( LRRK2 ) cause late-onset Parkinson's disease (PD) with a clinical appearance indistinguishable from idiopathic PD. Initial studies suggest that LRRK2 mutations are the most common yet identified determinant of PD susceptibility, transmitted in an autosomal-dominant mode of inheritance. Herein, we characterize the LRRK2 gene and transcript in human brain and subclone the predominant ORF. Exogenously expressed LRRK2 protein migrates at ≈280 kDa and is present largely in the cytoplasm but also associates with the mitochondrial outer membrane. Familial-linked mutations G2019S or R1441C do not have an obvious effect on protein steady-state levels, turnover, or localization. However, in vitro kinase assays using full-length recombinant LRRK2 reveal an increase in activity caused by familial-linked mutations in both autophosphorylation and the phosphorylation of a generic substrate. These results suggest a gain-of-function mechanism for LRRK2 -linked disease with a central role for kinase activity in the development of PD.

A novel human G protein-coupled receptor named AXOR12, exhibiting 81% homology to the rat orphan receptor GPR54, was cloned from a human brain cDNA library. Heterologous expression of AXOR12 in mammalian cells permitted the identification of three surrogate agonist peptides, all with a common C-terminal amidated motif. High potency agonism, indicative of a cognate ligand, was evident from peptides derived from the gene KiSS-1, the expression of which prevents metastasis in melanoma cells. Quantitative reverse transcriptase-polymerase chain reaction was used to study the expression of AXOR12 and KiSS-1 in a variety of tissues. The highest levels of expression of AXOR12 mRNA were observed in brain, pituitary gland, and placenta. The highest levels ofKiSS-1 gene expression were observed in placenta and brain. A polyclonal antibody raised to the C terminus of AXOR12 was generated and used to show localization of the receptor to neurons in the cerebellum, cerebral cortex, and brainstem. The biological significance of these expression patterns and the nature of the putative cognate ligand for AXOR12 are discussed. A novel human G protein-coupled receptor named AXOR12, exhibiting 81% homology to the rat orphan receptor GPR54, was cloned from a human brain cDNA library. Heterologous expression of AXOR12 in mammalian cells permitted the identification of three surrogate agonist peptides, all with a common C-terminal amidated motif. High potency agonism, indicative of a cognate ligand, was evident from peptides derived from the gene KiSS-1, the expression of which prevents metastasis in melanoma cells. Quantitative reverse transcriptase-polymerase chain reaction was used to study the expression of AXOR12 and KiSS-1 in a variety of tissues. The highest levels of expression of AXOR12 mRNA were observed in brain, pituitary gland, and placenta. The highest levels ofKiSS-1 gene expression were observed in placenta and brain. A polyclonal antibody raised to the C terminus of AXOR12 was generated and used to show localization of the receptor to neurons in the cerebellum, cerebral cortex, and brainstem. The biological significance of these expression patterns and the nature of the putative cognate ligand for AXOR12 are discussed. G protein-coupled receptor reverse transcriptase polymerase chain reaction Chinese hamster ovary neuropeptide FF high pressure liquid chromatography The G protein-coupled receptors (GPCRs)1 form a large family of membrane bound proteins that share a unique structural feature comprising seven transmembrane α-helices. These molecules act as receptors for a diverse range of extracellular signaling molecules including small molecules (amino acids and biogenic amines), lipids, small bioactive peptides, and large polypeptides (1Wilson S. Bergsma D.J. Chambers J.K. Muir A.I. Fantom K.G.M. Ellis C. Murdock P.R. Herrity N.C. Stadel J.M. Br. J. Pharmacol. 1998; 125: 1387-1392Crossref PubMed Scopus (114) Google Scholar). They have been used successfully as drug targets by the pharmaceutical industry for a number of years. Attention has focused on a number of proteins that are known to be GPCRs through structural homology but for which no ligand has been identified: so-called orphan receptors. At the same time as the recent discovery of new GPCRs, there has been a renewed focus on discovering potential novel peptides that may act as endogenous ligands for these receptors. Here, we describe the cloning of a novel human orphan receptor, a class I GPCR with sequence similarity to receptors for the neuropeptide galanin. This receptor was given the name AXOR12 in accordance with its position in a series of receptors identified in our organization. AXOR12 has a high degree of homology to the rat orphan receptor GPR54 (2Lee D.K. Nguyen T. O'Neill G.P. Cheng R. Liu Y. Howard A.D. Coulombe N. Tan C.P. Tang-Nguyen A.T. George S.R. O'Dowd B.F. FEBS Lett. 1999; 446: 103-107Crossref PubMed Scopus (380) Google Scholar) (81% amino acid identity), which suggests that these two receptors may be orthologs. To identify a ligand for AXOR12, we expressed this receptor in mammalian cells and screened the transfected cells in a functional assay against a library rich in known and putative peptide transmitters. Although there was no activity in response to galanin, we identified three peptides that acted as low potency agonists of AXOR12. These peptides were all derived from invertebrates and shared a C-terminal LRF- or LRW-amide motif. During the preparation of this article, a search of patent literature revealed the existence of additional high potency agonists with sequence similarities to the surrogate agonists identified from the screen. These peptides were derived from a precursor known as KiSS-1. The KiSS-1 gene was identified originally as being up-regulated in melanoma cells that have lost the potential to metastasize after microcell-mediated transfer of human chromosome 6 (3Lee J.-H. Miele M.E. Hicks D.J. Phillips K.K. Trent J.M. Weissman B.E. Welch D.R. J. Natl. Cancer Inst. 1996; 88: 1731-1737Crossref PubMed Scopus (809) Google Scholar). Subsequent studies have shown that the exogenous expression ofKiSS-1 in other tumor cell lines also prevents metastasis (4Lee J.-H. Welch D.R. Cancer Res. 1997; 57: 2384-2387PubMed Google Scholar). The mechanism by which this occurs remains largely unknown; however, KiSS-1 has structural features that suggest that it may be the precursor of a secreted peptide with an LRF-amide motif at the C terminus. We synthesized the putative processed products of KiSS-1 and observed that they acted as high potency agonists of AXOR12. To gain insight into the physiological role of this receptor, we used quantitative RT-PCR to localize the mRNA expression of AXOR12 and KiSS-1 in a range of human tissues. We observed high levels of AXOR12 expression in the brain. Further RT-PCR analysis of brain expression revealed a distinctive pattern of mRNA localization that was further explored by immunohistochemistry using an antibody raised to the extreme C-terminal tail of the receptor. The human AXOR12 gene was identified initially within a genomic clone (GenBankTMaccession number AC005379) as five coding exons interrupted by four introns. The full-length cDNA was obtained by a modification of a previously described cDNA capture method (5Shepard A.R. Rae J.L. Nucleic Acids Res. 1997; 25: 3183-3185Crossref PubMed Scopus (30) Google Scholar). Briefly, 5 µg of plasmid DNA from a human brain cDNA library was screened with a biotinylated oligonucleotide (5′-biotin with an 18-atom spacer) corresponding to the 5′-coding region (5′-GATGCGGACCGTGACCAACTTCTAC-3′). Two additional 40-mers (5′-GGAACTCGCTGGTCATCTACGTCATCTGCCGCCACAAGCC-3′ and 5′-ATCGCCAACCTGGCGGCCACGGACGTGACCTTCCTCCTGTG-3′), corresponding to the immediate 5′ and 3′ regions of the biotinylated probe, were also used as blocking oligos. Bacterial colonies from the second round of selection were screened by PCR using AXOR12-specific primers. Five positive clones were identified, and the entire inserts were sequenced on both strands using an ABI sequencer. Two of the sequenced clones were identical to each other and to the full-length AXOR12 cDNA predicted from the genomic DNA sequence. The AXOR12 cDNA was subcloned into the mammalian expression vector pCDN (6Trill J.J. Shatzman A.R. Ganguly S. Curr. Opin. Biotech. 1995; 6: 553-560Crossref PubMed Scopus (94) Google Scholar) as described previously (7Aiyar N. Disa J. Stadel J.M. Lysko P.G. Mol. Cell. Biochem. 1999; 197: 179-185Crossref PubMed Scopus (55) Google Scholar). Transient transfections of HEK293 cells with AXOR12 alone or AXOR12 co-transfected with Gqi5 (a chimeric G protein α-subunit consisting of Gαq with the C-terminal five amino acids substituted with the corresponding amino acids from Gαi2, which facilitates GPCR signaling through phospholipase C) were prepared for functional studies. A Ca2+ mobilization assay using the microtiter plate-based Ca2+ mobilization fluorometric imaging plate reader was performed as described previously (8Chambers J. Ames R.S. Bergsma D. Muir A. Fitzgerald L.R. Hervieu G. Dytko G.M. Foley J.J. Martin J. Liu W.S. Park J. Ellis C. Ganguly S. Konchar S. Cluderay J. Leslie R. Wilson S. Sarau H.M. Nature. 1999; 400: 261-265Crossref PubMed Scopus (460) Google Scholar). HEK293 cells co-transfected with AXOR12 and Gqi5 were screened against a large library of over 1500 known and putative GPCR agonists including all available mammalian neuropeptides as described previously (9Szekeres P.G. Muir A.I. Spinage L.D. Miller J.E. Butler S.I. Smith A. Rennie G.I. Murdock P.R. Fitzgerald L.R. Wu H. McMillan L.J. Guerrera S. Vawter L. Elshourbagy N.A. Mooney J.L. Bergsma D.J. Wilson S. Chambers J.K. J. Biol. Chem. 2000; 275: 20247-20250Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Peptides in this library were tested at a final concentration of >100 nm and other potential small molecule agonists at >1 µm. To obtain mammalian cells stably expressing AXOR12, the cDNA was subcloned into the vector pCD (a derivative of pCDN lacking the gene for neomycin resistance) and co-transfected with pCDN Gqi5 (10Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (599) Google Scholar) into Chinese hamster ovary cells using LipofectAMINE PLUS (Life Technologies, Inc.) at a DNA ratio of 10:1 (CHO/AXOR12:Gqi5 cells). 48 h later the cells were seeded into 96-well plates at 103cells/well and selected with G418 (Life Technologies, Inc.) (400 µg/ml) and in the absence of nucleosides. Doubly selected cells were screened by Northern blotting to confirm AXOR12 expression, and positive clones were screened functionally in the fluorometric imaging plate reader calcium assay. The clone that responded most sensitively to surrogate agonists was used in all future experiments. The peptides KiSS-1-(107–121), KiSS-1-(112–121), KiSS-1-(114–121), NPFF, neuropeptide AF, RF-amide-like peptide-1 and -3, and galanin-like peptide were synthesized by conventional solid-phase techniques using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry (11Chan W.C. White P.D. Fmoc Solid Phase Peptide Synthesis: A Practical Approach. Oxford University Press, Oxford2000Google Scholar), and purification was conducted by preparative reverse phase HPLC. All final products showed a purity of >95% by analytical reverse phase HPLC, and peptide identities were confirmed by electrospray mass spectrometry. The peptides KiSS-1-(58–65), KiSS-1-(68–91), KiSS-1-(68–80), KiSS-1-(68–121), KiSS-1-(96–121), and KiSS-1-(125–144) were prepared by California Peptide Research Inc., CA. Antho-RW-amides I and II, Peptide F1, and galanin were purchased from Bachem. RNA purification and TaqMan RT-PCR analysis of human tissue were performed as described previously (12Sarau H.M. Ames R.S. Chambers J. Ellis C. Elshourbagy N. Foley J.J. Schmidt D.B. Muccitelli R.M. Jenkins O. Murdock P.R. Herrity N.C. Halsey W. Sathe G. Muir A.I. Nuthulaganti P. Dytko G.M. Buckley P.T. Wilson S. Bergsma D.J. Hay D.W.P. Mol. Pharmacol. 1999; 56: 657-663Crossref PubMed Scopus (301) Google Scholar). TaqMan primer and probe sets for AXOR12, KiSS-1, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase were designed using Primer Express V1.0 software (Applied Biosystems). Primer and probe sequences (forward primer, reverse primer, TaqMan probe) were; AXOR12, 5′-TGGCACCCACGCAGCTA-3′, 5′-AGTTGCTGTAGGACATGCAGTGA-3′, 5′-CCGCCTACGCGCTTAAGACCTGG-3′; KiSS-1, 5′-ACTCACTGGTTTCTTGGCAGCT-3′, 5′-CAGAGGCCACCTTTTCTAATGG-3′, 5′-CTTTTCCTCTGTGCCACCCACTTTGG-3′; and glyceraldehyde-3-phosphate dehydrogenase, 5′-CAAGGTCATCCATGACAACTTTG-3′, 5′-GGGCCATCCACAGTCTTCTG-3′, 5′-ACCACAGTCCATGCCATCACTGCCA-3′. The levels of mRNA measured were calculated as copies of mRNA detected per ng of reverse transcribed poly(A)+ RNA. A unique synthetic peptide (CVLGEDNAPL) located at the extreme C terminus of the human AXOR12 receptor sequence, corresponding to amino acids 389–398, was synthesized (Bio Synthesis Inc.). Polyclonal antibodies were produced as described in detail elsewhere (13Moore D. Chambers J. Waldvogel H. Faull R. Emson P. J. Comp. Neurol. 2000; 421: 374-384Crossref PubMed Scopus (124) Google Scholar). In brief, New Zealand White rabbits were injected with a peptide-purified protein derivative of tuberculin conjugate and boosted at regular intervals. Crude bleeds were tested for antibody titer using a standard enzyme-linked immunosorbent assay protocol. AXOR12 antiserum was purified from crude rabbit serum by immunoaffinity chromatography on peptide-coupled Sulfolink™ gel (Pierce). Western blotting was carried out essentially as described elsewhere (13Moore D. Chambers J. Waldvogel H. Faull R. Emson P. J. Comp. Neurol. 2000; 421: 374-384Crossref PubMed Scopus (124) Google Scholar). In brief, membranes were prepared from selected tissue regions of human brain (frontal cortex, hippocampus, and basal ganglia) and CHO AXOR12:Gqi5 cells or nontransfected CHO cells. Protein concentrations were determined using the BCA protein assay kit (Pierce) according to manufacturer instructions. For SDS-polyacrylamide gel electrophoresis, 10 µg of membrane protein was denatured in Laemmli sample buffer (14Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). After electrophoresis, the proteins were blotted onto 0.45-µm nitrocellulose membranes, blocked in 5% milk solution in Tris-buffered saline/0.1% Tween 20, and incubated with affinity-purified AXOR12 antiserum (1:5000). Immunoreactivity was detected and visualized by incubating the membrane with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000, Promega, Madison, WI) followed by chemiluminescent detection (ECL, Amersham Pharmacia Biotech). Controls included pre-absorption of AXOR12 antibody with immunogenic peptide (10 µm) prior to incubation with the blot and omission of the primary antibody. Immunocytochemistry was performed on stably transfected CHO AXOR12:Gqi5 cells. The cells were fixed in 4% paraformaldehyde/PBS for 20 min at 4 °C, permeabilized for 5 min in 0.5% Triton X-100/PBS, and blocked for 30 min in 50% fetal calf serum. The fixed cells were incubated in a 1:250 dilution of affinity-purified AXOR12 antiserum overnight at 4 °C, washed three times in PBS, and incubated for 30 min in anti-rabbit secondary antibody conjugated to fluorescein isothiocyanate. Coverslips were mounted with vectashield containing 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories). Immunohistochemistry was carried out on perfusion-fixed post-mortem human brain tissue obtained from the New Zealand Neurological Foundation Human Brain Bank (University of Auckland) with consent from the University of Auckland Human Subjects Ethics Committee. All tissues used in this study were from cases with no previous history of neurological disorders or abnormalities after thorough pathological examinations. The post-mortem delay from death until tissue fixation ranged from 10 to 18 h. Coronal 50-µm-thick brain sections were cut on a freezing sledge microtome and collected in 0.1 mPBS, pH 7.4. Endogenous peroxidase activity was quenched for 30 min. Sections were pre-incubated with normal goat serum (1%) in buffer A (0.1 m PBS/0.3% (v/v) Triton X-100) for 1 h followed by incubation with affinity-purified AXOR12 antiserum (1:2000) overnight at 4 °C. Sections were then incubated with biotinylated goat anti-rabbit secondary antibody (1:200, Vector Laboratories) for 2 h followed by incubation with ABC reagent (1:200, Vector Laboratories) for 45 min. Sections were visualized using 0.5 mg/ml 3′3-diaminobenzidine as a substrate and 0.03% H2O2. Stained tissue sections were mounted onto microscope slides, air-dried, and coverslipped with Depex (BDH Laboratory Supplies). Controls included pre-absorption of primary antibody with 50 µm peptide antigen overnight at 4 °C prior to incubation in addition to the omission of the primary antibody or the use of pre-immune serum. As part of an ongoing effort to identify ligands for novel human orphan GPCRs, we cloned a novel GPCR, originally identified within a genomic clone, using a cDNA capture method. As shown in Fig.1 this gene, AXOR12, encodes a 398-amino acid protein. TMHMM, a program that uses a hidden Markov model for predicting transmembrane helices (15Sonnhammer E.L.L. von Heijne G. Krogh A. Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology. American Association for Artificial Intelligence Press, Menlo Park, CA1998: 175-182Google Scholar), predicted that the protein contained seven hydrophobic putative transmembrane domains. AXOR12 has sequence homology with class I members of the GPCR superfamily, exhibiting the highest sequence homology (81% amino acid identity) to GPR54, a rat receptor previously characterized as a galanin receptor-like orphan (2Lee D.K. Nguyen T. O'Neill G.P. Cheng R. Liu Y. Howard A.D. Coulombe N. Tan C.P. Tang-Nguyen A.T. George S.R. O'Dowd B.F. FEBS Lett. 1999; 446: 103-107Crossref PubMed Scopus (380) Google Scholar). The human GPCRs to which AXOR12 has closest homology are members of the galanin receptor family, sharing 28, 30, and 30% amino acid identity with human GalR1, GalR2, and GalR3, respectively. We adopted a “reverse pharmacological” strategy (16Stadel J.M. Wilson S. Bergsma D.J. Trends Pharmacol. Sci. 1997; 18: 430-437Abstract Full Text PDF PubMed Scopus (141) Google Scholar) to identify agonists for AXOR12. Thus, HEK293 cells co-transfected with AXOR12 and Gqi5 were challenged with a large library of more than 1500 putative ligands, and responses were measured in a microtiter plate-based (fluorometric imaging plate reader) calcium mobilization assay. We observed that specific responses were elicited by two neuropeptides originally isolated from the sea anemone, Anthopleura elegantissima (antho-RW-amide I (17Graff D. Grimmelikhuijzen C.J. Brain Res. 1988; 442: 354-358Crossref PubMed Scopus (29) Google Scholar) and antho-RW-amide II (18Graff D. Grimmelikhuijzen C.J. FEBS Lett. 1988; 239: 137-140Crossref PubMed Scopus (21) Google Scholar)), and a neuropeptide from the lobster, peptide F1 (19Trimmer B.A. Kobierski L.A. Kravitz E.A. J. Comp. Neurol. 1987; 266: 16-26Crossref PubMed Scopus (125) Google Scholar) (sequences shown in Table I). Further experiments demonstrated that these responses were not dependent upon co-transfection of the recombinant chimeric G protein and were concentration-dependent with EC50 values in the low micromolar range (Fig. 2). For further studies, CHO AXOR12:Gqi5 cells were used. The CHO parental cells did not respond to the peptides studied here, but the transfected cell line responded to the three surrogate agonists (Fig. 4 and TableI). Galanin and galanin-like peptide, when tested at concentrations up to 1 µm, did not activate CHO AXOR12:Gqi5 cells (data not shown). The common feature of all activating peptides is the presence of an amidated LRF or LRW motif at the C terminus. This suggests that the cognate ligand for this receptor is likely to have a similar structure at the C terminus.Table IpEC50 of peptides tested on CHO AXOR12:Gqi5 cells as determined from dose-dependent changes in intracellular Ca2+PeptideSequenceSpeciesMean pEC50S.E.M.Antho-RW-amide IIpESLRW-amideSea anemone5.730.28Antho-RW-amide IpEGLRW-amideSea anemone6.420.28Peptide F1TNRNFLRF-amideLobster5.630.08KiSS-1-(114–121)WNSFGLRF-amideHuman8.030.51KiSS-1-(112–121)YNWNSFGLRF-amideHuman9.300.39KiSS-1-(107–121)KDLPNYNWNSFGLRF-amideHuman8.920.37KiSS-1-(94–121)IPAPQGAVLVQREKDLPNYNWNSFGLRF-amideHuman8.760.43KiSS-1-(68–121)GTSLSPPPESSGSRQQPGLSAPHSRQ—Human8.000.16—IPAPQGAVLVQREKDLPNYNWNSFGLRF-amideKiSS-1-(68–91)GTSLSPPPESSGSRQQPGLSAPHSHuman<4N/AKiSS-1-(68–80)GTSLSPPPESSGSHuman<5N/AKiSS-1-(58–65)PAATLRSHuman<4N/AKiSS-1-(125–144)EAAPGNHGRSAGRGWGAGAGQHuman<4N/AThe peptide sequences and source species of the peptides are shown along with the mean pEC50 and S.E. derived from three experiments. N/A, not applicable. Open table in a new tab Figure 4CHO AXOR12:Gqi5 cells responded to nonmammalian peptides containing the LRF- or LRW-amide motif, but peptides deduced from KiSS-1, containing LRF-amide, were more potent activators as measured by mobilization of intracellular Ca2+. The data are expressed as the change in fluorescent intensity units (FIU) over background and are from a single experiment, representative of three such experiments. Each point was determined in triplicate and is given as a mean. ○, KiSS-1-(112–121); ■, KiSS-1-(94–121); ●, KiSS-1-(107–121); ▪, KiSS-1-(114–121); ▾, Antho-RW-amide I; ▵, Antho-RW-amide II; ▴, peptide F1.View Large Image Figure ViewerDownload (PPT) The peptide sequences and source species of the peptides are shown along with the mean pEC50 and S.E. derived from three experiments. N/A, not applicable. From a search of the patent literature, KiSS-1, a potential ligand for AXOR12, was identified (patent number WO200024890, Takeda Chemical Industries, Ltd). An analysis of the peptide sequence of KiSS-1 showed it to have features typical of secreted neuropeptides including a signal sequence, as predicted by signalP (20Nielsen H. Engelbrecht J. Brunak S. von Heijne G. Protein Eng. 1997; 10: 1-6Crossref PubMed Scopus (4911) Google Scholar), several potential dibasic cleavage sites, and a cleavage/amidation site (Fig.3). This would result in a putative 54-amino acid-secreted peptide product corresponding to residues 68–121 of the full-length KiSS-1. Most interestingly, this contains a C-terminal LRF-amide sequence, as predicted from our studies with nonmammalian peptides. A range of peptides of different lengths were synthesized, corresponding to the C-terminal end of the putative secreted segment of KiSS-1, and were tested in CHO AXOR12:Gqi5 cells using the fluorometric imaging plate reader assay as described above. A comparison of the peptides derived from the putative secreted segment of KiSS-1 (68–121, 94–121, 107–121, 112–121, and 114–121) (Table I) revealed that these peptides were substantially more potent than the nonmammalian LRF- or LRW-amide peptides tested (Fig.4, Table I). Of the four truncated KiSS-1 sequences, the decapeptide 112–121 possessed the highest potency. Further N-terminal deletion of this peptide resulted in nearly a 20-fold drop in functional potency. Chain elongation to the pentadecapeptide 107–121 or the longer fragment 96–121 afforded smaller reductions in potency (Fig. 4). Likewise, elongation to the 54-amino acid peptide corresponding to the entire putative secreted segment of KiSS-1, tested in separate experiments, resulted in a further reduction in potency (Table I). Peptides corresponding to the N terminus of the putative secreted segment and also to putatively nonsecreted segments of KiSS-1 were inactive (pEC50 < 5) (Table I). NPFF, neuropeptide AF, and the RF-amide like peptides active at the type I neuropeptide FF receptor, NPFFR1 (21Hinuma S. Shintani Y. Fukusumi S. Iijima N. Matsumoto Y. Hosoya M. Fujii R. Watanabe T. Kikuchi K. Terao Y. Yano T. Yamamoto T. Kawamata Y. Habata Y. Asada M. Kitada C. Kurokawa T. Onda H. Nishimura O. Tanaka M. Ibata Y. Fujino M. Nat. Cell Biol. 2000; 2: 703-708Crossref PubMed Scopus (498) Google Scholar), were also tested, and all failed to generate a response (pEC50 < 5, data not shown). The calcium mobilization response seen after the activation of AXOR12 when transiently transfected without additional G protein α-subunits into HEK293 cells (Fig. 2) suggests that this receptor is coupled to G proteins of the Gq/11 subfamily. In agreement with this hypothesis, KiSS-1-(112–121) caused identical calcium mobilization in both control and pertussis toxin-treated HEK293 cells transiently expressing AXOR12 (data not shown), suggesting that activation of G proteins from the Gi/o family and subsequent Gβγ-mediated activation of phospholipase Cβ does not contribute to the functional response observed. In addition, neither basal nor forskolin-elevated levels of intracellular cAMP were modulated by KiSS-1-(112–121) in HEK293 cells transiently expressing AXOR12 (data not shown), suggesting that this receptor does not couple strongly to G proteins of the Gs and/or Gi/o subfamilies. To characterize the expression pattern of both the receptor AXOR12 and its putative ligand KiSS-1 we carried out quantitative RT-PCR analysis on a broad range of human tissues. AXOR12 was widely expressed in human tissues including the placenta, brain, and pituitary at high levels (Fig. 5), with lower levels detected in lymphocytes, pancreas, and adipose tissue. Within the human central nervous system, AXOR12 mRNA was widespread in its expression including the amygdala, nucleus accumbens, hippocampus, and cingulate gyrus (Fig. 6). KiSS-1 mRNA was detected predominantly in the placenta (Fig. 5) but was also widespread throughout the central nervous system, although at much lower levels (Fig. 6).Figure 6Localization by quantitative RT-PCR of AXOR12 and KiSS-1 in the human central nervous system together with expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. Bars indicate mean number of copies detected per ng of reverse transcribed poly(A)+ RNA from two male and two female individuals. Error barsindicate standard error.View Large Image Figure ViewerDownload (PPT) To gain a greater understanding of the specific cellular localization and level of protein expression of AXOR12, we generated an antiserum directed against a C-terminal peptide from AXOR12. Western blot analysis of membranes from CHO AXOR12:Gqi5 cells revealed a broad immunoreactive band of ∼75 kDa, which was absent in membranes from untransfected CHO cells (Fig.7 A). Similarly, analysis of human brain membrane proteins revealed a specific band of ∼75 kDa (as well as a larger band at ∼125 kDa) in hippocampus, basal ganglia, and frontal cortex (Fig. 7 B). These immunoreactive bands were competed out by the immunizing peptide (data not shown). In both cases, the 75-kDa protein detected agrees with the predicted size of the native AXOR12 receptor together with a probable degree of glycosylation at extracellular consensus sites (see Fig. 1). The AXOR12 receptor-specific antiserum was used to localize receptor immunoreactivity in CHO AXOR12:Gqi5 cells. Expression of the receptor was detected both at the membrane and within the cells (Fig.7 C), although only a subset of the cells was immunoreactive for AXOR12 (Fig. 7, C and D). Immunohistochemical analysis of human brain sections revealed prominent neuronal expression in the regions sampled including the cerebral cortex, thalamus, pons-medulla, and cerebellum. In the cerebral cortex, AXOR12-specific staining was present on a large number of pyramidal neurones of layers III, V, and VI (Fig.8 A). However, in the basal ganglia (caudate nucleus, putamen, globus pallidus, and substantia nigra), staining was primarily localized to AXOR12-immunoreactive fibers and processes. In the cerebellum AXOR12 was strikingly localized to the surface of the Purkinje cells and their apical dendrites (Fig.8 B) and to a lesser extent the cells of the deep cerebellar nuclei. In the pons medulla AXOR12 immunoreactivity was widespread in a number of nuclei including the raphe nuclei, inferior olive, and hypoglossal nuclei (Fig. 8 C). AXOR12 staining was abolished when the antibody was pre-absorbed with 10 µm immunogenic peptide (Fig. 8, D and E) or when the primary antibody was omitted (data not shown). In this article we describe the cloning of a potential human ortholog of the rat G protein-coupled receptor GPR54, which we term AXOR12. A number of peptides possessing an LRW-amide or an LRF-amide motif at the C terminus are identified as surrogate ligands of AXOR12. The first RF-amide peptide to be described was FMRFamide, a cardioexcitatory neuropeptide isolated from the bivalve molluskMacrocallista nimbosa (22Price D. Greenberg M. Science. 1977; 197: 670-671Crossref PubMed Scopus (917) Google Scholar). FMRFamide and related RF-amides are widespread among invertebrates including Caenorhabditis elegans, which has at least 22 such peptides. These are expressed in the central and peripheral nervous systems and have a diverse range of functions including control of defecation, feeding, and reproduction (23Li C. Kim K. Nelson L.S. Brain Res. 1999; 848: 26-34Crossref PubMed Scopus (180) Google Scholar). More recently, several RF-amides have been discovered in mammalian species: neuropeptides FF and AF (NPFF and neuropeptide AF (NPAF)) (24Yang H.Y. Fratta W. Majane E.A. Costa E. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7757-7761Crossref PubMed Scopus (559) Google Scholar, 25Perry S.J. Huang E.Y.-K. Cronk D. Bagust J. Sharma R. Walker R.J. Wilson S. Burke J.F. FEBS Lett. 1997; 409: 426-430Crossref PubMed Scopus (197) Google Scholar), prolactin releasing peptide (26Hinuma S. Habata Y. Fujii R. Kawamata Y. Hosoya M. Fukusumi S. Kitada C. Masuo Y. Asano T. Matsumoto H. Sekiguchi M. Kurokawa T. Nishimura O. Onda H. Fujino M. Nature. 1998; 393: 272-276Crossref PubMed Scopus (526) Google Scholar), and RF-amide related peptides-1 and -3 (21Hinuma S. Shintani Y. Fukusumi S. Iijima N. Matsumoto Y. Hosoya M. Fujii R. Watanabe T. Kikuchi K. Terao Y. Yano T. Yamamoto T. Kawamata Y. Habata Y. Asada M. Kitada C. Kurokawa T. Onda H. Nishimura O. Tanaka M. Ibata Y. Fujino M. Nat. Cell Biol. 2000; 2: 703-708Crossref PubMed Scopus (498) Google Scholar). Some orphan receptors have been shown to be activated by naturally occurring peptides from lower organisms that have structural similarity to the cognate neuropeptide ligand for these receptors (27Elshourbagy N.A. Ames R.S. Fitzgerald L.R. Foley J.J. Chambers J.K. Szekeres P.G. Evans N.A. Schmidt D.B. Buckley P.T. Dytko G.M. Murdock P.R. Milligan G. Groarke D.A. Tan K.B. Shabon U. Nuthulaganti P. Wang D.Y. Wilson S. Bergsma D.J. Sarau H.M. J. Biol. Chem. 2000; 275: 25965-25971Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). For this reason we screened AXOR12 against a library with a wide diversity of mammalian and nonmammalian peptides. On the basis of our initial screen we reasoned that the natural ligand for AXOR12 was likely to contain a LRF- or LRW-amide motif at its C terminus. KiSS-1, a putative human neuropeptide with an LRF-amide motif identified from the patent literature, was therefore a candidate cognate ligand for this receptor. Structurally, KiSS-1 bears all of the hallmarks of a secreted neuropeptide, with a putative signal sequence and several sites amenable to cleavage including an amidation/cleavage site that would result in a number of amidated peptide fragments of various lengths. We synthesized a number of putative N-terminally truncated products of this peptide and tested them against AXOR12. The results showed that the C-terminal decapeptide from the putative full-length secreted segment of KiSS-1 possessed sub-nanomolar activity at AXOR12. A reduction in chain length to the octapeptide 114–121 resulted in a significant drop in functional activity, and it is postulated that Tyr112 and Asn113 may each play an important role in receptor interaction and activation. Chain elongation resulted in minor decreases in activity, suggesting that the most relevant pharmacophore resides in the C-terminal fragment 112–121. AXOR12 failed to be activated by other RF-amide peptides, NPFF, neuropeptide AF, and RF-amide-like peptide-1 and -3, indicating the selectivity of activity of the KiSS-1 peptides. Expression of KiSS-1 in normal tissues was detected initially only in placenta (3Lee J.-H. Miele M.E. Hicks D.J. Phillips K.K. Trent J.M. Weissman B.E. Welch D.R. J. Natl. Cancer Inst. 1996; 88: 1731-1737Crossref PubMed Scopus (809) Google Scholar). Our own observations have shown that although KiSS-1 mRNA is extremely abundant in the placenta, low levels of message are also found in the brain and more specifically in the hypothalamus and basal ganglia. This distribution pattern is consistent with the mRNA localization of AXOR12, which is also in placenta, several central nervous system regions, and pituitary. Our data on AXOR12 localization broadly correspond to the published data for the putative rat ortholog GPR54 (2Lee D.K. Nguyen T. O'Neill G.P. Cheng R. Liu Y. Howard A.D. Coulombe N. Tan C.P. Tang-Nguyen A.T. George S.R. O'Dowd B.F. FEBS Lett. 1999; 446: 103-107Crossref PubMed Scopus (380) Google Scholar). Immunohistochemical data indicate that expression of the receptor occurs specifically on a number of neuronal cell types in the human central nervous system regions that were examined. Indeed, neuronal localization of AXOR12 in many regions of the cerebral cortex, cerebellum, and medulla fits well with the observed mRNA expression pattern in similar regions of the human central nervous system. The prominent and widespread expression of AXOR12 throughout the central nervous system, especially on a number of projection neurones including pyramidal cells in the cerebral cortex and cerebellar Purkinje cells, indicates that ligands acting on AXOR12 would be able to influence a wide range of central nervous system functions ranging from cognition through to movement and balance. The mapping of AXOR12 to human chromosome 19p13.3 (2Lee D.K. Nguyen T. O'Neill G.P. Cheng R. Liu Y. Howard A.D. Coulombe N. Tan C.P. Tang-Nguyen A.T. George S.R. O'Dowd B.F. FEBS Lett. 1999; 446: 103-107Crossref PubMed Scopus (380) Google Scholar) corresponds with a number of inherited neurological diseases including familial febrile convulsions (28Johnson E.W. Dubovsky J. Rich S.S. O'Donovan C.A. Orr H.T. Anderson V.E. Gil-Nagel A. Ahmann P. Dokken C.G. Schneider D.T. Weber J.L. Hum. Mol. Genet. 1998; 7: 63-67Crossref PubMed Scopus (157) Google Scholar), vacuolar neuromyopathy (29Servidei S. Capon F. Spinazzola A. Mirabella M. Semprini S. de Rosa G. Gennarelli M. Sangiuolo F. Ricci E. Mohrenweiser H.W. Dallapiccola B. Tonali P. Novelli G. Neurology. 1999; 53: 830-837Crossref PubMed Google Scholar), and cayman-type cerebellar ataxia (30Nystuen A. Benke P.J. Merren J. Stone E.M. Sheffield V.C. Hum. Mol. Genet. 1996; 5: 525-531Crossref PubMed Scopus (80) Google Scholar). Furthermore, the syntenic region on mouse chromosome 10 is associated with the allelic mutationsjittery and hesitant, which have neuropathic phenotypes characterized by ataxia, dystonia, and seizures (31Kapfhamer D. Sweet H.O. Sufalko D. Warren S. Johnson K.R. Burmeister M. Genomics. 1996; 35: 533-538Crossref PubMed Scopus (22) Google Scholar). The KiSS-1 peptide was identified originally as being differentially up-regulated in C8161 melanoma cells that have been rendered nonmetastatic by microcell-mediated transfer of human chromosome 6 (3Lee J.-H. Miele M.E. Hicks D.J. Phillips K.K. Trent J.M. Weissman B.E. Welch D.R. J. Natl. Cancer Inst. 1996; 88: 1731-1737Crossref PubMed Scopus (809) Google Scholar). Transfection of KiSS-1 into human breast carcinoma cells also prevents these cells from metastasizing (4Lee J.-H. Welch D.R. Cancer Res. 1997; 57: 2384-2387PubMed Google Scholar). The role of AXOR12 in these systems has not been explored, although expressed sequence tags corresponding to AXOR12 have been identified in a number of tumor cDNA libraries (GenBankTM accession numbers AI823800, AI819198, andAA887801). Interestingly, several neuropeptides are known to have functional roles in tumor biology including galanin, vasopressin, cholecystokinin, and neurotensin by autocrine and paracrine mechanisms (32Seufferlein T. Rozengurt E. Cancer Res. 1996; 56: 5758-5764PubMed Google Scholar, 33Ormandy C.J. Lee C.S.L. Ormandy H.F. Fantl V. Shine J. Peters G. Sutherland R.L. Cancer Res. 1998; 58: 1353-1357PubMed Google Scholar). The high levels of expression of both KiSS-1 andAXOR12 in placenta are also noteworthy in this respect. The placenta is also an invasive tissue, and there are similarities in the behavior of invading cancer cells and that of invading placenta cells (34Murray M.J. Lessey B.A. Semin. Reprod. Endocrinol. 1999; 17: 275-290Crossref PubMed Scopus (219) Google Scholar). It is possible that KiSS-1 and AXOR12 may form part of a mechanism that is common to both of these processes. In conclusion, AXOR12 constitutes a new human G protein-coupled receptor that has now been paired with its neuropeptide ligand, KiSS-1. Although there is still much to be discovered about both the receptor and its ligand, the biological evidence thus far suggests that they may have important physiological roles both in the central nervous system and in tumor biology. As such, they represent an intriguing target for novel therapies in the fields of both neurology and oncology.

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

Since the discovery and isolation of α-synuclein (α-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that α-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in α-helical structure (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107-110; Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A., Nguyen, L. T. T., Liao, J., Auclair, J. R., Johnson, D., Landeru, A., Simorellis, A. K., Ju, S., Cookson, M. R., Asturias, F. J., Agar, J. N., Webb, B. N., Kang, C., Ringe, D., Petsko, G. A., Pochapsky, T. C., and Hoang, Q. Q. (2011) Proc. Natl. Acad. Sci. 108, 17797-17802). However, it remains unknown whether or not this putative tetramer is the main physiological form of α-syn in the brain. In this study, we investigated the oligomeric state of α-syn in mouse, rat, and human brains. To assess the conformational and oligomeric state of native α-syn in complex mixtures, we generated α-syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed α-syn to these standards using native and denaturing gel electrophoresis techniques, size-exclusion chromatography, and an oligomer-specific ELISA. Our findings demonstrate that both human and rodent α-syn expressed in the central nervous system exist predominantly as an unfolded monomer. Similar results were observed when human α-syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293, HeLa, and SH-SY5Y). Furthermore, we show that α-syn expressed in E. coli and purified under denaturing or nondenaturing conditions, whether as a free protein or as a fusion construct with GST, is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that α-syn becomes structured upon interaction with other proteins and/or biological membranes.

The PARK8 gene responsible for late-onset autosomal dominant Parkinson's disease encodes a large novel protein of unknown biological function termed leucine-rich repeat kinase 2 (LRRK2). The studies herein explore the localization of LRRK2 in the mammalian brain.Polyclonal antibodies generated against the amino or carboxy termini of LRRK2 were used to examine the biochemical, subcellular, and immunohistochemical distribution of LRRK2.LRRK2 is detected in rat brain as an approximate 280kDa protein by Western blot analysis. Subcellular fractionation demonstrates the presence of LRRK2 in microsomal, synaptic vesicle-enriched and synaptosomal cytosolic fractions from rat brain, as well as the mitochondrial outer membrane. Immunohistochemical analysis of rat and human brain tissue and primary rat cortical neurons, with LRRK2-specific antibodies, shows widespread neuronal-specific labeling localized exclusively to punctate structures within perikarya, dendrites, and axons. Confocal colocalization analysis of primary cortical neurons shows partial yet significant overlap of LRRK2 immunoreactivity with markers specific for mitochondria and lysosomes. Furthermore, ultrastructural analysis in rodent basal ganglia detects LRRK2 immunoreactivity associated with membranous and vesicular intracellular structures, including lysosomes, endosomes, transport vesicles, and mitochondria.The association of LRRK2 with a variety of membrane and vesicular structures, membrane-bound organelles, and microtubules suggests an affinity of LRRK2 for lipids or lipid-associated proteins and may suggest a potential role in the biogenesis and/or regulation of vesicular and membranous intracellular structures within the mammalian brain.

Parkinson's disease (PD) is a disorder of movement, cognition, and emotion, and it is characterized pathologically by neuronal degeneration with Lewy bodies, which are cytoplasmic inclusion bodies containing deposits of aggregated proteins. Most PD cases appear to be sporadic, but genetic forms of the disease, caused by mutations in alpha-synuclein, parkin, and other genes, have helped elucidate pathogenesis. Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant Parkinsonism with clinical features of PD and with pleomorphic pathology including deposits of aggregated protein. To study expression and interactions of LRRK2, we synthesized cDNAs and generated expression constructs coding for human WT and mutant LRRK2 proteins. Expression of full-length LRRK2 in cells in culture suggests that the protein is predominately cytoplasmic, as is endogenous protein by subcellular fractionation. Using coimmunoprecipitation, we find that LRRK2, expressed in cells in culture, interacts with parkin but not with alpha-synuclein, DJ-1, or tau. A small proportion of the cells overexpressing LRRK2 contain protein aggregates, and this proportion is greatly increased by coexpression of parkin. In addition, parkin increases ubiquitination of aggregated protein. Also, mutant LRRK2 causes neuronal degeneration in both SH-SY5Y cells and primary neurons. This cell model may be useful for studies of PD cellular pathogenesis and therapeutics. These findings suggest a gain-of-function mechanism in the pathogenesis of LRRK2-linked PD and suggest that LRRK2 may be involved in a pathogenic pathway with other PD-related proteins such as parkin, which may help illuminate both familial and sporadic PD.

Both homozygous (L166P, M26I, deletion) and heterozygous mutations (D149A, A104T) in the DJ-1 gene have been identified in Parkinson's disease (PD) patients. The biochemical function and subcellular localization of DJ-1 protein have not been clarified. To date the localization of DJ-1 protein has largely been described in studies over-expressing tagged DJ-1 protein in vitro. It is not known whether the subcellular localization of over-expressed DJ-1 protein is identical to that of endogenously expressed DJ-1 protein both in vitro and in vivo. To clarify the subcellular localization and function of DJ-1, we generated three highly specific antibodies to DJ-1 protein and investigated the subcellular localization of endogenous DJ-1 protein in both mouse brain tissues and human neuroblastoma cells. We have found that DJ-1 is widely distributed and is highly expressed in the brain. By cell fractionation and immunogold electron microscopy, we have identified an endogenous pool of DJ-1 in mitochondrial matrix and inter-membrane space. To further investigate whether pathogenic mutations might prevent the distribution of DJ-1 to mitochondria, we generated human neuroblastoma cells stably transfected with wild-type (WT) or mutant (M26I, L166P, A104T, D149A) DJ-1 and performed mitochondrial fractionation and confocal co-localization imaging studies. When compared with WT and other mutants, L166P mutant exhibits largely reduced protein level. However, the pathogenic mutations do not alter the distribution of DJ-1 to mitochondria. Thus, DJ-1 is an integral mitochondrial protein that may have important functions in regulating mitochondrial physiology. Our findings of DJ-1's mitochondrial localization may have important implications for understanding the pathogenesis of PD.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s) through which familial mutations precipitate neuronal degeneration and PD.

J. Neurochem. (2011) 118 , 636–645. Abstract Mutations in the parkin gene cause early‐onset, autosomal recessive Parkinson’s disease. Parkin functions as an E3 ubiquitin ligase to mediate the covalent attachment of ubiquitin monomers or linked chains to protein substrates. Substrate ubiquitination can target proteins for proteasomal degradation or can mediate a number of non‐degradative functions. Parkin has been shown to preserve mitochondrial integrity in a number of experimental systems through the regulation of mitochondrial fission. Upon mitochondrial damage, parkin translocates to mitochondria to mediate their selective elimination by autophagic degradation. The mechanism underlying this process remains unclear. Here, we demonstrate that parkin interacts with and selectively mediates the atypical poly‐ubiquitination of the mitochondrial fusion factor, mitofusin 1, leading to its enhanced turnover by proteasomal degradation. Our data supports a model whereby the translocation of parkin to damaged mitochondria induces the degradation of mitofusins leading to impaired mitochondrial fusion. This process may serve to selectively isolate damaged mitochondria for their removal by autophagy.

Misfolded alpha-synuclein (αSyn) is a major constituent of Lewy bodies and Lewy neurites, which are pathological hallmarks of Parkinson's disease (PD). The contribution of αSyn to PD is well established, but the detailed mechanism remains obscure. Using a model in which αSyn aggregation in primary neurons was seeded by exogenously added, preformed αSyn amyloid fibrils (PFF), we found that a majority of pathogenic αSyn (indicated by serine 129 phosphorylated αSyn, ps-αSyn) was membrane-bound and associated with mitochondria. In contrast, only a minuscule amount of physiological αSyn was mitochondrial bound. In vitro, αSyn PFF displayed a stronger binding to purified mitochondria than did αSyn monomer, revealing a preferential mitochondria binding by aggregated αSyn. This selective mitochondrial ps-αSyn accumulation was confirmed in other neuronal and animal αSyn aggregation models that do not require exogenously added PFF and, more importantly, in postmortem brain tissues of patients suffering from PD and other neurodegenerative diseases with αSyn aggregation (α-synucleinopathies). We also showed that the mitochondrial ps-αSyn accumulation was accompanied by defects in cellular respiration in primary neurons, suggesting a link to mitochondrial dysfunction. Together, our results show that, contrary to physiological αSyn, pathogenic αSyn aggregates preferentially bind to mitochondria, indicating mitochondrial dysfunction as the common downstream mechanism for α-synucleinopathies. Our findings suggest a plausible model explaining the formation and the peculiar morphology of Lewy body and reveal that disrupting the interaction between ps-αSyn and the mitochondria is a therapeutic target for α-synucleinopathies.

The identification of rare monogenic forms of Parkinson's disease (PD) has provided tremendous insight into the molecular pathogenesis of this disorder. Heritable mutations in alpha-synuclein, parkin, DJ-1 and PINK1 cause familial forms of PD. In the more common sporadic form of PD, oxidative stress and derangements in mitochondrial complex-I function are considered to play a prominent role in disease pathogenesis. However, the relationship of DJ-1 with other PD-linked genes and oxidative stress has not been explored. Here, we show that pathogenic mutant forms of DJ-1 specifically but differentially associate with parkin, an E3 ubiquitin ligase. Chemical cross-linking shows that pathogenic DJ-1 mutants exhibit impairments in homo-dimer formation, suggesting that parkin may bind to monomeric DJ-1. Parkin fails to specifically ubiquitinate and enhance the degradation of L166P and M26I mutant DJ-1, but instead promotes their stability in cultured cells. The interaction of parkin with L166P DJ-1 may involve a larger protein complex that contains CHIP and Hsp70, perhaps accounting for the lack of parkin-mediated ubiquitination. Oxidative stress also promotes an interaction between DJ-1 and parkin, but this does not result in the ubiquitination or degradation of DJ-1. Parkin-mediated alterations in DJ-1 protein stability may be pathogenically relevant as DJ-1 levels are dramatically increased in the detergent-insoluble fraction from sporadic PD/DLB brains, but are reduced in the insoluble fraction from parkin-linked autosomal recessive juvenile-onset PD brains. These data potentially link DJ-1 and parkin in a common molecular pathway at multiple levels that may have important implications for understanding the pathogenesis of inherited and sporadic PD.

The identification of genetic mutations responsible for rare familial forms of Parkinson's disease (PD) have provided tremendous insight into the molecular pathogenesis of this disorder. Mutations in the DJ-1 gene cause autosomal recessive early onset PD in two European families. A Dutch kindred displays a large homozygous genomic deletion encompassing exons 1-5 of the DJ-1 gene, whereas an Italian kindred harbors a single homozygous L166P missense mutation. A homozygous M26I missense mutation was also recently reported in an Ashkenazi Jewish patient with early onset PD. Mutations in DJ-1 are predicted to be loss of function. The recent determination of the crystal structure of human DJ-1 demonstrates that it exists in a homo-dimeric form in vitro, whereas the L166P mutant exists only as a monomer. Here, we examine the in vivo effects of the pathogenic L166P and M26I mutations on the properties of DJ-1 in cell culture. We report that the L166P mutation confers markedly reduced protein stability to DJ-1, which results from enhanced degradation by the 20S/26S proteasome but not from a loss of mRNA expression. Furthermore, the L166P mutant protein exhibits an impaired ability to self-interact to form homo-oligomers. In contrast, the M26I mutation does not appear to adversely affect either protein stability, turnover by the proteasome, or the capacity of DJ-1 to form homo-oligomers. These properties of the L166P mutation may contribute to the loss of normal DJ-1 function and are likely to be the underlying cause of early onset PD in affected members of the Italian kindred.

Mutation in leucine-rich repeat kinase-2 (LRRK2) is the most common cause of late-onset Parkinson's disease (PD). Although most cases of PD are sporadic, some are inherited, including those caused by LRRK2 mutations. Because these mutations may be associated with a toxic gain of function, controlling the expression of LRRK2 may decrease its cytotoxicity. Here we show that the carboxyl terminus of HSP70-interacting protein (CHIP) binds, ubiquitinates, and promotes the ubiquitin proteasomal degradation of LRRK2. Overexpression of CHIP protects against and knockdown of CHIP exacerbates toxicity mediated by mutant LRRK2. Moreover, HSP90 forms a complex with LRRK2, and inhibition of HSP90 chaperone activity by 17AAG leads to proteasomal degradation of LRRK2, resulting in increased cell viability. Thus, increasing CHIP E3 ligase activity and blocking HSP90 chaperone activity can prevent the deleterious effects of LRRK2. These findings point to potential treatment options for LRRK2-associated PD.

Parkinson's disease (PD), a progressive neurodegenerative disease characterized by bradykinesia, rigidity, and resting tremor, is the most common neurodegenerative movement disorder. Although the majority of PD cases are sporadic, some are inherited, including those caused by leucine-rich repeat kinase 2 (LRRK2) mutations. The substitution of serine for glycine at position 2019 (G2019S) in the kinase domain of LRRK2 represents the most prevalent genetic mutation in both familial and apparently sporadic cases of PD. Because mutations in LRRK2 are likely associated with a toxic gain of function, destabilization of LRRK2 may be a novel way to limit its detrimental effects. Here we show that LRRK2 forms a complex with heat shock protein 90 (Hsp90) in vivo and that inhibition of Hsp90 disrupts the association of Hsp90 with LRRK2 and leads to proteasomal degradation of LRRK2. Hsp90 inhibitors may therefore limit the mutant LRRK2-elicited toxicity to neurons. As a proof of principle, we show that Hsp90 inhibitors rescue the axon growth retardation caused by overexpression of the LRRK2 G2019S mutation in neurons. Therefore, inhibition of LRRK2 kinase activity can be achieved by blocking Hsp90-mediated chaperone activity and Hsp90 inhibitors may serve as potential anti-PD drugs.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with late-onset, autosomal-dominant, familial Parkinson's disease (PD) and also contribute to sporadic disease. The LRRK2 gene encodes a large protein with multiple domains, including functional Roc GTPase and protein kinase domains. Mutations in LRRK2 most likely cause disease through a toxic gain-of-function mechanism. The expression of human LRRK2 variants in cultured primary neurons induces toxicity that is dependent on intact GTP binding or kinase activities. However, the mechanism(s) underlying LRRK2-induced neuronal toxicity is poorly understood, and the contribution of GTPase and/or kinase activity to LRRK2 pathobiology is not well defined. To explore the pathobiology of LRRK2, we have developed a model of LRRK2 cytotoxicity in the baker's yeast Saccharomyces cerevisiae. Protein domain analysis in this model reveals that expression of GTPase domain-containing fragments of human LRRK2 are toxic. LRRK2 toxicity in yeast can be modulated by altering GTPase activity and is closely associated with defects in endocytic vesicular trafficking and autophagy. These truncated LRRK2 variants induce similar toxicity in both yeast and primary neuronal models and cause similar vesicular defects in yeast as full-length LRRK2 causes in primary neurons. The toxicity induced by truncated LRRK2 variants in yeast acts through a mechanism distinct from toxicity induced by human alpha-synuclein. A genome-wide genetic screen identified modifiers of LRRK2-induced toxicity in yeast including components of vesicular trafficking pathways, which can also modulate the trafficking defects caused by expression of truncated LRRK2 variants. Our results provide insight into the basic pathobiology of LRRK2 and suggest that the GTPase domain may contribute to the toxicity of LRRK2. These findings may guide future therapeutic strategies aimed at attenuating LRRK2-mediated neurodegeneration.

Mutations in the LRRK2 gene cause autosomal dominant, late-onset parkinsonism, which presents with pleomorphic pathology including alpha-synucleopathy. To promote our understanding of the biological role of LRRK2 in the brain we examined the distribution of LRRK2 mRNA and protein in postmortem human brain tissue from normal and neuropathological subjects. In situ hybridization and immunohistochemical analysis demonstrate the expression and localization of LRRK2 to various neuronal populations in brain regions implicated in Parkinson's disease (PD) including the cerebral cortex, caudate-putamen and substantia nigra pars compacta. Immunofluorescent double labeling studies additionally reveal the prominent localization of LRRK2 to cholinergic-, calretinin- and GABA(B) receptor 1-positive, dopamine-innervated, neuronal subtypes in the caudate-putamen. The distribution of LRRK2 in brain tissue from sporadic PD and dementia with Lewy bodies (DLB) subjects was also examined. In PD brains, LRRK2 immunoreactivity localized to nigral neuronal processes is dramatically reduced which reflects the disease-associated loss of dopaminergic neurons in this region. However, surviving nigral neurons occasionally exhibit LRRK2 immunostaining of the halo structure of Lewy bodies. Moreover, LRRK2 immunoreactivity is not associated with Lewy neurites or with cortical Lewy bodies in sporadic PD and DLB brains. These observations indicate that LRRK2 is not a primary component of Lewy bodies and does not co-localize with mature fibrillar alpha-synuclein to a significant extent. The localization of LRRK2 to key neuronal populations throughout the nigrostriatal dopaminergic pathway is consistent with the involvement of LRRK2 in the molecular pathogenesis of familial and sporadic parkinsonism.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset Parkinson's disease (PD). Emerging evidence suggests a role for LRRK2 in the endocytic pathway. Here, we show that LRRK2 is released in extracellular microvesicles (i.e. exosomes) from cells that natively express LRRK2. LRRK2 localizes to collecting duct epithelial cells in the kidney that actively secrete exosomes into urine. Purified urinary exosomes contain LRRK2 protein that is both dimerized and phosphorylated. We provide a quantitative proteomic profile of 1673 proteins in urinary exosomes and find that known LRRK2 interactors including 14-3-3 are some of the most abundant exosome proteins. Disruption of the 14-3-3 LRRK2 interaction with a 14-3-3 inhibitor or through acute LRRK2 kinase inhibition potently blocks LRRK2 release in exosomes, but familial mutations in LRRK2 had no effect on secretion. LRRK2 levels were overall comparable but highly variable in urinary exosomes derived from PD cases and age-matched controls, although very high LRRK2 levels were detected in some PD affected cases. We further characterized LRRK2 exosome release in neurons and macrophages in culture, and found that LRRK2-positive exosomes circulate in cerebral spinal fluid (CSF). Together, these results define a pathway for LRRK2 extracellular release, clarify one function of the LRRK2 14-3-3 interaction and provide a foundation for utilization of LRRK2 as a biomarker in clinical trials.

Mutations in the ATP13A2 gene (PARK9, OMIM 610513) cause autosomal recessive, juvenile-onset Kufor-Rakeb syndrome and early-onset parkinsonism. ATP13A2 is an uncharacterized protein belonging to the P(5)-type ATPase subfamily that is predicted to regulate the membrane transport of cations. The physiological function of ATP13A2 in the mammalian brain is poorly understood. Here, we demonstrate that ATP13A2 is localized to intracellular acidic vesicular compartments in cultured neurons. In the human brain, ATP13A2 is localized to pyramidal neurons within the cerebral cortex and dopaminergic neurons of the substantia nigra. ATP13A2 protein levels are increased in nigral dopaminergic and cortical pyramidal neurons of Parkinson's disease brains compared with normal control brains. ATP13A2 levels are increased in cortical neurons bearing Lewy bodies (LBs) compared with neurons without LBs. Using short hairpin RNA-mediated silencing or overexpression to explore the function of ATP13A2, we find that modulating the expression of ATP13A2 reduces the neurite outgrowth of cultured midbrain dopaminergic neurons. We also find that silencing of ATP13A2 expression in cortical neurons alters the kinetics of intracellular pH in response to cadmium exposure. Furthermore, modulation of ATP13A2 expression leads to reduced intracellular calcium levels in cortical neurons. Finally, we demonstrate that silencing of ATP13A2 expression induces mitochondrial fragmentation in neurons. Oppositely, overexpression of ATP13A2 delays cadmium-induced mitochondrial fragmentation in neurons consistent with a neuroprotective effect. Collectively, this study reveals a number of intriguing neuronal phenotypes due to the loss- or gain-of-function of ATP13A2 that support a role for this protein in regulating intracellular cation homeostasis and neuronal integrity.

Mutations within the leucine-rich repeat kinase 2 (LRRK2) gene account for a significant proportion of autosomal-dominant and some late-onset sporadic Parkinson's disease. Elucidation of LRRK2 protein function in health and disease provides an opportunity for deciphering molecular pathways important in neurodegeneration. In mammals, LRRK1 and LRRK2 protein comprise a unique family encoding a GTPase domain that controls intrinsic kinase activity. The expression profiles of the murine LRRK proteins have not been fully described and insufficiently characterized antibodies have produced conflicting results in the literature.Herein, we comprehensively evaluate twenty-one commercially available antibodies to the LRRK2 protein using mouse LRRK2 and human LRRK2 expression vectors, wild-type and LRRK2-null mouse brain lysates and human brain lysates. Eleven antibodies detect over-expressed human LRRK2 while four antibodies detect endogenous human LRRK2. In contrast, two antibodies recognize over-expressed mouse LRRK2 and one antibody detected endogenous mouse LRRK2. LRRK2 protein resides in both soluble and detergent soluble protein fractions. LRRK2 and the related LRRK1 genes encode low levels of expressed mRNA species corresponding to low levels of protein both during development and in adulthood with largely redundant expression profiles.Despite previously published results, commercially available antibodies generally fail to recognize endogenous mouse LRRK2 protein; however, several antibodies retain the ability to detect over-expressed mouse LRRK2 protein. Over half of the commercially available antibodies tested detect over-expressed human LRRK2 protein and some have sufficient specificity to detect endogenous LRRK2 in human brain. The mammalian LRRK proteins are developmentally regulated in several tissues and coordinated expression suggest possible redundancy in the function between LRRK1 and LRRK2.

Mutations in the vacuolar protein sorting 35 ortholog (VPS35) gene encoding a core component of the retromer complex, have recently emerged as a new cause of late-onset, autosomal dominant familial Parkinson's disease (PD). A single missense mutation, AspD620Asn (D620N), has so far been unambiguously identified to cause PD in multiple individuals and families worldwide. The exact molecular mechanism(s) by which VPS35 mutations induce progressive neurodegeneration in PD are not yet known. Understanding these mechanisms, as well as the perturbed cellular pathways downstream of mutant VPS35, is important for the development of appropriate therapeutic strategies. In this review, we focus on the current knowledge surrounding VPS35 and its role in PD. We provide a critical discussion of the emerging data regarding the mechanisms underlying mutant VPS35-mediated neurodegeneration gleaned from genetic cell and animal models and highlight recent advances that may provide insight into the interplay between VPS35 and several other PD-linked gene products (i.e. α-synuclein, LRRK2 and parkin) in PD. Present data support a role for perturbed VPS35 and retromer function in the pathogenesis of PD.

The G2019S mutation in the leucine-rich repeat kinase 2 (LRRK2) gene is the most common genetic cause of Parkinson's disease (PD), accounting for a significant proportion of both autosomal dominant familial and sporadic PD cases. Our aim in the present study is to generate a mammalian model of mutant G2019S LRRK2 pathogenesis, which reproduces the robust nigral neurodegeneration characteristic of PD. We developed adenoviral vectors to drive neuron-specific expression of full-length wild-type or mutant G2019S human LRRK2 in the nigrostriatal system of adult rats. Wild-type LRRK2 did not induce any significant neuronal loss. In contrast, under the same conditions and levels of expression, G2019S mutant LRRK2 causes a progressive degeneration of nigral dopaminergic neurons. Our data provide a novel rat model of PD, based on a prevalent genetic cause, that reproduces a cardinal feature of the disease within a rapid time frame suitable for testing of neuroprotective strategies.

Mutations in the vacuolar protein sorting 35 homolog (VPS35) gene at the PARK17 locus, encoding a key component of the retromer complex, were recently identified as a new cause of late-onset, autosomal dominant Parkinson's disease (PD). Here we explore the pathogenic consequences of PD-associated mutations in VPS35 using a number of model systems. VPS35 exhibits a broad neuronal distribution throughout the rodent brain, including within the nigrostriatal dopaminergic pathway. In the human brain, VPS35 protein levels and distribution are similar in tissues from control and PD subjects, and VPS35 is not associated with Lewy body pathology. The common D620N missense mutation in VPS35 does not compromise its protein stability or localization to endosomal and lysosomal vesicles, or the vesicular sorting of the retromer cargo, sortilin, SorLA and cation-independent mannose 6-phosphate receptor, in rodent primary neurons or patient-derived human fibroblasts. In yeast we show that PD-linked VPS35 mutations are functional and can normally complement VPS35 null phenotypes suggesting that they do not result in a loss-of-function. In rat primary cortical cultures the overexpression of human VPS35 induces neuronal cell death and increases neuronal vulnerability to PD-relevant cellular stress. In a novel viral-mediated gene transfer rat model, the expression of D620N VPS35 induces the marked degeneration of substantia nigra dopaminergic neurons and axonal pathology, a cardinal pathological hallmark of PD. Collectively, these studies establish that dominant VPS35 mutations lead to neurodegeneration in PD consistent with a gain-of-function mechanism, and support a key role for VPS35 in the development of PD.

Mutations in the LRRK2 gene cause autosomal dominant Parkinson's disease. LRRK2 encodes a multi-domain protein containing a Ras-of-complex (Roc) GTPase domain, a C-terminal of Roc domain and a protein kinase domain. LRRK2 can function as a GTPase and protein kinase, although the interplay between these two enzymatic domains is poorly understood. Although guanine nucleotide binding is critically required for the kinase activity of LRRK2, the contribution of GTP hydrolysis is not known. In general, the molecular determinants regulating GTPase activity and how the GTPase domain contributes to the properties of LRRK2 remain to be clarified. Here, we identify a number of synthetic missense mutations in the GTPase domain that functionally modulate GTP binding and GTP hydrolysis and we employ these mutants to comprehensively explore the contribution of GTPase activity to the kinase activity and cellular phenotypes of LRRK2. Our data demonstrate that guanine nucleotide binding and, to a lesser extent, GTP hydrolysis are required for maintaining normal kinase activity and both activities contribute to the GTP-dependent activation of LRRK2 kinase activity. Guanine nucleotide binding but not GTP hydrolysis regulates the dimerization, structure and stability of LRRK2. Furthermore, GTP hydrolysis regulates the LRRK2-dependent inhibition of neurite outgrowth in primary cortical neurons but is unable to robustly modulate the effects of the familial G2019S mutation. Our study elucidates the role of GTPase activity in regulating kinase activity and cellular phenotypes of LRRK2 and has important implications for the validation of the GTPase domain as a molecular target for attenuating LRRK2-mediated neurodegeneration.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 encodes a large multi-domain protein with GTPase and kinase activity. Initial data indicates that an intact functional GTPase domain is critically required for LRRK2 kinase activity. PD-associated mutations in LRRK2, including the most common G2019S variant, have variable effects on enzymatic activity but commonly alter neuronal process morphology. The mechanisms underlying the intrinsic and extrinsic regulation of LRRK2 GTPase and kinase activity, and the pathogenic effects of familial mutations, are incompletely understood. Here, we identify a novel functional interaction between LRRK2 and ADP-ribosylation factor GTPase-activating protein 1 (ArfGAP1). LRRK2 and ArfGAP1 interact in vitro in mammalian cells and in vivo in brain, and co-localize in the cytoplasm and at Golgi membranes. PD-associated and functional mutations that alter the GTPase activity of LRRK2 modulate the interaction with ArfGAP1. The GTP hydrolysis activity of LRRK2 is markedly enhanced by ArfGAP1 supporting a role for ArfGAP1 as a GTPase-activating protein for LRRK2. Unexpectedly, ArfGAP1 promotes the kinase activity of LRRK2 suggesting a potential role for GTP hydrolysis in kinase activation. Furthermore, LRRK2 robustly and directly phosphorylates ArfGAP1 in vitro. Silencing of ArfGAP1 expression in primary cortical neurons rescues the neurite shortening phenotype induced by G2019S LRRK2 overexpression, whereas the co-expression of ArfGAP1 and LRRK2 synergistically promotes neurite shortening in a manner dependent upon LRRK2 GTPase activity. Neurite shortening induced by ArfGAP1 overexpression is also attenuated by silencing of LRRK2. Our data reveal a novel role for ArfGAP1 in regulating the GTPase activity and neuronal toxicity of LRRK2; reciprocally, LRRK2 phosphorylates ArfGAP1 and is required for ArfGAP1 neuronal toxicity. ArfGAP1 may represent a promising target for interfering with LRRK2-dependent neurodegeneration in familial and sporadic PD.

Mutations in LRRK2 cause autosomal dominant Parkinson's disease (PD). LRRK2 encodes a multi-domain protein containing GTPase and kinase domains, and putative protein–protein interaction domains. Familial PD mutations alter the GTPase and kinase activity of LRRK2 in vitro. LRRK2 is suggested to regulate a number of cellular pathways although the underlying mechanisms are poorly understood. To explore such mechanisms, it has proved informative to identify LRRK2-interacting proteins, some of which serve as LRRK2 kinase substrates. Here, we identify common interactions of LRRK2 with members of the dynamin GTPase superfamily. LRRK2 interacts with dynamin 1–3 that mediate membrane scission in clathrin-mediated endocytosis and with dynamin-related proteins that mediate mitochondrial fission (Drp1) and fusion (mitofusins and OPA1). LRRK2 partially co-localizes with endosomal dynamin-1 or with mitofusins and OPA1 at mitochondrial membranes. The subcellular distribution and oligomeric complexes of dynamin GTPases are not altered by modulating LRRK2 in mouse brain, whereas mature OPA1 levels are reduced in G2019S PD brains. LRRK2 enhances mitofusin-1 GTP binding, whereas dynamin-1 and OPA1 serve as modest substrates of LRRK2-mediated phosphorylation in vitro. While dynamin GTPase orthologs are not required for LRRK2-induced toxicity in yeast, LRRK2 functionally interacts with dynamin-1 and mitofusin-1 in cultured neurons. LRRK2 attenuates neurite shortening induced by dynamin-1 by reducing its levels, whereas LRRK2 rescues impaired neurite outgrowth induced by mitofusin-1 potentially by reversing excessive mitochondrial fusion. Our study elucidates novel functional interactions of LRRK2 with dynamin-superfamily GTPases that implicate LRRK2 in the regulation of membrane dynamics important for endocytosis and mitochondrial morphology.

Specificity and mechanism-of-action of the JAK2 tyrosine kinase inhibitors ruxolitinib and SAR302503 (TG101348)

Mutations in the vacuolar protein sorting 35 ortholog (VPS35) gene represent a cause of late-onset, autosomal dominant familial Parkinson's disease (PD). A single missense mutation, D620N, is considered pathogenic based upon its segregation with disease in multiple families with PD. At present, the mechanism(s) by which familial VPS35 mutations precipitate neurodegeneration in PD are poorly understood. Here, we employ a germline D620N VPS35 knockin (KI) mouse model of PD to formally establish the age-related pathogenic effects of the D620N mutation at physiological expression levels. Our data demonstrate that a heterozygous or homozygous D620N mutation is sufficient to reproduce key neuropathological hallmarks of PD as indicated by the progressive degeneration of nigrostriatal pathway dopaminergic neurons and widespread axonal pathology. Unexpectedly, endogenous D620N VPS35 expression induces robust tau-positive somatodendritic pathology throughout the brain as indicated by abnormal hyperphosphorylated and conformation-specific tau, which may represent an important and early feature of mutant VPS35-induced neurodegeneration in PD. In contrast, we find no evidence for α-synuclein-positive neuropathology in aged VPS35 KI mice, a hallmark of Lewy body pathology in PD. D620N VPS35 expression also fails to modify the lethal neurodegenerative phenotype of human A53T-α-synuclein transgenic mice. Finally, by crossing VPS35 KI and null mice, our data demonstrate that a single D620N VPS35 allele is sufficient for survival and early maintenance of dopaminergic neurons, indicating that the D620N VPS35 protein is fully functional. Our data raise the tantalizing possibility of a pathogenic interplay between mutant VPS35 and tau for inducing neurodegeneration in PD.

Animal models that accurately recapitulate the accumulation of alpha-synuclein (α-syn) inclusions, progressive neurodegeneration of the nigrostriatal system and motor deficits can be useful tools for Parkinson's disease (PD) research. The preformed fibril (PFF) synucleinopathy model in rodents generally displays these PD-relevant features, however, the magnitude and predictability of these events is far from established. We therefore sought to optimize the magnitude of α-syn accumulation and nigrostriatal degeneration, and to understand the time course of both. Rats were injected unilaterally with different quantities of α-syn PFFs (8 or 16 μg of total protein) into striatal sites selected to concentrate α-syn inclusion formation in the substantia nigra pars compacta (SNpc). Rats displayed an α-syn PFF quantity-dependent increase in the magnitude of ipsilateral SNpc inclusion formation at 2 months and bilateral loss of nigral dopamine neurons at 6 months. Unilateral 16 μg PFF injection also resulted in modest sensorimotor deficits in forelimb adjusting steps associated with degeneration at 6 months. Bilateral injection of 16 μg α-syn PFFs resulted in symmetric bilateral degeneration equivalent to the ipsilateral nigral degeneration observed following unilateral 16 μg PFF injection (~50% loss). Bilateral PFF injections additionally resulted in alterations in several gait analysis parameters. These α-syn PFF parameters can be applied to generate a reproducible synucleinopathy model in rats with which to study pathogenic mechanisms and vet potential disease-modifying therapies.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant familial Parkinson’s disease (PD), with pathogenic mutations enhancing LRRK2 kinase activity. There is a growing body of evidence indicating that LRRK2 contributes to neuronal damage and pathology both in familial and sporadic PD, making it of particular interest for understanding the molecular pathways that underlie PD. Although LRRK2 has been extensively studied to date, our understanding of the seemingly diverse functions of LRRK2 throughout the cell remains incomplete. In this review, we discuss the functions of LRRK2 within the endolysosomal pathway. Endocytosis, vesicle trafficking pathways, and lysosomal degradation are commonly disrupted in many neurodegenerative diseases, including PD. Additionally, many PD-linked gene products function in these intersecting pathways, suggesting an important role for the endolysosomal system in maintaining protein homeostasis and neuronal health in PD. LRRK2 activity can regulate synaptic vesicle endocytosis, lysosomal function, Golgi network maintenance and sorting, vesicular trafficking and autophagy, with alterations in LRRK2 kinase activity serving to disrupt or regulate these pathways depending on the distinct cell type or model system. LRRK2 is critically regulated by at least two proteins in the endolysosomal pathway, Rab29 and VPS35, which may serve as master regulators of LRRK2 kinase activity. Investigating the function and regulation of LRRK2 in the endolysosomal pathway in diverse PD models, especially in vivo models, will provide critical insight into the cellular and molecular pathophysiological mechanisms driving PD and whether LRRK2 represents a viable drug target for disease-modification in familial and sporadic PD.

More than 200 years after James Parkinsondescribed a clinical syndrome based on his astute observations, Parkinson's disease (PD) has evolved into a complex entity, akin to the heterogeneity of other complex human syndromes of the central nervous system such as dementia, motor neuron disease, multiple sclerosis, and epilepsy. Clinicians, pathologists, and basic science researchers evolved arrange of concepts andcriteria for the clinical, genetic, mechanistic, and neuropathological characterization of what, in their best judgment, constitutes PD. However, these specialists have generated and used criteria that are not necessarily aligned between their different operational definitions, which may hinder progress in solving the riddle of the distinct forms of PD and ultimately how to treat them.This task force has identified current in consistencies between the definitions of PD and its diverse variants in different domains: clinical criteria, neuropathological classification, genetic subtyping, biomarker signatures, and mechanisms of disease. This initial effort for "defining the riddle" will lay the foundation for future attempts to better define the range of PD and its variants, as has been done and implemented for other heterogeneous neurological syndromes, such as stroke and peripheral neuropathy. We strongly advocate for a more systematic and evidence-based integration of our diverse disciplines by looking at well-defined variants of the syndrome of PD.Accuracy in defining endophenotypes of "typical PD" across these different but interrelated disciplines will enable better definition of variants and their stratification in therapeutic trials, a prerequisite for breakthroughs in the era of precision medicine. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

The biological actions of extracellular nucleotides are exerted via two families of P2 receptors, P2X and P2Y. The metabotropic P2Y receptors comprise at least 7 distinct subtypes, which have been cloned from a number of species. However, none of the P2Y receptor proteins have been visualised yet in human brain. In the present study, the regional and cellular distribution of the P2Y(1) receptor was investigated in the human brain by using immunohistochemistry. Polyclonal antibodies were raised against a synthetic peptide from the C-terminus of the P2Y(1) protein. Immunoblot analysis demonstrated that P2Y(1) antiserum specifically recognised a 63-kDa band in human and rat brain membranes. Similarly, the antiserum specifically detected the human P2Y(1) receptor in transfected 1321N1 cells. Immunohistochemical analysis on perfusion-fixed human brain tissue showed a widespread distribution for this receptor throughout the brain. At the cellular level, the P2Y(1) receptor was strikingly localised to neuronal structures of the cerebral cortex, cerebellar cortex, hippocampus, caudate nucleus, putamen, globus pallidus, subthalamic nucleus, red nucleus, and midbrain. Expression of the P2Y(1) receptor was not detected in other non-neuronal cell types. These results provide the first characterisation of the cellular distribution of a P2Y receptor in the human brain. The widespread and abundant distribution of the P2Y(1) receptor suggests its involvement in a number of important functions within the human brain. The neuronal localisation of this receptor points towards a possible role in neurotransmission, and also highlights a major role for extracellular nucleotides as signaling molecules within the brain.

The diverse biological actions of extracellular nucleotides in tissues and cells are mediated by two distinct classes of P2 receptor, P2X and P2Y. The G protein-coupled P2Y receptors comprise at least six mammalian subtypes (P2Y(1,2,4,6,11,12)), all of which have been cloned from human tissues, as well as other species. The P2Y receptor subtypes differ in their pharmacological selectivity for various adenosine and uridine nucleotides, which overlap in some cases. Data concerning the mRNA expression patterns of five P2Y receptors (P2Y(1,2,4,6,11)) in different human tissues and cells are currently quite limited, while P2Y mRNA distribution in the human brain has not previously been studied. In this study, we have addressed this deficiency in receptor expression data by using a quantitative reverse transcription-polymerase chain reaction approach to measure the precise mRNA expression pattern of each P2Y receptor subtype in a number of human peripheral tissues and brain regions, from multiple individuals, as well as numerous human cell lines and primary cells. All five P2Y receptors exhibited widespread yet subtype-selective mRNA expression profiles throughout the human tissues, brain regions and cells used. Our extensive expression data indicate the many potentially important roles of P2Y receptors throughout the human body, and will help in elucidating the physiological function of each receptor subtype in a wide variety of human systems.

Mutations in the parkin gene are a common cause of autosomal recessive early-onset parkinsonism. Parkin functions as an E3 ubiquitin ligase where it can polyubiquitinate a number of its protein substrates, thus targeting them for degradation by the 26 S proteasomal complex. Recent studies have demonstrated that alternative modes of parkin-mediated ubiquitination may serve other non-degradative regulatory roles. In addition, parkin appears to function as a multipurpose neuroprotectant in a number of toxic paradigms. Coupled with these observations, parkin may integrate other gene products associated with parkinsonism, including alpha-synuclein, LRRK2 (leucine-rich repeat kinase 2), DJ-1 and PINK1 [PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1], into a common biochemical pathway of potential relevance to disease pathogenesis. Parkin therefore represents a unique multifaceted ubiquitin ligase consistent with an important housekeeping role in maintaining the integrity or survival of dopaminergic neurons.

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) have been identified as the cause of familial Parkinson's disease (PD) at the PARK8 locus. To begin to understand the physiological role of LRRK2 and its involvement in PD, we have investigated the distribution of LRRK2 mRNA and protein in the adult mouse brain. In situ hybridization studies indicate sites of mRNA expression throughout the mouse brain, with highest levels of expression detected in forebrain regions, including the cerebral cortex and striatum, intermediate levels observed in the hippocampus and cerebellum, and low levels in the thalamus, hypothalamus and substantia nigra. Immunohistochemical studies demonstrate localization of LRRK2 protein to neurones in the cerebral cortex and striatum, and to a variety of interneuronal subtypes in these regions. Furthermore, expression of LRRK2 mRNA in the striatum of VMAT2-deficient mice is unaltered relative to wild-type littermate controls despite extensive dopamine depletion in this mouse model of parkinsonism. Collectively, our results demonstrate that LRRK2 is present in anatomical brain regions of direct relevance to the pathogenesis of PD, including the nigrostriatal dopaminergic pathway, in addition to other regions unrelated to PD pathology, and is likely to play an important role in the normal function of telencephalic forebrain neurones and other neuronal populations.

Mutations in the parkin gene cause autosomal recessive, juvenile-onset parkinsonism. Parkin is an E3 ubiquitin ligase that mediates the ubiquitination of protein substrates. Disease-associated mutations cause a loss-of-function of parkin which may compromise the poly-ubiquitination and proteasomal degradation of specific protein substrates, potentially leading to their deleterious accumulation. Here, we identify the molecular chaperones, Hsp70 and Hsc70, as substrates for parkin. Parkin mediates the ubiquitination of Hsp70 both in vitro and in cultured cells. Parkin interacts with Hsp70 via its second RING finger domain and mutations in/near this domain compromise Hsp70 ubiquitination. Ubiquitination of Hsp70 fails to alter its steady-state levels or turnover, nor does it promote its proteasomal degradation. Consistent with this observation, Hsp70 levels remain unaltered in brains from parkin-deficient autosomal recessive, juvenile-onset parkinsonism subjects, whereas alternatively, Hsp70 levels are elevated in the detergent-insoluble fraction of sporadic Parkinson's disease/dementia with Lewy bodies brains. Parkin mediates the multiple mono-ubiquitination of Hsp70/Hsc70 consistent with a degradation-independent role for this ubiquitin modification. Our observations support a novel functional relationship between parkin and Hsc/Hsp70 and support the notion that parkin is a multi-purpose E3 ubiquitin ligase capable of modifying proteins either via attachment of alternatively linked poly-ubiquitin chains or through multiple mono-ubiquitination to achieve alternate biological outcomes.

Mutations in the genes encoding LRRK2 and α-synuclein cause autosomal dominant forms of familial Parkinson's disease (PD). Fibrillar forms of α-synuclein are a major component of Lewy bodies, the intracytoplasmic proteinaceous inclusions that are a pathological hallmark of idiopathic and certain familial forms of PD. LRRK2 mutations cause late-onset familial PD with a clinical, neurochemical and, for the most part, neuropathological phenotype that is indistinguishable from idiopathic PD. Importantly, α-synuclein-positive Lewy bodies are the most common pathology identified in the brains of PD subjects harboring LRRK2 mutations. These observations may suggest that LRRK2 functions in a common pathway with α-synuclein to regulate its aggregation. To explore the potential pathophysiological interaction between LRRK2 and α-synuclein in vivo, we modulated LRRK2 expression in a well-established human A53T α-synuclein transgenic mouse model with transgene expression driven by the hindbrain-selective prion protein promoter. Deletion of LRRK2 or overexpression of human G2019S-LRRK2 has minimal impact on the lethal neurodegenerative phenotype that develops in A53T α-synuclein transgenic mice, including premature lethality, pre-symptomatic behavioral deficits and human α-synuclein or glial neuropathology. We also find that endogenous or human LRRK2 and A53T α-synuclein do not interact together to influence the number of nigrostriatal dopaminergic neurons. Taken together, our data suggest that α-synuclein-related pathology, which occurs predominantly in the hindbrain of this A53T α-synuclein mouse model, occurs largely independently from LRRK2 expression. These observations fail to provide support for a pathophysiological interaction of LRRK2 and α-synuclein in vivo, at least within neurons of the mouse hindbrain.

Missense mutations and multiplications of the alpha-synuclein gene cause autosomal dominant familial Parkinson's disease (PD). alpha-Synuclein protein is also a major component of Lewy bodies, the hallmark pathological inclusions of PD. Therefore, alpha-synuclein plays an important role in the pathogenesis of familial and sporadic PD. To model alpha-synuclein-linked disease in vivo, transgenic mouse models have been developed that express wild-type or mutant human alpha-synuclein from a variety of neuronal-selective heterologous promoter elements. These models exhibit a variety of behavioral and neuropathological features resembling some aspects of PD. However, an important deficiency of these models is the observed lack of robust or progressive nigrostriatal dopaminergic neuronal degeneration that is characteristic of PD.We have developed conditional alpha-synuclein transgenic mice that can express A53T, E46K or C-terminally truncated (1-119) human alpha-synuclein pathological variants from the endogenous murine ROSA26 promoter in a Cre recombinase-dependent manner. Using these mice, we have evaluated the expression of these alpha-synuclein variants on the integrity and viability of nigral dopaminergic neurons with age. Expression of A53T alpha-synuclein or truncated alphaSyn119 selectively in nigrostriatal pathway dopaminergic neurons for up to 12 months fails to precipitate dopaminergic neuronal loss in these mice. However, alphaSyn119 expression in nigral dopaminergic neurons for up to 12 months causes a marked reduction in the levels of striatal dopamine and its metabolites together with other subtle neurochemical alterations.We have developed and evaluated novel conditional alpha-synuclein transgenic mice with transgene expression directed selectively to nigrostriatal dopaminergic neurons as a potential new mouse model of PD. Our data support the pathophysiological relevance of C-terminally truncated alpha-synuclein species in vivo. The expression of alphaSyn119 in the mouse nigrostriatal dopaminergic pathway may provide a useful model of striatal dopamine depletion and could potentially provide a presymptomatic model of PD perhaps representative of the earliest derangements in dopaminergic neuronal function observed prior to neuronal loss. These conditional alpha-synuclein transgenic mice provide novel tools for evaluating and dissecting the age-related effects of alpha-synuclein pathological variants on the function of the nigrostriatal dopaminergic pathway or other specific neuronal populations.

Mitochondria are highly dynamic, multifunctional organelles. Aside from their major role in energy metabolism, they are also crucial for many cellular processes including neurotransmission, synaptic maintenance, calcium homeostasis, cell death, and neuronal survival. Significance: Increasing evidence supports a role for abnormal mitochondrial function in the molecular pathophysiology of Parkinson's disease (PD). For three decades we have known that mitochondrial toxins are capable of producing clinical parkinsonism in humans. PD is the most common neurodegenerative movement disorder that is characterized by the progressive loss of substantia nigra dopaminergic neurons leading to a deficiency of striatal dopamine. Although the neuropathology underlying the disease is well defined, it remains unclear why nigral dopaminergic neurons degenerate and die. Recent Advances: Most PD cases are idiopathic, but there are rare familial cases. Mutations in five genes are known to unambiguously cause monogenic familial PD: α-synuclein, parkin, DJ-1, PTEN-induced kinase 1 (PINK1), and leucine-rich repeat kinase 2 (LRRK2). These key molecular players are proteins of seemingly diverse function, but with potentially important roles in mitochondrial maintenance and function. Cell and animal-based genetic models have provided indispensable tools for understanding the molecular basis of PD, and have provided additional evidence implicating mitochondrial dysfunction as a primary pathogenic pathway leading to the demise of dopaminergic neurons in PD. Critical Issues: Here, we critically discuss the evidence for mitochondrial dysfunction in genetic animal models of PD, and evaluate whether abnormal mitochondrial function represents a cause or consequence of disease pathogenesis. Future Directions: Mitochondria may represent a potential target for the development of disease-modifying therapies. Antioxid. Redox Signal. 16, 896–919.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant Parkinson's disease (PD). The clinical and neurochemical features of LRRK2-linked PD are similar to idiopathic disease although neuropathology is somewhat heterogeneous. Dominant mutations in LRRK2 precipitate neurodegeneration through a toxic gain-of-function mechanism which can be modeled in transgenic mice overexpressing human LRRK2 variants. A number of LRRK2 transgenic mouse models have been developed that display abnormalities in dopaminergic neurotransmission and alterations in tau metabolism yet without consistently inducing dopaminergic neurodegeneration. To directly explore the impact of mutant LRRK2 on the nigrostriatal dopaminergic pathway, we developed conditional transgenic mice that selectively express human R1441C LRRK2 in dopaminergic neurons from the endogenous murine ROSA26 promoter. The expression of R1441C LRRK2 does not induce the degeneration of substantia nigra dopaminergic neurons or striatal dopamine deficits in mice up to 2years of age, and fails to precipitate abnormal protein inclusions containing alpha-synuclein, tau, ubiquitin or autophagy markers (LC3 and p62). Furthermore, mice expressing R1441C LRRK2 exhibit normal motor activity and olfactory function with increasing age. Intriguingly, the expression of R1441C LRRK2 induces age-dependent abnormalities of the nuclear envelope in nigral dopaminergic neurons including reduced nuclear circularity and increased invaginations of the nuclear envelope. In addition, R1441C LRRK2 mice display increased neurite complexity of cultured midbrain dopaminergic neurons. Collectively, these novel R1441C LRRK2 conditional transgenic mice reveal altered dopaminergic neuronal morphology with advancing age, and provide a useful tool for exploring the pathogenic mechanisms underlying the R1441C LRRK2 mutation in PD.

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

Mutations in a number of genes cause familial forms of Parkinson’s disease (PD), including mutations in the vacuolar protein sorting 35 ortholog (VPS35) and parkin genes. In this study, we identify a novel functional interaction between parkin and VPS35. We demonstrate that parkin interacts with and robustly ubiquitinates VPS35 in human neural cells. Familial parkin mutations are impaired in their ability to ubiquitinate VPS35. Parkin mediates the attachment of an atypical poly-ubiquitin chain to VPS35 with three lysine residues identified within the C-terminal region of VPS35 that are covalently modified by ubiquitin. Notably, parkin-mediated VPS35 ubiquitination does not promote the proteasomal degradation of VPS35. Furthermore, parkin does not influence the steady-state levels or turnover of VPS35 in neural cells and VPS35 levels are normal in the brains of parkin knockout mice. These data suggest that ubiquitination of VPS35 by parkin may instead serve a non-degradative cellular function potentially by regulating retromer-dependent sorting. Accordingly, we find that components of the retromer-associated WASH complex are markedly decreased in the brain of parkin knockout mice, suggesting that parkin may modulate WASH complex-dependent retromer sorting. Parkin gene silencing in primary cortical neurons selectively disrupts the vesicular sorting of the autophagy receptor ATG9A, a WASH-dependent retromer cargo. Parkin is not required for dopaminergic neurodegeneration induced by the expression of PD-linked D620N VPS35 in mice, consistent with VPS35 being located downstream of parkin function. Our data reveal a novel functional interaction of parkin with VPS35 that may be important for retromer-mediated endosomal sorting and PD.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset, autosomal dominant familial Parkinson`s disease (PD) and variation at the LRRK2 locus contributes to the risk for idiopathic PD. LRRK2 can function as a protein kinase and mutations lead to increased kinase activity. To elucidate the pathophysiological mechanism of the R1441C mutation in the GTPase domain of LRRK2, we expressed human wild-type or R1441C LRRK2 in dopaminergic neurons of Drosophila and observe reduced locomotor activity, impaired survival and an age-dependent degeneration of dopaminergic neurons thereby creating a new PD-like model. To explore the function of LRRK2 variants in vivo, we performed mass spectrometry and quantified 3,616 proteins in the fly brain. We identify several differentially-expressed cytoskeletal, mitochondrial and synaptic vesicle proteins (SV), including synaptotagmin-1, syntaxin-1A and Rab3, in the brain of this LRRK2 fly model. In addition, a global phosphoproteome analysis reveals the enhanced phosphorylation of several SV proteins, including synaptojanin-1 (pThr1131) and the microtubule-associated protein futsch (pSer4106) in the brain of R1441C hLRRK2 flies. The direct phosphorylation of human synaptojanin-1 by R1441C hLRRK2 could further be confirmed by in vitro kinase assays. A protein–protein interaction screen in the fly brain confirms that LRRK2 robustly interacts with numerous SV proteins, including synaptojanin-1 and EndophilinA. Our proteomic, phosphoproteomic and interactome study in the Drosophila brain provides a systematic analyses of R1441C hLRRK2-induced pathobiological mechanisms in this model. We demonstrate for the first time that the R1441C mutation located within the LRRK2 GTPase domain induces the enhanced phosphorylation of SV proteins in the brain.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease (PD). However, the precise function of LRRK2 remains unclear. We report an interaction between LRRK2 and VPS52, a subunit of the Golgi-associated retrograde protein (GARP) complex that identifies a function of LRRK2 in regulating membrane fusion at the trans-Golgi network (TGN). At the TGN, LRRK2 further interacts with the Golgi SNAREs VAMP4 and Syntaxin-6 and acts as a scaffolding platform that stabilizes the GARP-SNAREs complex formation. Therefore, LRRK2 influences both retrograde and post-Golgi trafficking pathways in a manner dependent on its GTP binding and kinase activity. This action is exaggerated by mutations associated with Parkinson’s disease and can be blocked by kinase inhibitors. Disruption of GARP sensitizes dopamine neurons to mutant LRRK2 toxicity in C. elegans, showing that these pathways are interlinked in vivo and suggesting a link in PD.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of late-onset, autosomal-dominant familial Parkinson's disease (PD). LRRK2 functions as both a kinase and GTPase, and PD-linked mutations are known to influence both enzymatic activities. While PD-linked LRRK2 mutations can commonly induce neuronal damage in culture models, the mechanisms underlying these pathogenic effects remain uncertain. Rodent models containing familial LRRK2 mutations often lack robust PD-like neurodegenerative phenotypes. Here, we develop a robust preclinical model of PD in adult rats induced by the brain delivery of recombinant adenoviral vectors with neuronal-specific expression of human LRRK2 harboring the most common G2019S mutation. In this model, G2019S LRRK2 induces the robust degeneration of substantia nigra dopaminergic neurons, a pathological hallmark of PD. Introduction of a stable kinase-inactive mutation or administration of the selective kinase inhibitor, PF-360, attenuates neurodegeneration induced by G2019S LRRK2. Neuroprotection provided by pharmacological kinase inhibition is mediated by an unusual mechanism involving the robust destabilization of human LRRK2 protein in the brain relative to endogenous LRRK2. Our study further demonstrates that G2019S LRRK2-induced dopaminergic neurodegeneration critically requires normal GTPase activity, as hypothesis-testing mutations that increase GTP hydrolysis or impair GTP-binding activity provide neuroprotection although via distinct mechanisms. Taken together, our data demonstrate that G2019S LRRK2 induces neurodegeneration in vivo via a mechanism that is dependent on kinase and GTPase activity. Our study provides a robust rodent preclinical model of LRRK2-linked PD and nominates kinase inhibition and modulation of GTPase activity as promising disease-modifying therapeutic targets.

We have recently shown that UDP-glucose, and some related UDP-sugars, are potent agonists of the novel G protein-coupled receptor GPR105 (recently re-named P2Y(14)). GPR105 is widely expressed throughout many brain regions and peripheral tissues of human and rodents, and couples to a pertussis toxin-sensitive G protein. To further characterise the role of GPR105, we demonstrate by immunohistochemistry with receptor-specific antiserum that GPR105 protein is widely distributed throughout the post mortem human brain where it is localised to glial cells, and specifically co-localises with astrocytes. Using quantitative RT-PCR we also show that GPR105 mRNA exhibits a restricted expression profile in an array of human cell lines and primary cells, with prominent expression detected in immune cells including neutrophils, lymphocytes, and megakaryocytic cells. To investigate the G protein selectivity of GPR105, we used chimeric Galpha subunits (Galpha(qi5), Galpha(qo5), and Galpha(qs5)) and an intracellular Ca(2+) mobilisation assay to demonstrate that GPR105 couples to Galpha subunits of the G(i/o) family but not to G(s) family proteins or to endogenous G(q/11) proteins in HEK-293 cells. Finally, we show that expression of GPR105 mRNA in the rat brain is up-regulated by immunologic challenge with lipopolysaccharide. Based on these observations, we propose that G(i/o)-coupled GPR105 might play an important role in peripheral and neuroimmune function in response to extracellular UDP-sugars.

Missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common causes of both familial and sporadic forms of Parkinson disease and are also associated with diverse pathological alterations. The mechanisms whereby LRRK2 mutations cause these pathological phenotypes are unknown. We used immunohistochemistry with 3 distinct anti-LRRK2 antibodies to characterize the expression of LRRK2 in the brains of 21 subjects with various neurodegenerative disorders and 7 controls. The immunoreactivity of LRRK2 was localized in a subset of brainstem-type Lewy bodies (LBs) but not in cortical-type LBs, tau-positive inclusions, or TAR-DNA-binding protein-43-positive inclusions. The immunoreactivity of LRRK2 frequently appeared as enlarged granules or vacuoles within neurons of affected brain regions, including the substantia nigra, amygdala, and entorhinal cortex in patients with Parkinson disease or dementia with LBs. The volumes of LRRK2-positive granular structures in neurons of the entorhinal cortex were significantly increased in dementia with LBs brains compared with age-matched control brains (p < 0.05). Double immunolabeling demonstrated that these LRRK2-positive granular structures frequently colocalized with the late-endosomal marker Rab7B and occasionally with the lysosomal marker, the lysosomal-associated membrane protein 2. These results suggest that LRRK2 normally localizes to the endosomal-lysosomal compartment within morphologically altered neurons in neurodegenerative diseases, particularly in the brains of patients with LB diseases.

Mutations in the ATP13A2 gene (PARK9) cause autosomal recessive, juvenile-onset Kufor-Rakeb syndrome (KRS), a neurodegenerative disease characterized by parkinsonism. KRS mutations produce truncated forms of ATP13A2 with impaired protein stability resulting in a loss-of-function. Recently, homozygous and heterozygous missense mutations in ATP13A2 have been identified in subjects with early-onset parkinsonism. The mechanism(s) by which missense mutations potentially cause parkinsonism are not understood at present. Here, we demonstrate that homozygous F182L, G504R and G877R missense mutations commonly impair the protein stability of ATP13A2 leading to its enhanced degradation by the proteasome. ATP13A2 normally localizes to endosomal and lysosomal membranes in neurons and the F182L and G504R mutations disrupt this vesicular localization and promote the mislocalization of ATP13A2 to the endoplasmic reticulum. Heterozygous T12M, G533R and A746T mutations do not obviously alter protein stability or subcellular localization but instead impair the ATPase activity of microsomal ATP13A2 whereas homozygous missense mutations disrupt the microsomal localization of ATP13A2. The overexpression of ATP13A2 missense mutants in SH-SY5Y neural cells does not compromise cellular viability suggesting that these mutant proteins lack intrinsic toxicity. However, the overexpression of wild-type ATP13A2 may impair neuronal integrity as it causes a trend of reduced neurite outgrowth of primary cortical neurons, whereas the majority of disease-associated missense mutations lack this ability. Finally, ATP13A2 overexpression sensitizes cortical neurons to neurite shortening induced by exposure to cadmium or nickel ions, supporting a functional interaction between ATP13A2 and heavy metals in post-mitotic neurons, whereas missense mutations influence this sensitizing effect. Collectively, our study provides support for common loss-of-function effects of homozygous and heterozygous missense mutations in ATP13A2 associated with early-onset forms of parkinsonism.

There is emerging evidence implicating a role for the autophagy-lysosome pathway in the pathogenesis of Lewy body disease. We investigated potential neuropathologic and biochemical alterations of autophagy-lysosome pathway-related proteins in the brains of patients with dementia with Lewy bodies (DLB), Alzheimer disease (AD), and control subjects using antibodies against Ras-related protein Rab-7B (Rab7B), lysosomal-associated membrane protein 2 (LAMP2), and microtubule-associated protein 1A/1B light chain 3 (LC3). In DLB, but not in control brains, there were large Rab7B-immunoreactive endosomal granules. LC3 immunoreactivity was increased in vulnerable areas of DLB brains relative to that in control brains; computerized cell counting analysis revealed that LC3 levels were greater in the entorhinal cortex and amygdala of DLB brains than in controls. Rab7B levels were increased, and LAMP2 levels were decreased in the entorhinal cortex of DLB brains. In contrast, only a decrease in LAMP2 levels versus controls was found in AD brains. LC3 widely colocalized with several types of Lewy pathology; LAMP2 localized to the periphery or outside of brainstem-type Lewy bodies; Rab7B did not colocalize with Lewy pathology. Immunoblot analysis demonstrated specific accumulation of the autophagosomal LC3-II isoform in detergent-insoluble fractions from DLB brains. These results support apotential role for the autophagy-lysosome pathway in the pathogenesis of DLB.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene represent the most common cause of familial Parkinson’s disease (PD), whereas common variation at the LRRK2 locus is associated with an increased risk of idiopathic PD. Considerable progress has been made toward understanding the biological functions of LRRK2 and the molecular mechanisms underlying the pathogenic effects of disease-associated mutations. The development of neuronal culture models and transgenic or viral-based rodent models have proved useful for identifying a number of emerging pathways implicated in LRRK2-dependent neuronal damage, including the microtubule network, actin cytoskeleton, autophagy, mitochondria, vesicular trafficking, and protein quality control. However, many important questions remain to be posed and answered. Elucidating the molecular mechanisms and pathways underlying LRRK2-mediated neurodegeneration is critical for the identification of new molecular targets for therapeutic intervention in PD. In this review we discuss recent advances and unanswered questions in understanding the pathophysiology of LRRK2.

Mutations in the leucine-rich repeat kinase 2 (LRRK2, PARK8, OMIM 607060) gene represent the most common known cause of hereditary Parkinson's disease (PD) with late-onset and dominant inheritance. LRRK2 protein is composed of multiple domains including two distinct enzymatic domains, a kinase and a Ras-of-complex (Roc) GTPase, connected by a C-terminal-of-Roc (COR) domain, and belongs to the ROCO protein family. Disease-causing mutations located in the kinase domain enhance kinase activity (i.e., G2019S) whereas mutations clustering within the Roc-COR tandem domain impair GTPase activity (i.e., R1441C/G and Y1699C). Familial LRRK2 mutations commonly induce neuronal toxicity that, at least for the frequent G2019S variant, is dependent on kinase activity. The contribution of GTPase activity to LRRK2-dependent neuronal toxicity is not yet clear. Therefore, both GTPase and kinase activity may be important for mediating neurodegeneration in PD due to familial LRRK2 mutations. At present, the physiological function of LRRK2 in the mammalian brain and the regulation of its enzymatic activity are incompletely understood. In this review, we focus on the GTPase domain of LRRK2 and discuss the recent advances in elucidating its function and its interplay with the kinase domain for the regulation of LRRK2 activity and toxicity. GTPase activity is an important feature of LRRK2 biology and pathophysiology and represents an underexplored yet potentially tractable therapeutic target for treating LRRK2-associated PD.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most frequent cause of Parkinson’s disease (PD) with late-onset and autosomal-dominant inheritance. LRRK2 belongs to the ROCO superfamily of proteins, characterized by a Ras-of-complex (Roc) GTPase domain in tandem with a C-terminal-of-Roc (COR) domain. LRRK2 also contains a protein kinase domain adjacent to the Roc-COR tandem domain in addition to multiple repeat domains. Disease-causing familial mutations cluster within the Roc-COR tandem and kinase domains of LRRK2, where they act to either impair GTPase activity or enhance kinase activity. Familial LRRK2 mutations share in common the capacity to induce neuronal toxicity in cultured cells. While the contribution of the frequent G2019S mutation, located within the kinase domain, to kinase activity and neurotoxicity has been extensively investigated, the contribution of GTPase activity has received less attention. The GTPase domain has been shown to play an important role in regulating kinase activity, in dimerization, and in mediating the neurotoxic effects of LRRK2. Accordingly, the GTPase domain has emerged as a potential therapeutic target for inhibiting the pathogenic effects of LRRK2 mutations. Many important mechanisms remain to be elucidated, including how the GTPase cycle of LRRK2 is regulated, whether GTPase effectors exist for LRRK2, and how GTPase activity contributes to the overall functional output of LRRK2. In this review, we discuss the importance of the GTPase domain for LRRK2-linked PD focusing in particular on its regulation, function, and contribution to neurotoxic mechanisms.

Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). The most prevalent LRRK2 mutation is the G2019S coding change, located in the kinase domain of this complex multi-domain protein. The majority of G2019S autopsy cases feature typical Lewy Body pathology with a clinical phenotype almost indistinguishable from idiopathic PD (iPD). Here we have investigated the biochemical characteristics of α-synuclein in G2019S LRRK2 PD post-mortem material, in comparison to pathology-matched iPD. Immunohistochemistry with pS129 α-synuclein antibody showed that the medulla is heavily affected with pathology in G2019S PD whilst the basal ganglia (BG), limbic and frontal cortical regions demonstrated comparable pathology scores between G2019S PD and iPD. Significantly lower levels of the highly aggregated α-synuclein species in urea-SDS fractions were observed in G2019S cases compared to iPD in the BG and limbic cortex. Our data, albeit from a small number of cases, highlight a difference in the biochemical properties of aggregated α-synuclein in G2019S linked PD compared to iPD, despite a similar histopathological presentation. This divergence in solubility is most notable in the basal ganglia, a region that is affected preclinically and is damaged before overt dopaminergic cell death.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial Parkinson's disease (PD) with autosomal dominant inheritance. Accordingly, LRRK2 has emerged as a promising therapeutic target for disease modification in PD. Since the first discovery of LRRK2 mutations some 12 years ago, LRRK2 has been the subject of intense investigation. It has been established that LRRK2 can function as a protein kinase, with many putative substrates identified, and can also function as a GTPase that may serve in part to regulate kinase activity. Familial mutations influence both of these enzymatic activities, suggesting that they may be important for the development of PD. Many LRRK2 models have been established to understand the pathogenic effects and mechanisms of familial mutations. Here, we provide a focused discussion of the evidence supporting a role for kinase and GTPase activity in mediating the pathogenic effects of familial LRRK2 mutations in different model systems, with an emphasis on rodent models of PD. We also critically discuss the contribution and relevance of protein aggregation, namely of α-synuclein and tau-proteins, which are known to form aggregates in PD brains harboring LRRK2 mutations, to neurodegeneration in LRRK2 rodent models. We aim to provide a clear and unbiased review of some of the key mechanisms that are important for LRRK2-dependent neurodegeneration in PD.

Mutations in LRRK2 are one of the primary genetic causes of Parkinson's disease (PD). LRRK2 contains a kinase and a GTPase domain, and familial PD mutations affect both enzymatic activities. However, the signaling mechanisms regulating LRRK2 and the pathogenic effects of familial mutations remain unknown. Identifying the signaling proteins that regulate LRRK2 function and toxicity remains a critical goal for the development of effective therapeutic strategies. In this study, we apply systems biology tools to human PD brain and blood transcriptomes to reverse-engineer a LRRK2-centered gene regulatory network. This network identifies several putative master regulators of LRRK2 function. In particular, the signaling gene RGS2, which encodes for a GTPase-activating protein (GAP), is a key regulatory hub connecting the familial PD-associated genes DJ-1 and PINK1 with LRRK2 in the network. RGS2 expression levels are reduced in the striata of LRRK2 and sporadic PD patients. We identify RGS2 as a novel interacting partner of LRRK2 in vivo. RGS2 regulates both the GTPase and kinase activities of LRRK2. We show in mammalian neurons that RGS2 regulates LRRK2 function in the control of neuronal process length. RGS2 is also protective against neuronal toxicity of the most prevalent mutation in LRRK2, G2019S. We find that RGS2 regulates LRRK2 function and neuronal toxicity through its effects on kinase activity and independently of GTPase activity, which reveals a novel mode of action for GAP proteins. This work identifies RGS2 as a promising target for interfering with neurodegeneration due to LRRK2 mutations in PD patients.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant Parkinson's disease (PD). LRRK2 contains functional GTPase and kinase domains. The most common G2019S mutation enhances the kinase activity of LRRK2 in vitro whereas G2019S LRRK2 expression in cultured neurons induces toxicity in a kinase-dependent manner. These observations suggest a potential role for kinase activity in LRRK2-associated PD. We have recently developed a novel rodent model of PD with progressive neurodegeneration induced by the adenoviral-mediated expression of G2019S LRRK2. In the present study, we further characterize this LRRK2 model and determine the contribution of kinase activity to LRRK2-mediated neurodegeneration. Recombinant human adenoviral vectors were employed to deliver human wild-type, G2019S or kinase-inactive G2019S/D1994N LRRK2 to the rat striatum. LRRK2-dependent pathology was assessed in the striatum, a region where LRRK2 protein is normally enriched in the mammalian brain. Human LRRK2 variants are robustly expressed throughout the rat striatum. Expression of G2019S LRRK2 selectively induces the accumulation of neuronal ubiquitin-positive inclusions accompanied by neurite degeneration and the altered distribution of axonal phosphorylated neurofilaments. Importantly, the introduction of a kinase-inactive mutation (G2019S/D1994N) completely ameliorates the pathological effects of G2019S LRRK2 in the striatum supporting a kinase activity-dependent mechanism for this PD-associated mutation. Collectively, our study further elucidates the pathological effects of the G2019S mutation in the mammalian brain and supports the development of kinase inhibitors as a potential therapeutic approach for treating LRRK2-associated PD. This adenoviral rodent model provides an important tool for elucidating the molecular basis of LRRK2-mediated neurodegeneration.

Perturbations of the endolysosomal pathway have been suggested to play an important role in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease (PD) and Alzheimer's disease (AD). Specifically, VPS35 and the retromer complex play an important role in the endolysosomal system and are implicated in the pathophysiology of these diseases. A single missense mutation in VPS35, Asp620Asn (D620N), is known to cause late-onset, autosomal dominant familial PD. In this review, we focus on the emerging role of the PD-linked D620N mutation in causing retromer dysfunction and dissect its implications in neurodegeneration. Additionally, we will discuss how VPS35 and the retromer are linked to AD, amyotrophic lateral sclerosis, and primary tauopathies. Interestingly, reduced levels of VPS35 and other retromer components have been observed in post-mortem brain tissue, suggesting a role for the retromer in the pathophysiology of these diseases. This review will provide a comprehensive dive into the mechanisms of VPS35 dysfunction in neurodegenerative diseases. Furthermore, we will highlight outstanding questions in the field and the retromer as a therapeutic target for neurodegenerative disease at large.

The vacuolar protein sorting 35 ortholog (VPS35) gene encodes a core component of the retromer complex essential for the endosomal sorting and recycling of transmembrane cargo. Endo-lysosomal pathway deficits are suggested to play a role in the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD). Mutations in VPS35 cause a late-onset, autosomal dominant form of PD, with a single missense mutation (D620N) shown to segregate with disease in PD families. Understanding how the PD-linked D620N mutation causes retromer dysfunction will provide valuable insight into the pathophysiology of PD and may advance the identification of therapeutics. D620N VPS35 can induce LRRK2 hyperactivation and impair endosomal recruitment of the WASH complex but is also linked to mitochondrial and autophagy-lysosomal pathway dysfunction and altered neurotransmitter receptor transport. The clinical similarities between VPS35-linked PD and sporadic PD suggest that defects observed in cellular and animal models with the D620N VPS35 mutation may provide valuable insights into sporadic disease. In this review, we highlight the current knowledge surrounding VPS35 and its role in retromer dysfunction in PD. We provide a critical discussion of the mechanisms implicated in VPS35-mediated neurodegeneration in PD, as well as the interplay between VPS35 and other PD-linked gene products. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

It has recently been shown that UDP-glucose is a potent agonist of the orphan G-protein-coupled receptor (GPCR) KIAA0001. Here we report cloning and analysis of the rat and mouse orthologs of this receptor. In accordance with GPCR nomenclature, we have renamed the cDNA clone, KIAA0001, and its orthologs GPR105 to reflect their functionality as G-protein-coupled receptors. The rat and mouse orthologs show 80% and 83% amino acid identity, respectively, to the human GPR105 protein. We demonstrate by genomic Southern blot analysis that there are no genes in the mouse or rat genomes with higher sequence similarity. Chromosomal mapping shows that the mouse and human genes are located on syntenic regions of chromosome 3. Further analyses of the rat and mouse GPR105 proteins show that they are activated by the same agonists as the human receptor, responding to UDP-glucose and closely related molecules with similar affinities. The mouse and rat receptors are widely expressed, as is the human receptor. Thus we conclude that we have identified the rat and mouse orthologs of the human gene GPR105.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified as an important cause of late-onset, autosomal dominant familial Parkinson disease and contribute to sporadic Parkinson disease. LRRK2 is a large complex protein with multiple functional domains, including a Roc-GTPase, protein kinase, and multiple protein-protein interaction domains. Previous studies have suggested an important role for kinase activity in LRRK2-induced neuronal toxicity and inclusion body formation. Disease-associated mutations in LRRK2 also tend to increase kinase activity. Thus, enhanced kinase activity may therefore underlie LRRK2-linked disease. Similar to the closely related mixed-lineage kinases, LRRK2 can undergo autophosphorylation in vitro. Three putative autophosphorylation sites (Thr-2031, Ser-2032, and Thr-2035) have been identified within the activation segment of the LRRK2 kinase domain based on sequence homology to mixed-lineage kinases. Phosphorylation at one or more of these sites is critical for the kinase activity of LRRK2. Sensitive phospho-specific antibodies to each of these three sites have been developed and validated by ELISA, dot-blot, and Western blot analysis. Using these antibodies, we have found that all three putative sites are phosphorylated in LRRK2, and Ser-2032 and Thr-2035 are the two important sites that regulate LRRK2 kinase activity.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of autosomal dominant familial Parkinson's disease (PD). LRRK2 encodes a multi-domain protein containing GTPase and kinase enzymatic domains. Disease-associated mutations in LRRK2 variably influence enzymatic activity with the common G2019S variant leading to enhanced kinase activity. Mutant LRRK2 induces neuronal toxicity through a kinase-dependent mechanism suggesting that kinase activity is important for mediating the pathogenic effects of LRRK2 mutations. A number of LRRK2 kinase substrates have been identified in vitro but whether they represent authentic physiological substrates in mammalian cells or tissues is not yet clear. The eukaryotic initiation factor 4E (eIF4E)-binding protein, 4E-BP1, was recently identified as a potential substrate of LRRK2 kinase activity in vitro and in Drosophila with phosphorylation occurring at Thr37 and Thr46. Here, we explore a potential interaction of LRRK2 and 4E-BP1 in mammalian cells and brain. We find that LRRK2 can weakly phosphorylate 4E-BP1 in vitro but LRRK2 overexpression is not able to alter endogenous 4E-BP1 phosphorylation in mammalian cells. In mammalian neurons LRRK2 and 4E-BP1 display minimal co-localization, whereas the subcellular distribution, protein complex formation and covalent post-translational modification of endogenous 4E-BP1 are not altered in the brains of LRRK2 knockout or mutant LRRK2 transgenic mice. In the brain, the phosphorylation of 4E-BP1 at Thr37 and Thr46 does not change in LRRK2 knockout or mutant LRRK2 transgenic mice, nor is 4E-BP1 phosphorylation altered in idiopathic or G2019S mutant PD brains. Collectively, our results suggest that 4E-BP1 is neither a major nor robust physiological substrate of LRRK2 in mammalian cells or brain.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant Parkinson’s disease (PD). LRRK2 mutations typically give rise to Lewy pathology in the brains of PD subjects yet can induce tau-positive neuropathology in some cases. The pathological interaction between LRRK2 and tau remains poorly defined. To explore this interaction in vivo, we crossed a well-characterized human P301S-tau transgenic mouse model of tauopathy with human G2019S-LRRK2 transgenic mice or LRRK2 knockout (KO) mice. We find that endogenous or pathogenic LRRK2 expression has minimal effects on the steady-state levels, solubility and abnormal phosphorylation of human P301S-tau throughout the mouse brain. We next developed a new model of tauopathy by delivering AAV2/6 vectors expressing human P301S-tau to the hippocampal CA1 region of G2019S-LRRK2 transgenic or LRRK2 KO mice. P301S-tau expression induces hippocampal tau pathology and marked degeneration of CA1 pyramidal neurons in mice, however, this occurs independently of endogenous or pathogenic LRRK2 expression. We further developed new AAV2/6 vectors co-expressing human WT-tau and GFP to monitor the neuron-to-neuron transmission of tau within defined hippocampal neuronal circuits. While endogenous LRRK2 is not required for tau transmission, we find that G2019S-LRRK2 markedly enhances the neuron-to-neuron transmission of tau in mice. Our data suggest that mutant tau-induced neuropathology occurs independently of LRRK2 expression in two mouse models of tauopathy but identifies a novel pathogenic role for G2019S-LRRK2 in promoting the neuronal transmission of WT-tau protein. These findings may have important implications for understanding the development of tau neuropathology in LRRK2-linked PD brains.

To understand the cause of Parkinson's disease (PD), it is important to determine the functional interactions between factors linked to the disease. Parkin is associated with autosomal recessive early-onset PD, and controls the transcription of PGC-1α, a master regulator of mitochondrial biogenesis. These two factors functionally interact to regulate the turnover and quality of mitochondria, by increasing both mitophagic activity and mitochondria biogenesis. In cortical neurons, co-expressing PGC-1α and Parkin increases the number of mitochondria, enhances maximal respiration, and accelerates the recovery of the mitochondrial membrane potential following mitochondrial uncoupling. PGC-1α enhances Mfn2 transcription, but also leads to increased degradation of the Mfn2 protein, a key ubiquitylation target of Parkin on mitochondria. In vivo, Parkin has significant protective effects on the survival and function of nigral dopaminergic neurons in which the chronic expression of PGC-1α is induced. Ultrastructural analysis shows that these two factors together control the density of mitochondria and their interaction with the endoplasmic reticulum. These results highlight the combined effects of Parkin and PGC-1α in the maintenance of mitochondrial homeostasis in dopaminergic neurons. These two factors synergistically control the quality and function of mitochondria, which is important for the survival of neurons in Parkinson's disease.

The biological actions of extracellular nucleotides are mediated by two distinct classes of P2 receptor, P2X and P2Y. The G protein-coupled P2Y receptors comprise five mammalian subtypes, P2Y(1-11). The P2Y1 subtype is expressed abundantly throughout the human brain and is specifically localized to neuronal structures. In the present study, the distribution of the P2Y1 receptor was investigated in Alzheimer's disease (AD) brains. In contrast to control human brain, the P2Y1 receptor was localized to a number of characteristic AD structures such as neurofibrillary tangles, neuritic plaques and neuropil threads. Immunoblot analysis showed that this specific immunostaining observed over tangles was not a result of cross-reactivity between the anti-P2Y1 antiserum and abnormal tau protein, the major constituent of tangles. The significance of this altered P2Y1 cellular distribution in AD brains is at present unclear.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized pathologically by the degeneration of nigrostriatal pathway dopaminergic neurons and other neuronal systems and the appearance of Lewy bodies that contain α-synuclein. PD is generally a sporadic disease, but a small proportion of cases have a clear genetic component. Mutations have been identified in six genes that clearly segregate with disease in rare families with PD. Transgenic, knockout, and virus-based models of disease have been developed in rodents to further understand how these genes contribute to the pathogenesis of PD. In general, these animal models recapitulate many key features of the disease, including derangements in dopaminergic synaptic transmission, selective neurodegeneration, neurochemical deficits, α-synuclein-positive neuropathology, and motor deficits. However, a genetic model with all or most of these pathogenic features has proved difficult to create. In this article, we discuss these mammalian genetic models of PD and what they have revealed about the cause and mechanisms of this disease.

Neurodegenerative diseases are generally characterized by the selective degeneration of particular neuronal populations and the accumulation of abnormal or aggregated proteins within, but occasionally external to, neurons in affected brain regions. These diseases can be broadly classified as disorders of cognition and memory or movement, and both features can often coexist in a single disease. In recent years, the identification of genetic mutations that cause rare monogenic familial disease has revolutionized our understanding of the molecular basis of neurodegenerative disease and has provided new targets for the development of disease-modifying therapies. An essential part of this process has been the development of genetic animal models that accurately recapitulate the essential features of each disease, with particular emphasis on the use of mouse models. Such mouse models have provided unique insight into the molecular mechanism(s) through which genetic mutations precipitate neurodegeneration and produce associated clinical and pathological phenotypes. In this review, we provide an overview of the current status, uses and limitations of genetic mouse models for understanding major neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's disease and amyotrophic lateral sclerosis.

Mutations in the ATP13A2 (PARK9) gene cause early-onset, autosomal recessive Parkinson's disease (PD) and Kufor–Rakeb syndrome. ATP13A2 mRNA is spliced into three distinct isoforms encoding a P5-type ATPase involved in regulating heavy metal transport across vesicular membranes. Here, we demonstrate that three ATP13A2 mRNA isoforms are expressed in the normal human brain and are modestly increased in the cingulate cortex of PD cases. ATP13A2 can mediate protection toward a number of stressors in mammalian cells and can protect against α-synuclein-induced toxicity in cellular and invertebrate models of PD. Using a primary cortical neuronal model combined with lentiviral-mediated gene transfer, we demonstrate that human ATP13A2 isoforms 1 and 2 display selective neuroprotective effects toward toxicity induced by manganese and hydrogen peroxide exposure through an ATPase-independent mechanism. The familial PD mutations, F182L and G504R, abolish the neuroprotective effects of ATP13A2 consistent with a loss-of-function mechanism. We further demonstrate that the AAV-mediated overexpression of human ATP13A2 is not sufficient to attenuate dopaminergic neurodegeneration, neuropathology, and striatal dopamine and motoric deficits induced by human α-synuclein expression in a rat model of PD. Intriguingly, the delivery of an ATPase-deficient form of ATP13A2 (D513N) to the substantia nigra is sufficient to induce dopaminergic neuronal degeneration and motor deficits in rats, potentially suggesting a dominant-negative mechanism of action. Collectively, our data demonstrate a distinct lack of ATP13A2-mediated protection against α-synuclein-induced neurotoxicity in the rat nigrostriatal dopaminergic pathway, and limited neuroprotective capacity overall, and raise doubts about the potential of ATP13A2 as a therapeutic target for PD.

Coding variation in the Leucine rich repeat kinase 2 gene linked to Parkinson's disease (PD) promotes enhanced activity of the encoded LRRK2 kinase, particularly with respect to autophosphorylation at S1292 and/or phosphorylation of the heterologous substrate RAB10.To determine the inter-laboratory reliability of measurements of cellular LRRK2 kinase activity in the context of wildtype or mutant LRRK2 expression using published protocols.Benchmark western blot assessments of phospho-LRRK2 and phospho-RAB10 were performed in parallel with in situ immunological approaches in HEK293T, mouse embryonic fibroblasts, and lymphoblastoid cell lines. Rat brain tissue, with or without adenovirus-mediated LRRK2 expression, and human brain tissues from subjects with or without PD, were also evaluated for LRRK2 kinase activity markers.Western blots were able to detect extracted LRRK2 activity in cells and tissue with pS1292-LRRK2 or pT73-RAB10 antibodies. However, while LRRK2 kinase signal could be detected at the cellular level with over-expressed mutant LRRK2 in cell lines, we were unable to demonstrate specific detection of endogenous cellular LRRK2 activity in cell culture models or tissues that we evaluated.Further development of reliable methods that can be deployed in multiple laboratories to measure endogenous LRRK2 activities are likely required, especially at cellular resolution.

Neurodegenerative diseases are characterized by the selective degeneration of neuronal populations in different brain regions and frequently the formation of distinct protein aggregates that often overlap between diseases. While the causes of many sporadic neurodegenerative diseases are unclear, genes associated with familial or sporadic forms of disease and the underlying cellular pathways involved tend to support common disease mechanisms. Underscoring this concept, mutations in the Vacuolar Protein Sorting 35 Orthologue (VPS35) gene have been identified to cause late-onset, autosomal dominant familial Parkinson's disease, whereas reduced VPS35 protein levels are reported in vulnerable brain regions of subjects with Alzheimer's disease, neurodegenerative tauopathies such as progressive supranuclear palsy and Pick's disease, and amyotrophic lateral sclerosis. Therefore, VPS35 is commonly implicated in many neurodegenerative diseases. VPS35 plays a critical role in the retromer complex that mediates the retrieval and recycling of transmembrane protein cargo from endosomes to the trans-Golgi network or plasma membrane. VPS35 and retromer function are highly conserved in eukaryotic cells, with the homozygous deletion of VPS35 inducing early embryonic lethality in mice that has hindered an understanding of its role in the brain. Here, we develop conditional knockout mice with the selective deletion of VPS35 in neurons to better elucidate its role in neuronal viability and its connection to neurodegenerative diseases. Surprisingly, the pan-neuronal deletion of VPS35 induces a progressive and rapid disease with motor deficits and early post-natal lethality. Underlying this neurological phenotype is the relatively selective and robust degeneration of motor neurons in the spinal cord. Neuronal loss is accompanied and preceded by the formation of p62-positive protein inclusions and robust reactive astrogliosis. Our study reveals a critical yet unappreciated role for VPS35 function in the normal maintenance and survival of motor neurons during post-natal development that has important implications for neurodegenerative diseases, particularly amyotrophic lateral sclerosis.

The identification of Parkinson's disease (PD)-associated genes has created a powerful platform to begin to understand and nominate pathophysiological disease mechanisms. Herein, we discuss the genetic and experimental evidence supporting endolysosomal dysfunction as a major pathway implicated in PD. Well-studied familial PD-linked gene products, including LRRK2, VPS35, and α-synuclein, demonstrate how disruption of different aspects of endolysosomal sorting pathways by disease-causing mutations may manifest into PD-like phenotypes in many disease models. Newly-identified PD-linked genes, including auxilin, synaptojanin-1 and Rab39b, as well as putative risk genes for idiopathic PD (endophilinA1, Rab29, GAK), further support endosomal sorting deficits as being central to PD. LRRK2 may represent a nexus by regulating many distinct features of endosomal sorting, potentially via phosphorylation of key endocytosis machinery (i.e., auxilin, synaptojanin-1, endoA1) and Rab GTPases (i.e., Rab29, Rab8A, Rab10) that function within these pathways. In turn, LRRK2 kinase activity is critically regulated by Rab29 at the Golgi complex and retromer-associated VPS35 at endosomes. Taken together, the known functions of PD-associated gene products, the impact of disease-linked mutations, and the emerging functional interactions between these proteins points to endosomal sorting pathways as a key point of convergence in the pathogenesis of PD.

As individuals enter their 80s, they are inevitably confronted with the problem of neuronal loss in the brain. The incidence of the common movement disorder 'mild parkinsonian signs' (MPS) is approximately 50% over the age of 85 years. It has long been known that the loss of dopaminergic neurons in the substantia nigra pars compacta is a neuropathological hallmark of Parkinson's disease (PD). Recently, two papers present clear evidence for a high burden of mitochondrial DNA deletions within substantia nigra neurons in aged individuals and individuals with PD, pointing towards a common pathway inevitably leading to neuronal dysfunction and death.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are an important cause of late-onset, familial and sporadic Parkinson's disease. LRRK2 is a large unique protein containing both GTPase and kinase enzymatic domains together with multiple protein-protein interaction domains. LRRK2 initially appears to function as a GTPase-regulated protein kinase. The majority of pathogenic mutations lead to enhanced kinase activity of LRRK2. Disease-associated mutations in LRRK2 also promote the formation of cytoplasmic inclusions and induce neuronal toxicity in cultured cells in a kinase-dependent manner. These and other important aspects of LRRK2 biology and pathophysiology are discussed in detail in this review.

Parkinson disease (PD) is a relatively common neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra. About 5%-10% of PD cases are inherited. Mutations in the Parkin gene, which encodes a protein that can function as an E3 ubiquitin ligase, are a common cause of familial PD. Such mutations act in a loss-of-function manner and impair the ability of the encoded protein to mediate substrate ubiquitination, although the subsequent molecular pathway that precipitates neuronal degeneration is poorly defined. In this issue of the JCI, Kim and colleagues describe painstaking evidence using a number of dissecting approaches in intact animals and cultured cells to functionally link Parkin and the class B scavenger receptor CD36, suggesting a novel and complex connection between PD and fatty acid metabolism.

Mutations in the leucine-rich repeat kinase 2 (LRRK2, PARK8 ) gene represent the most common cause of familial Parkinson’s diseaseParkinson’s disease (PD) with autosomal dominant inheritance, whereas common variation at the LRRK2 genomic locus influences the risk of developing idiopathic PD. LRRK2 is a member of the ROCO protein family and contains multiple domains, including Ras-of-Complex (ROC) GTPase, kinase, and protein-protein interaction domains. In the last decade, the biochemical characterization of LRRK2LRRK2 and the development of animal modelanimal model s have provided important insight into the pathobiology of LRRK2. In this review, we comprehensively describe the different models employed to understand LRRK2-associated PD, including yeast, invertebrates, transgenic and viral-based rodents, and patient-derived induced pluripotent stem cells. We discuss how these models have contributed to understanding LRRK2 pathobiology and the advantages and limitations of each model for exploring aspects of LRRK2-associated PD.

Leucine-rich repeat kinase 2 (LRRK2) and α-synuclein share enigmatic roles in the pathobiology of Parkinson's disease (PD). LRRK2 mutations are a common genetic cause of PD which, in addition to neurodegeneration, often present with abnormal deposits of α-synuclein in the form of Lewy-related pathology. As Lewy-related pathology is a prominent neuropathologic finding in sporadic PD, the relationship between LRRK2 and α-synuclein has garnered considerable interest. However, whether and how LRRK2 might influence the accumulation of Lewy-related pathology remains poorly understood. Through stereotactic injection of mouse α-synuclein pre-formed fibrils (PFF), we modeled the spread of Lewy-related pathology within forebrain regions where LRRK2 is most highly expressed. The impact of LRRK2 genotype on the formation of α-synuclein inclusions was evaluated at 1-month post-injection. Neither deletion of LRRK2 nor G2019S LRRK2 knockin appreciably altered the burden of α-synuclein pathology at this early timepoint. These observations fail to provide support for a robust pathophysiologic interaction between LRRK2 and α-synuclein in the forebrain in vivo. There was, however, a modest reduction in microglial activation induced by PFF delivery in the hippocampus of LRRK2 knockout mice, suggesting that LRRK2 may contribute to α-synuclein-induced neuroinflammation. Collectively, our data indicate that the pathological accumulation of α-synuclein in the mouse forebrain is largely independent of LRRK2.

Cognitive dysfunction is a salient feature of Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). The onset of dementia reflects the spread of Lewy pathology throughout forebrain structures. The mere presence of Lewy pathology, however, provides limited indication of cognitive status. Thus, it remains unclear whether Lewy pathology is the de facto substrate driving cognitive dysfunction in PD and DLB. Through application of α-synuclein fibrils in vivo, we sought to examine the influence of pathologic inclusions on cognition. Following stereotactic injection of α-synuclein fibrils within the mouse forebrain, we measured the burden of α-synuclein pathology at 1-, 3-, and 6-months post-injection within subregions of the hippocampus and cortex. Under this paradigm, the hippocampal CA2/3 subfield was especially susceptible to α-synuclein pathology. Strikingly, we observed a drastic reduction of pathology in the CA2/3 subfield across time-points, consistent with the consolidation of α-synuclein pathology into dense somatic inclusions followed by neurodegeneration. Silver-positive degenerating neurites were observed prior to neuronal loss, suggesting that this might be an early feature of fibril-induced neurotoxicity and a precursor to neurodegeneration. Critically, mice injected with α-synuclein fibrils developed progressive deficits in spatial learning and memory. These findings support that the formation of α-synuclein inclusions in the mouse forebrain precipitate neurodegenerative changes that recapitulate features of Lewy-related cognitive dysfunction.

Although most cases of Parkinson's disease (PD) are sporadic, mutations in over 20 genes are known to cause heritable forms of PD. A surprising number of familial PD-linked genes and PD risk genes are involved in intracellular trafficking and protein degradation. Recessive loss-of-function mutations in ATP13A2 , a lysosomal transmembrane P5B-type ATPase and polyamine exporter, can cause early-onset familial PD. Familial ATP13A2 mutations are also linked to related neurodegenerative diseases, including Kufor-Rakeb syndrome (KRS), hereditary spastic paraplegias (HSPs), neuronal ceroid lipofuscinosis, and amyotrophic lateral sclerosis (ALS). Given the severe effects of ATP13A2 mutations in humans, it is surprising that ATP13A2 knockout (KO) mice fail to exhibit neurodegeneration even at advanced ages. This discrepancy between human subjects and rodents makes it challenging to study the neuropathological effects of ATP13A2 loss in vivo. Germline deletion of ATP13A2 in rodents may trigger the upregulation of compensatory pathways during embryonic development that mask the full neurotoxic effects of ATP13A2 loss in the brain. To explore this idea, we selectively deleted ATP13A2 in the adult mouse brain by the unilateral delivery of an AAV-Cre vector into the substantia nigra of young adult mice carrying conditional loxP -flanked ATP13A2 KO alleles. We observe a progressive loss of striatal dopaminergic nerve terminals at 3 and 10 months after AAV-Cre delivery. Cre-injected mice also exhibit robust dopaminergic neuronal degeneration in the substantia nigra at 10 months. Adult-onset ATP13A2 KO also recreates many of the phenotypes observed in aged germline ATP13A2 KO mice, including lysosomal abnormalities, p62-positive inclusions, and neuroinflammation. Our study demonstrates that the adult-onset homozygous deletion of ATP13A2 in the nigrostriatal pathway produces robust and progressive dopaminergic neurodegeneration that serves as a useful in vivo model of ATP13A2 -related neurodegenerative diseases.

In 2011, the UK medical research charity Cure Parkinson’s set up the international Linked Clinical Trials (iLCT) committee to help expedite the clinical testing of potentially disease modifying therapies for Parkinson’s disease (PD). The first committee meeting was held at the Van Andel Institute in Grand Rapids, Michigan in 2012. This group of PD experts has subsequently met annually to assess and prioritize agents that may slow the progression of this neurodegenerative condition, using a systematic approach based on preclinical, epidemiological and, where possible, clinical data. Over the last 12 years, 171 unique agents have been evaluated by the iLCT committee, and there have been 21 completed clinical studies and 20 ongoing trials associated with the initiative. In this review, we briefly outline the iLCT process as well as the clinical development and outcomes of some of the top prioritized agents. We also discuss a few of the lessons that have been learnt, and we conclude with a perspective on what the next decade may bring, including the introduction of multi-arm, multi-stage clinical trial platforms and the possibility of combination therapies for PD.

Mutations in the DJ-1 gene are associated with rare forms of autosomal recessive early-onset Parkinson's disease (PD). Although the precise physiological function of DJ-1 remains obscure, accumulating evidence suggests that DJ-1 may normally function as a redox-sensitive molecular chaperone that can protect against the deleterious effects of oxidative stress, particularly in mitochondria. Recent studies in the fruit fly, Drosophila melanogaster, have shed further light on the biological role of DJ-1. DJ-1-deficient Drosophila models exhibit distinct phenotypes but collectively highlight a prominent neuroprotective role for DJ-1 against oxidative insult. However, Drosophila lacking DJ-1 do not consistently produce a useful PD-like phenotype (that is, they generally fail to exhibit degeneration of neurons that contain the neurotransmitter dopamine), which may reflect putative compensatory neuroprotective mechanisms. DJ-1-deficient fly models further highlight the utility of Drosophila as an important tool for elucidating protein function and for modeling neurodegenerative disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) instigate an autosomal dominant form of Parkinson’s disease (PD). Despite the neuropathological heterogeneity observed in LRRK2-PD, accumulating evidence suggests that alpha-synuclein and tau pathology are observed in a vast majority of cases. Intriguingly, the presence of protein aggregates spans both LRRK2-PD and idiopathic disease, supportive of a common pathologic mechanism. Thus, it is important to consider how LRRK2 mutations give rise to such pathology, and whether targeting LRRK2 might modify the accumulation, transmission, or toxicity of protein aggregates. Likewise, it is not clear how LRRK2 mutations drive PD pathogenesis, and whether protein aggregates are implicated in LRRK2-dependent neurodegeneration. While animal models have been instrumental in furthering our understanding of a potential interaction between LRRK2 and protein aggregation, the biology is far from clear. We aim to provide a thoughtful overview of the evidence linking LRRK2 to protein aggregation in animal models.

Abstract Background LRRK2-targeting therapeutics that inhibit LRRK2 kinase activity have advanced to clinical trials in idiopathic Parkinson’s disease (iPD). LRRK2 phosphorylates Rab10 on endolysosomes in phagocytic cells to promote some types of immunological responses. The identification of factors that regulate LRRK2-mediated Rab10 phosphorylation in iPD, and whether phosphorylated-Rab10 levels change in different disease states, or with disease progression, may provide insights into the role of Rab10 phosphorylation in iPD and help guide therapeutic strategies targeting this pathway. Methods Capitalizing on past work demonstrating LRRK2 and phosphorylated-Rab10 interact on vesicles that can shed into biofluids, we developed and validated a high-throughput single-molecule array assay to measure extracellular pT73-Rab10. Ratios of pT73-Rab10 to total Rab10 measured in biobanked serum samples were compared between informative groups of transgenic mice, rats, and a deeply phenotyped cohort of iPD cases and controls. Multivariable and weighted correlation network analyses were used to identify genetic, transcriptomic, clinical, and demographic variables that predict the extracellular pT73-Rab10 to total Rab10 ratio. Results pT73-Rab10 is absent in serum from Lrrk2 knockout mice but elevated by LRRK2 and VPS35 mutations, as well as SNCA expression. Bone-marrow transplantation experiments in mice show that serum pT73-Rab10 levels derive primarily from circulating immune cells. The extracellular ratio of pT73-Rab10 to total Rab10 is dynamic, increasing with inflammation and rapidly decreasing with LRRK2 kinase inhibition. The ratio of pT73-Rab10 to total Rab10 is elevated in iPD patients with greater motor dysfunction, irrespective of disease duration, age, sex, or the usage of PD-related or anti-inflammatory medications. pT73-Rab10 to total Rab10 ratios are associated with neutrophil activation, antigenic responses, and the suppression of platelet activation. Conclusions The extracellular ratio of pT73-Rab10 to total Rab10 in serum is a novel pharmacodynamic biomarker for LRRK2-linked innate immune activation associated with disease severity in iPD. We propose that those iPD patients with higher serum pT73-Rab10 levels may benefit from LRRK2-targeting therapeutics to mitigate associated deleterious immunological responses.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most frequent cause of late-onset, familial Parkinson's disease (PD), and LRRK2 variants are associated with increased risk for sporadic PD. While advanced age represents the strongest risk factor for disease development, it remains unclear how different age-related pathways interact to regulate LRRK2-driven late-onset PD. In this study, we employ a C. elegans model expressing PD-linked G2019S LRRK2 to examine the interplay between age-related pathways and LRRK2-induced dopaminergic neurodegeneration. We find that multiple genetic pathways that regulate lifespan extension can provide robust neuroprotection against mutant LRRK2. However, the level of neuroprotection does not strictly correlate with the magnitude of lifespan extension, suggesting that lifespan can be experimentally dissociated from neuroprotection. Using tissue-specific RNAi, we demonstrate that lifespan-regulating pathways, including insulin/insulin-like growth factor-1 (IGF-1) signaling, target of rapamycin (TOR), and mitochondrial respiration, can be directly manipulated in neurons to mediate neuroprotection. We extend this finding for AGE-1/PI3K, where pan-neuronal versus dopaminergic neuronal restoration of AGE-1 reveals both cell-autonomous and non-cell-autonomous neuroprotective mechanisms downstream of insulin signaling. Our data demonstrate the importance of distinct lifespan-regulating pathways in the pathogenesis of LRRK2-linked PD, and suggest that extended longevity is broadly neuroprotective via the actions of these pathways at least in part within neurons. This study further highlights the complex interplay that occurs between cells and tissues during organismal aging and disease manifestation.

Parkinson's disease is a sporadic and common neurodegenerative movement disorder resulting from the complex interplay between genetic risk, aging and environmental exposure. Familial forms of PD account for ~10% of cases and are known to result from the inheritance of mutations in at least 15 genes. Mutations in the vacuolar protein sorting 35 ortholog (VPS35) gene cause late-onset, autosomal dominant familial PD. VPS35 is a key suunit of the pentameric retromer complex that plays a role in the retrograde sorting and recycling of transmembrane cargo proteins from endosomes to the plasma membrane and trans-Golgi network. A single heterozygous Asp620Asn (D620N) mutation in VPS35 has been identified in multiple families that segregates with PD, and a number of experimental cellular and animal models have been developed to understand its pathogenic effects. At the molecular level, the D620N mutation has been shown to impair the interaction of VPS35 with the WASH complex, that plays an accessory function in retromer-dependent sorting. In addition, the D620N mutation has been linked to the abnormal sorting of retromer cargo, including CI-M6PR, AMPA receptor subunits, MUL1, LAMP2a and ATG9A, as well as to LRRK2 hyperactivation. At the cellular level, data support an impact of D620N VPS35 on mitochondrial function, the autophagy-lysosomal pathway, Wnt signaling and neurotransmission via altered endosomal sorting. The relevance of abnormal retromer sorting and cellular pathways to PD-related neurodegenerative phenotypes induced by D620N VPS35 in rodent models is not yet clear. There is also uncertainty regarding the mechanism-of-action of the D620N mutation and whether it manifests pathogenic effects in animal models and PD through a gain-of-function and/or a partial dominant-negative mechanism. Here, we discuss the emerging molecular and cellular mechanisms underlying PD induced by familial VPS35 mutations, going from structure to cellular function to neuropathology. We further discuss studies linking reduced retromer function to other neurodegenerative diseases and potential therapeutic strategies to normalize retromer function to mitigate disease.

Cognitive dysfunction is a salient feature of Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). The onset of dementia reflects the spread of Lewy pathology throughout forebrain structures. The mere presence of Lewy pathology, however, provides limited indication of cognitive status. Thus, it remains unclear whether Lewy pathology is the de facto substrate driving cognitive dysfunction in PD and DLB. Through application of α-synuclein fibrils in vivo , we sought to examine the influence of pathologic inclusions on cognition. Following stereotactic injection of α-synuclein fibrils within the mouse forebrain, we measured the burden of α-synuclein pathology at 1-, 3-, and 6-months post-injection within subregions of the hippocampus and cortex. Under this paradigm, the hippocampal CA2/3 subfield was especially susceptible to α- synuclein pathology. Strikingly, we observed a drastic reduction of pathology in the CA2/3 subfield across time-points, consistent with the consolidation of α-synuclein pathology into dense somatic inclusions followed by neurodegeneration. Silver-positive degenerating neurites were observed prior to neuronal loss, suggesting that this might be an early feature of fibril-induced neurotoxicity and a precursor to neurodegeneration. Critically, mice injected with α-synuclein fibrils developed progressive deficits in spatial learning and memory. These findings support that the formation of α-synuclein inclusions in the mouse forebrain precipitate neurodegenerative changes that recapitulate features of Lewy-related cognitive dysfunction.Mice injected with α-synuclein fibrils develop hippocampal and cortical α- synuclein pathology with a dynamic regional burden at 1-, 3-, and 6-months post-injection.Silver-positive neuronal processes are an early and enduring degenerative feature of the fibril model, while extensive neurodegeneration of the hippocampal CA2/3 subfield is detected at 6-months post-injection.Mice exhibit progressive hippocampal-dependent spatial learning and memory deficits.Forebrain injection of α-synuclein fibrils may be used to model aspects of Lewy-related cognitive dysfunction.

Mutations in the vacuolar protein sorting 35 ortholog (VPS35) gene cause late-onset, autosomal dominant Parkinson's disease (PD), with a single missense mutation (Asp620Asn, D620N) known to segregate with disease in families with PD. The VPS35 gene encodes a core component of the retromer complex, involved in the endosomal sorting and recycling of transmembrane cargo proteins. VPS35-linked PD is clinically indistinguishable from sporadic PD, although it is not yet known whether VPS35-PD brains exhibit α-synuclein-positive brainstem Lewy pathology that is characteristic of sporadic cases. Prior studies have suggested a functional interaction between VPS35 and the PD-linked gene product α-synuclein in lower organisms, where VPS35 deletion enhances α-synuclein-induced toxicity. In mice, VPS35 overexpression is reported to rescue hippocampal neuronal loss in human α-synuclein transgenic mice, potentially suggesting a retromer deficiency in these mice.Here, we employ multiple well-established genetic rodent models to explore a functional or pathological interaction between VPS35 and α-synuclein in vivo.We find that endogenous α-synuclein is dispensable for nigrostriatal pathway dopaminergic neurodegeneration induced by the viral-mediated delivery of human D620N VPS35 in mice, suggesting that α-synuclein does not operate downstream of VPS35. We next evaluated retromer levels in affected brain regions from human A53T-α-synuclein transgenic mice, but find normal levels of the core subunits VPS35, VPS26 or VPS29. We further find that heterozygous VPS35 deletion fails to alter the lethal neurodegenerative phenotype of these A53T-α-synuclein transgenic mice, suggesting the absence of retromer deficiency in this PD model. Finally, we explored the neuroprotective capacity of increasing VPS35 expression in a viral-based human wild-type α-synuclein rat model of PD. However, we find that the overexpression of wild-type VPS35 is not sufficient for protection against α-synuclein-induced nigral dopaminergic neurodegeneration, α-synuclein pathology and reactive gliosis.Collectively, our data suggest a limited interaction of VPS35 and α-synuclein in neurodegenerative models of PD, and do not provide support for their interaction within a common pathophysiological pathway.

Leucine-rich repeat kinase 2 (LRRK2) and α-synuclein share enigmatic roles in the pathobiology of Parkinson's disease (PD). LRRK2 mutations are a common genetic cause of PD which, in addition to neurodegeneration, often present with abnormal deposits of α-synuclein in the form of Lewy-related pathology. As Lewy-related pathology is a prominent neuropathologic finding in sporadic PD, the relationship between LRRK2 and α-synuclein has garnered considerable interest. However, whether and how LRRK2 might influence the accumulation of Lewy-related pathology remains poorly understood. Through stereotactic injection of mouse α-synuclein pre-formed fibrils (PFF), we modeled the spread of Lewy-related pathology within forebrain regions where LRRK2 is most highly expressed. The impact of LRRK2 genotype on the formation of α-synuclein inclusions was evaluated at 1-month post-injection. Neither deletion of LRRK2 nor G2019S LRRK2 knockin appreciably altered the burden of α-synuclein pathology at this early timepoint. These observations fail to provide support for a robust pathophysiologic interaction between LRRK2 and α-synuclein in the forebrain in vivo. There was, however, a modest reduction in microglial activation induced by PFF delivery in the hippocampus of LRRK2 knockout mice, suggesting that LRRK2 may contribute to α-synuclein-induced neuroinflammation. Collectively, our data indicate that the pathological accumulation of α-synuclein in the mouse forebrain is largely independent of LRRK2.

We report here that human Ntera-2/D1 (NT-2) cells, an undifferentiated committed neuronal progenitor cell line, endogenously express a functional P2Y(1) receptor, while other P2Y subtypes, except perhaps P2Y(4), are not functionally expressed. Quantitative RT-PCR analysis showed that NT-2 cells abundantly express mRNA for P2Y(1) and P2Y(11) receptors, while P2Y(2) and P2Y(4) receptors were detected at considerably lower levels. Western blot analysis also demonstrated expression of P2Y(1) receptors and Galpha(q/11) subunits. Various nucleotides induced intracellular Ca(2+) mobilisation in NT-2 cells in a concentration-dependent manner with a rank order potency of 2-MeSADP > 2-MeSATP > ADP > ATP > UTP > ATPgammaS, a profile resembling that of human P2Y(1) receptors. Furthermore, P2Y(1) receptor-specific (A3P5P) and P2Y-selective (PPADS, suramin) antagonists inhibited adenine nucleotide-induced Ca(2+) responses in a concentration-dependent manner, consistent with expression of a P2Y(1) receptor. Moreover, of seven adenine nucleotides tested, only Bz-ATP and ATPgammaS elicited small increases in cAMP formation suggesting that few, if any, functional P2Y(11) receptors were expressed. P2Y(1) receptor-selective adenine nucleotides, including 2-MeSADP and ADP, also induced concentration-dependent phosphorylation and hence, activation of the extracellular-signal regulated protein kinases (ERK1/2). NT-2 cells, therefore, provide a useful neuronal-like cellular model for studying the precise signalling pathways and physiological responses mediated by a native P2Y(1) receptor.

Annals of NeurologyVolume 54, Issue 3 p. 281-282 Editorial Genetics of Parkinson's disease: What do mutations in DJ-1 tell us? Darren J. Moore PhD, Darren J. Moore PhD Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD Departments of Neurology, Johns Hopkins University School of Medicine, Baltimore, MDSearch for more papers by this authorValina L. Dawson PhD, Valina L. Dawson PhD Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD Departments of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD Neuroscience Johns Hopkins University School of Medicine, Baltimore, MD Physiology, Johns Hopkins University School of Medicine, Baltimore, MDSearch for more papers by this authorTed M. Dawson MD, PhD, Ted M. Dawson MD, PhD Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD Departments of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD Neuroscience Johns Hopkins University School of Medicine, Baltimore, MDSearch for more papers by this author Darren J. Moore PhD, Darren J. Moore PhD Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD Departments of Neurology, Johns Hopkins University School of Medicine, Baltimore, MDSearch for more papers by this authorValina L. Dawson PhD, Valina L. Dawson PhD Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD Departments of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD Neuroscience Johns Hopkins University School of Medicine, Baltimore, MD Physiology, Johns Hopkins University School of Medicine, Baltimore, MDSearch for more papers by this authorTed M. Dawson MD, PhD, Ted M. Dawson MD, PhD Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD Departments of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD Neuroscience Johns Hopkins University School of Medicine, Baltimore, MDSearch for more papers by this author First published: 28 August 2003 https://doi.org/10.1002/ana.10740Citations: 8Read 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 No abstract is available for this article. References 1 Giasson BI, Lee VM. Are ubiquitination pathways central to Parkinson's disease? Cell 2003; 114: 1– 8. 2 Dawson TM, Dawson VL. Rare genetic mutations shed light on the pathogenesis of Parkinson disease. J Clin Invest 2003; 111: 145– 151. 3 Lansbury PT, Jr., Brice A. Genetics of Parkinson's disease and biochemical studies of implicated gene products. Curr Opin Genet Dev 2002; 12: 299– 306. 4 Hardy J, Cookson MR, Singleton A. Genes and parkinsonism. Lancet Neurol 2003; 2: 221– 228. 5 Cookson MR. Pathways to Parkinsonism. Neuron 2003; 37: 7– 10. 6 Bonifati V, Rizzu P, Van Baren MJ, et al. Mutations in the DJ–1 Gene Associated with Autosomal Recessive Early–Onset Parkinsonism. Science 2002; 299: 256– 259. 7 Lucking CB, Durr A, Bonifati V et al. Association between early–onset Parkinson's disease and mutations in the parkin gene. French Parkinson's Disease Genetics Study Group. N Engl J Med 2000; 342: 1560– 1567. 8 Wilson MA, Collins JL, Hod Y, et al. The 1.1–A resolution crystal structure of DJ–1, the protein mutated in autosomal recessive early onset Parkinson's disease. Proc Natl Acad Sci USA 2003; in press. 9 Tao X, Tong L. Crystal structure of human DJ–1, a protein associated with early–onset Parkinson's diseasec. J Biol Chem 2003; in press. 10 Honbou K, Suzuki NN, Horiuchi M, et al. The crystal structure of DJ–1, a protein related to male fertility and Parkinson's disease. J Biol Chem 2003; in press. 11 Abou–Sleiman PM, Healy DG, Quinn N, et al. The role of pathogenic DJ–1 mutations in Parkinson's disease. Ann Neurol 2003; 54: 283– 286. 12 Hague S, Rogaeva E, Hernandez D, et al. Early–onset Parkinson's disease caused by a compound heterozygous DJ–1 mutation. Ann Neurol 2003; 54: 271– 274. Citing Literature Volume54, Issue3September 2003Pages 281-282 ReferencesRelatedInformation

Reference EPFL-ARTICLE-159659doi:10.1016/j.bbadis.2009.06.001View record in Web of Science Record created on 2010-11-30, modified on 2016-08-09

Parkinson's disease (PD), the most common neurodegenerative movement disorder, affects about 2% of the population over the age of 65. That percentage increases to about 5% in people over the age of 85 (Lang & Lozano, 1998a, 1998b). PD is clinically characterized by its motor symptoms including resting tremor, rigidity, bradykinesia, and postural instability. Neuropathologically, PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta resulting in reduced levels of dopamine in the striatum (Lang & Lozano, 1998a). Dopaminergic cell loss is also accompanied by the accumulation of intracellular inclusions termed Lewy bodies, composed of aggregated protein, of which α-synuclein is a major component (Lang & Lozano, 1998a; Spillantini et al., 1997). Currently, no disease-modifying treatments for PD exist, and symptomatic treatments do not offer long-term disease control. The lack of disease-modifying treatments can be attributed to the fact that the mechanism(s) by which dopaminergic neurons die remain unclear. Although PD is largely an idiopathic disease, 5–10% of cases are hereditary. To date, mutations in at least 13 genes have been shown to cause familial PD, including autosomal dominant mutations in the vacuolar protein sorting 35 ortholog (VPS35, PARK17) gene (Hernandez, Reed, & Singleton, 2016; Vilariño-Güell et al., 2011; Zimprich et al., 2011). A single missense mutation in VPS35 (D620N) was originally reported in 2011 in Swiss and Austrian families by two independent groups. Since its initial discovery, the D620N mutation has been shown to be present in PD subjects worldwide (Vilariño-Güell et al., 2011; Zimprich et al., 2011). Although the D620N mutation has been shown to clearly segregate with disease, several other rare putative pathogenic mutations in VPS35 have been identified (i.e. P316S, R524W, L774M, etc.) but their disease association remains unclear. Clinically, VPS35-associated PD presents like that of idiopathic PD being progressive, tremor-predominant and responsive to levodopa treatment. The neuropathological characteristics of VPS35-associated PD have yet to be established (Williams, Chen, & Moore, 2017). The VPS35 protein functions as a subunit of the retromer complex, a heteropentameric complex composed of VPS35, VPS26, VPS29, and a SNX-BAR dimer, to facilitate endosome-to-Golgi complex and endosome-to-plasma membrane transport of transmembrane protein cargo (Williams et al., 2017). Interestingly, the D620N mutation in VPS35 does not disrupt its interaction with other components of the retromer or disrupt the transport of canonical protein cargo. Studies from our laboratory have demonstrated that overexpression of D620N VPS35 induces toxicity in primary rodent cortical neurons and in vivo using viral-mediated gene transfer (Tsika et al., 2014). Additionally, overexpression of the D620N mutant has been shown to induce neurodegeneration in a Drosophila model (H. S. Wang et al., 2014). However, the molecular mechanism through which the D620N mutation in VPS35 acts to induce neurodegeneration in experimental models and human disease remains unclear. To date, three cellular mechanisms have been identified that are disrupted by the D620N mutation in VPS35. Those mechanisms include WASH complex binding defects, AMPA receptor trafficking defects, and impaired mitochondrial function and dynamics (Williams et al., 2017). Briefly, the D620N mutation has been shown by two independent groups to impair binding to the WASH complex (McGough et al., 2014; Zavodszky et al., 2014). The WASH complex is an accessory protein complex that can bind to a small percentage of the retromer and facilitate the formation of F-actin patches that help guide cargo for transport (Williams et al., 2017). Interestingly, impaired WASH complex binding results in macroautophagy defects through abnormal trafficking of the autophagy receptor ATG9A (Zavodszky et al., 2014). The D620N mutation has also been shown to impair AMPA receptor trafficking both through overexpression of D620N VPS35 and depletion of VPS35 protein (Munsie et al., 2014; Tian et al., 2015). When overexpressed, the D620N mutation causes defects in the trafficking of GluR1 which causes downstream synaptic transmission defects (Munsie et al., 2014). Interestingly, neurons with depletion of VPS35 also exhibit abnormal synaptic transmission and trafficking of both GluR1 and GluR2 (Tian et al., 2015). Finally, the D620N mutation has also been implicated in mitochondrial dysfunction by increasing mitochondrial fragmentation when overexpressed through abnormal recycling of the fission factor, DLP1 (W. Wang et al., 2016). Additionally, VPS35 knockout in mice is reported to impair mitochondrial fusion through reducing the levels of fusion factor, MFN2 (Tang et al., 2015). While intriguing, none of these disrupted pathways have been shown yet to directly impact neurodegeneration therefore leaving the tantalizing possibility that the true cause of D620N VPS35-induced neurotoxicity has yet to be identified. With this in mind, we are actively using several different methods to dissect the protein interactome of VPS35 (WT and D620N) to determine differential interactors that may underlie D620N VPS35-induced neurodegeneration. In addition to the above proposed cellular mechanisms of D620N VPS35-induced dysfunction, VPS35 has also been shown to interact with other PD-linked proteins including LRRK2, α-synuclein, and parkin, raising the interesting possibility that these proteins converge on common pathways, that when disrupted, could lead to neurodegeneration (Williams et al., 2017). Deciphering how VPS35 acts alone and in concert with other PD-linked proteins will be critical to our understanding of the pathogenic events that underlie PD pathophysiology. None declared.

A bstract Mutations in the leucine-rich repeat kinase 2 ( LRRK2 ) gene cause late-onset, autosomal dominant familial Parkinson’s disease (PD) and represent the most common known cause of PD. LRRK2 can function as both a protein kinase and GTPase and PD-linked mutations are known to influence both of these enzymatic activities. While PD-linked LRRK2 mutations can commonly induce neuronal damage and toxicity in cellular models, the mechanisms underlying these pathogenic effects remain uncertain. Rodent models based upon familial LRRK2 mutations often lack the hallmark features of PD and robust neurodegenerative phenotypes in general. Here, we develop a robust pre-clinical model of PD in adult rats induced by the brain delivery of recombinant adenoviral vectors with neuronal-specific expression of full-length human LRRK2 harboring the most common G2019S mutation. In this model, G2019S LRRK2 induces the robust degeneration of substantia nigra dopaminergic neurons, a pathological hallmark of PD. Introduction of a stable kinase-inactive mutation or in-diet dosing with the selective kinase inhibitor, PF-360, attenuates neurodegeneration induced by G2019S LRRK2. Neuroprotection provided by pharmacological kinase inhibition is mediated by an unusual mechanism involving the selective and robust destabilization of human LRRK2 protein in the rat brain relative to endogenous LRRK2. Our study further demonstrates that dopaminergic neurodegeneration induced by G2019S LRRK2 critically requires normal GTPase activity. The introduction of hypothesis-testing mutations that increase GTP hydrolysis or impair GTP binding activity provide neuroprotection against G2019S LRRK2 via distinct mechanisms. Taken together, our data demonstrate that G2019S LRRK2 induces neurodegeneration in vivo via a mechanism that is dependent on kinase and GTPase activity. Our study provides a robust rodent model of LRRK2 -linked PD and nominates kinase inhibition and modulation of GTPase activity as promising disease-modifying therapeutic targets.

Mutations in the leucine-rich repeat kinase 2 (LRRK2, PARK8) gene have been identified as an important cause of late-onset, autosomal dominant familial Parkinson's disease (PD) and also contribute to sporadic PD. The LRRK2 gene encodes a large multi-domain protein that functions as both a GTPase and a serine-/threonine-directed protein kinase. Disease-associated mutant forms of human LRRK2 induce neuronal toxicity through a mechanism dependent on both GTPase and kinase activity. However, the molecular mechanisms underlying LRRK2-linked disease are poorly understood. Here we describe two distinct models that we have developed to probe the pathophysiology of LRRK2. Firstly, we have developed a simple model of LRRK2-induced cytotoxicity in the baker's yeast, Saccharomyces cerevisiae. In this model, a key role for the GTPase domain is revealed in mediating the detrimental effects of LRRK2. LRRK2 toxicity in yeast can be modulated by altering GTPase activity and is associated with defects in vesicular trafficking pathways. LRRK2-induced toxicity acts through a mechanism distinct from toxicity induced by human alpha-synuclein in yeast. Genome-wide screening has identified a number of genetic interactions that enhance or suppress LRRK2-induced toxicity. This yeast model will provide novel insight into the molecular mechanisms underlying LRRK2-induced cellular toxicity. To model LRRK2-linked disease in vivo, we have also developed transgenic mice expressing full-length human LRRK2 variants from a hybrid CMV-enhanced human PDGF-beta promoter. These mice exhibit widespread transgene expression throughout the brain, including the nigrostriatal dopaminergic pathway. Mutant LRRK2 transgenic mice display subtle motoric and neurochemical abnormalities. However, G2019S mutant LRRK2 mice exhibit a loss of substantia nigra dopaminergic neurons and neuritic processes at advanced ages concomitant with ultrastructural cytopathology. These LRRK2 transgenic mice provide important tools for understanding the molecular mechanisms through which disease-associated LRRK2 mutations induce neurodegeneration in PD. The general biology and pathophysiology of LRRK2 will be discussed.

Reference EPFL-CONF-159572View record in Web of Science Record created on 2010-11-30, modified on 2017-05-12

Gay bear refers to a burly gay man with a hirsute body and face. Chinese gay bear men are highly homogeneous and strictly emphasize a uniform bear appearance; however, obesity is an obvious health issue in this population. This study aims to explore the Chinese gay bear men's inner conflicts between bear identity and health concerns. Eleven Chinese gay bear men including four Taiwanese, two mainland Chinese, two Hong Kong, two Malaysian, and one Singaporean were interviewed. The study used a thematic analysis approach and found three coping strategies including (a) Eat healthy but maintain a minimal bear standard; (b) Eat like a bear but go to gym and take physical exam; (c) Reframe the meaning of being a bear or reduce the need of being a bear. This study expects to increase health professionals' knowledge about Chinese gay bear men's inner conflicts between identity and health and to suggest coping strategies for health professionals when addressing this population's health issues.

Abstract Mutations in the vacuolar protein sorting 35 ortholog ( VPS35 ) gene cause late-onset, autosomal dominant Parkinson’s disease (PD), with a single missense mutation (Asp620Asn, D620N) known to segregate with disease in families with PD. The VPS35 gene encodes a core component of the retromer complex, involved in the endosomal sorting and recycling of transmembrane cargo proteins. VPS35 -linked PD is clinically indistinguishable from sporadic PD, although it is not yet known whether VPS35 -PD brains exhibit α-synuclein-positive brainstem Lewy pathology that is characteristic of sporadic cases. Prior studies have suggested a functional interaction between VPS35 and the PD-linked gene product α-synuclein in lower organisms, where VPS35 deletion enhances α-synuclein-induced toxicity. In mice, VPS35 overexpression is reported to rescue hippocampal neuronal loss in human α-synuclein transgenic mice, potentially suggesting a retromer deficiency in these mice. Here, we employ multiple well-established genetic rodent models to explore a functional or pathological interaction between VPS35 and α-synuclein in vivo . We find that endogenous α-synuclein is dispensable for nigrostriatal pathway dopaminergic neurodegeneration induced by the viral-mediated delivery of human D620N VPS35 in mice, suggesting that α-synuclein does not operate downstream of VPS35. We next evaluated retromer levels in affected brain regions from human A53T-α-synuclein transgenic mice, but find normal levels of the core subunits VPS35, VPS26 or VPS29. We further find that heterozygous VPS35 deletion fails to alter the lethal neurodegenerative phenotype of these A53T-α-synuclein transgenic mice, suggesting the absence of retromer deficiency in this PD model. Finally, we explored the neuroprotective capacity of increasing VPS35 expression in a viral-based human wild-type α-synuclein rat model of PD. However, we find that the overexpression of wild-type VPS35 is not sufficient for protection against α-synuclein-induced nigral dopaminergic neurodegeneration, α-synuclein pathology and reactive gliosis. Collectively, our data suggest a limited interaction of VPS35 and α-synuclein in neurodegenerative models of PD, and do not provide support for their interaction within a common pathophysiological pathway.