INTRODUCTION Parkinson’s disease (PD) is the second most common neurodegenerative disorder that leads to slowness of movement, tremor, rigidity and in the later stages of PD, cognitive impairment. Pathologically PD is characterized by the accumulation of α-synuclein in Lewy bodies and neurites. There is degeneration of neurons throughout the nervous system with the degeneration of dopamine neurons in the substantia nigra pars compacta leading to the major symptoms of PD. RATIONALE In the brains of PD patients, pathologic α-synuclein seems to spread from cell-to-cell via self-amplification, propagation, and transmission in a stereotypical and topographical pattern among neighboring cells and/or anatomically connected brain regions. The spread or transmission of pathologic α-synuclein is emerging as potentially important driver of PD pathogenesis. The underlying mechanisms and molecular entities responsible for the transmission of pathologic α-synuclein from cell-to-to cell are not known, but the entry of pathologic α-synuclein into neurons is thought to occur, in part through an active clathrin-dependent endocytic process. RESULTS Using recombinant α-synuclein pre-formed fibrils (PFF) as a model system to study the transmission of misfolded α-synuclein from neuron to neuron, we screened a library encoding transmembrane proteins for α-synuclein-biotin PFF binding candidates via detection by streptavidin-AP (alkaline phosphatase) staining. Three positive clones were identified that bind α-synuclein PFF and include lymphocyte-activation gene 3 (LAG3), neurexin 1β and amyloid beta precursor-like protein 1 (APLP1). Of these three transmembrane proteins, LAG3 demonstrated the highest ratio of selectivity for α-synuclein PFF over the α-synuclein monomer. α-Synuclein PFF binds to LAG3 in a saturable manner (Kd = 77 nM), while the α-synuclein monomer does not bind to LAG3. Co-immunoprecipitation also suggests that pathological α-synuclein PFF specifically binds to LAG3. Tau PFF, β-amyloid oligomer and β-amyloid PFF do not bind LAG3 indicating that LAG3 is specific for α-synuclein PFF. The internalization of α-synuclein PFF involves LAG3 since deletion of LAG3 reduces the endocytosis of α-synuclein PFF. LAG3 colocalizes with the endosomal GTPases, Rab5 and Rab7 and co-endocytoses with pathologic α-synuclein. Neuron-to-neuron transmission of pathologic α-synuclein and the accompanying pathology and neurotoxicity is substantially attenuated by deletion of LAG3 or by LAG3 antibodies. The lack of LAG3 also substantially delayed α-synuclein PFF induced loss of dopamine neurons, as well as biochemical and behavioral deficits in vivo. CONCLUSION We discovered that pathologic α-synuclein transmission and toxicity is initiated by binding to LAG3 and that neuron-to-neuron transmission of pathological α-synuclein involves the endocytosis of exogenous α-synuclein PFF by the engagement of LAG3 on neurons. Depletion of LAG3 or antibodies to LAG3 substantially reduce the pathology set in motion by the transmission of pathologic α-...

The pathologic accumulation and aggregation of α-synuclein (α-syn) underlies Parkinson’s disease (PD). The molecular mechanisms by which pathologic α-syn causes neurodegeneration in PD is not known. Here we found that pathologic α-syn activates poly (ADP-ribose) (PAR) polymerase-1 (PARP-1) and PAR generation accelerates the formation of pathologic α-syn resulting in cell death via parthanatos. PARP inhibitors or genetic deletion of PARP-1 prevented pathologic α-syn toxicity. In a feed-forward loop, PAR converted pathologic α-syn to a more toxic strain. PAR levels were increased in the cerebral spinal fluid and brains of PD patients suggesting that PARP activation plays a role in PD pathogenesis. Thus, strategies aimed at inhibiting PARP-1 activation could hold promise as a disease modifying therapy to prevent the loss of dopamine (DA) neurons in PD.

Excessive poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation kills cells via a cell-death process designated "parthanatos" in which PAR induces the mitochondrial release and nuclear translocation of apoptosis-inducing factor to initiate chromatinolysis and cell death. Accompanying the formation of PAR are the reduction of cellular NAD + and energetic collapse, which have been thought to be caused by the consumption of cellular NAD + by PARP-1. Here we show that the bioenergetic collapse following PARP-1 activation is not dependent on NAD + depletion. Instead PARP-1 activation initiates glycolytic defects via PAR-dependent inhibition of hexokinase, which precedes the NAD + depletion in N-methyl-N-nitroso-N-nitroguanidine (MNNG)-treated cortical neurons. Mitochondrial defects are observed shortly after PARP-1 activation and are mediated largely through defective glycolysis, because supplementation of the mitochondrial substrates pyruvate and glutamine reverse the PARP-1-mediated mitochondrial dysfunction. Depleting neurons of NAD + with FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, does not alter glycolysis or mitochondrial function. Hexokinase, the first regulatory enzyme to initiate glycolysis by converting glucose to glucose-6-phosphate, contains a strong PAR-binding motif. PAR binds to hexokinase and inhibits hexokinase activity in MNNG-treated cortical neurons. Preventing PAR formation with PAR glycohydrolase prevents the PAR-dependent inhibition of hexokinase. These results indicate that bioenergetic collapse induced by overactivation of PARP-1 is caused by PAR-dependent inhibition of glycolysis through inhibition of hexokinase.

Inhibition or genetic deletion of poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is protective against toxic insults in many organ systems. The molecular mechanisms underlying PARP-1–dependent cell death involve release of mitochondrial apoptosis-inducing factor (AIF) and its translocation to the nucleus, which results in chromatinolysis. We identified macrophage migration inhibitory factor (MIF) as a PARP-1–dependent AIF-associated nuclease (PAAN). AIF was required for recruitment of MIF to the nucleus, where MIF cleaves genomic DNA into large fragments. Depletion of MIF, disruption of the AIF-MIF interaction, or mutation of glutamic acid at position 22 in the catalytic nuclease domain blocked MIF nuclease activity and inhibited chromatinolysis, cell death induced by glutamate excitotoxicity, and focal stroke. Inhibition of MIF's nuclease activity is a potential therapeutic target for diseases caused by excessive PARP-1 activation.

The majority of ER-targeted tail-anchored (TA) proteins are inserted into membranes by the Guided Entry of Tail-anchored protein (GET) system. Disruption of this system causes a subset of TA proteins to mislocalize to mitochondria. We show that the AAA+ ATPase Msp1 limits the accumulation of mislocalized TA proteins on mitochondria. Deletion of MSP1 causes the Pex15 and Gos1 TA proteins to accumulate on mitochondria when the GET system is impaired. Likely as a result of failing to extract mislocalized TA proteins, yeast with combined mutation of the MSP1 gene and the GET system exhibit strong synergistic growth defects and severe mitochondrial damage, including loss of mitochondrial DNA and protein and aberrant mitochondrial morphology. Like yeast Msp1, human ATAD1 limits the mitochondrial mislocalization of PEX26 and GOS28, orthologs of Pex15 and Gos1, respectively. GOS28 protein level is also increased in ATAD1 À/À mouse tissues. Therefore, we propose that yeast Msp1 and mammalian ATAD1 are conserved members of the mitochondrial protein quality control system that might promote the extraction and degradation of mislocalized TA proteins to maintain mitochondrial integrity.

Accumulating evidence suggests that α-synuclein (α-syn) occurs physiologically as a helically folded tetramer that resists aggregation. However, the mechanisms underlying the regulation of formation of α-syn tetramers are still mostly unknown. Cellular membrane lipids are thought to play an important role in the regulation of α-syn tetramer formation. Since glucocerebrosidase 1 (GBA1) deficiency contributes to the aggregation of α-syn and leads to changes in neuronal glycosphingolipids (GSLs) including gangliosides, we hypothesized that GBA1 deficiency may affect the formation of α-syn tetramers. Here, we show that accumulation of GSLs due to GBA1 deficiency decreases α-syn tetramers and related multimers and increases α-syn monomers in CRISPR-GBA1 knockout (KO) SH-SY5Y cells. Moreover, α-syn tetramers and related multimers are decreased in N370S Parkinson's disease (PD) induced pluripotent stem cell (iPSC)-derived human dopaminergic (hDA) neurons and murine neurons carrying the heterozygous L444P mutation. Treatment with miglustat to reduce GSL accumulation and overexpression of GBA1 to augment GBA1 activity reverse the destabilization of α-syn tetramers and protect against α-syn preformed fibril-induced toxicity in hDA neurons. Taken together, these studies provide mechanistic insights into how GBA1 regulates the transition from monomeric α-syn to α-syn tetramers and multimers and suggest unique therapeutic opportunities for PD and dementia with Lewy bodies.

Mutations in PTEN-induced putative kinase 1 (PINK1) and parkin cause autosomal-recessive Parkinson’s disease through a common pathway involving mitochondrial quality control. Parkin inactivation leads to accumulation of the parkin interacting substrate (PARIS, ZNF746) that plays an important role in dopamine cell loss through repression of proliferator-activated receptor gamma coactivator-1α (PGC-1α) promoter activity. Here we show that PARIS, links PINK1 and parkin in a common pathway that regulates dopaminergic neuron survival. PINK1 interacts with and phosphorylates serines 322 and 613 of PARIS to control its ubiquitination and clearance by parkin. PINK1 phosphorylation of PARIS alleviates PARIS toxicity as well as repression of PGC-1α promoter activity. Conditional knockdown of PINK1 in adult mouse brains leads to a progressive loss of dopaminergic neurons in the substantia nigra that is dependent on PARIS. Together, these results uncover a function of PINK1 to direct parkin-PARIS regulated PGC-1α expression and dopaminergic neuronal survival.

The interaction between a 3,4-dihydroxylphenylalanine (DOPA) labeled analog of the tridecapeptide α-factor (W-H-W-L-Q-L-K-P-G-Q-P-M-Y) and Ste2p, a Saccharomyces cerevisiae model G protein-coupled receptor (GPCR), has been analyzed by periodate-mediated cross-linking. Chemically synthesized α-factor with DOPA substituting for tyrosine at position 13 and biotin tagged onto lysine 7 ([Lys 7 (BioACA),-Nle 12 ,DOPA 13 ]α-factor; Bio-DOPA-α-factor) was used for crosslinking into Ste2p. The biological activity of Bio-DOPA-α-factor was about one-third that of native α-factor as determined by growth arrest assay and exhibited about a ten-fold lower binding affinity to Ste2p. Bio-DOPA-α-factor cross-linked into Ste2p as demonstrated by western blot analysis using a Neutravidin-HRP conjugate to detect Bio-DOPA-α-factor. Cross-linking was inhibited by excess native α-factor and an α-factor antagonist. The Ste2p-ligand complex was purified using a metal ion affinity column, and after cyanogen bromide treatment, avidin affinity purification was used to capture Bio-DOPA-α-factor-Ste2p cross-linked peptides. MALDI-TOF spectrometric analyses of the cross-linked fragments showed that Bio-DOPA-α-factor reacted with the Phe 55 -Met 69 region of Ste2p. Cross-linking of Bio-DOPA-α-factor was reduced by 80% using a cysteine-less Ste2p (Cys59Ser). These results suggest an interaction between position thirteen of α-factor and residue Cys 59 of Ste2p. This study is the first to report DOPA cross-linking of a peptide hormone to a GPCR and the first to identify a residue-to-residue cross-link between Ste2p and α-factor thereby defining a specific contact point between the bound ligand and its receptor.G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins associated with signaling systems present in mammalians, plants, protozoans, fungi, and metazoans (1). Malfunctions of GPCRs contribute to diseases such as Alzheimer's, Parkinson's disease, diabetes, color blindness, asthma, depression, hypertension, stress, cardiovascular, and immune disorders (2,3). All GPCRs share a common structure consisting of seven transmembrane domains (TMDs) connected by three extracellular and three intracellular loops (1,4,5). Upon binding of their ligands, GPCRs undergo conformational changes that transduce NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 March 10. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the signal into the cell by activating a cascade of protein-protein interactions initiated through a heterotrimeric G protein complex (6).Ste2p, the Saccharomyces cerevisiae α-factor pheromone receptor, has been studied as a model for peptide-responsive GPCRs (7,8). Though Ste2p does not share recognizable sequence similarity with mammalian GPCRs or even with Ste3p, the a-factor pheromone receptor of yeast, all GPCRs have strong structural and functional similarities (9,10). For example, the packing and interactions between the fifth and sixth tr...