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α-Synuclein (αS) plays a key role in Parkinson's disease. Although Parkinson's disease is typically “sporadic,” inherited αS missense mutations provide crucial insights into molecular mechanisms. Here, we examine two clinical mutants, E46K and G51D, which are both in the conserved N-terminus that mediates transient αS-membrane interactions. However, E46K increases and G51D decreases αS-membrane interactions. Previously, we amplified E46K via the 11-residue repeat motifs, creating “3K” (E35K+E46K+E61K). Here, we engineered these motifs to amplify G51D (V40D+G51D+V66D = “3D”) and systematically compared E46K/3K versus G51D/3D. We found that G51D increased cytosolic αS in neural cells and 3D aggravates this. G51D, and 3D even more, reduced αS multimer-to-monomer (αS60:αS14) ratio. Both amplified variants caused cellular stress in rat primary neurons and reduced growth in human neuroblastoma cells. Importantly, both 3K- and 3D-induced stress was ameliorated by pharmacologically inhibiting stearoyl-CoA desaturase or by conditioning the cells in palmitic (16:0) or myristic (14:0) acid. SCD inhibition lowered lipid-droplet accumulation in both 3D- and 3K-expressing cells and benefitted G51D by normalizing multimer:monomer ratio, as reported previously for E46K. Our findings suggest that, despite divergent cytosol/membrane partitioning, both G51D and E46K neurotoxicity can be prevented by decreasing fatty-acid unsaturation as a common therapeutic approach.SIGNIFICANCE STATEMENTα-Synuclein (αS) dyshomeostasis is linked to Parkinson's disease. Here we focus on two contrasting familial-Parkinson's disease αS mutants, E46K and G51D, that alter αS membrane association in opposite directions (E46K increases, G51D decreases it). Taking advantage of αS repeat structure, here we designed αS “3D,” an amplified G51D variant (V40D+G51D+V66D). αS 3D further enhanced G51D's cytosolic enrichment. Systematic comparison of G51D/3D with membrane-enriched E46K/its amplified variant 3K revealed that both can elicit stress in human neural cells and primary rodent neurons. This toxicity can be ameliorated by inhibiting stearoyl-CoA desaturase or by saturated fatty acid conditioning. Thus, despite divergent membrane binding, both G51D and E46K αS dyshomeostasis are mitigated by altering fatty acid saturation as a shared target.

Intracellular inclusions accompanying neurodegeneration are histopathologically and ultrastructurally heterogeneous but the significance of this heterogeneity is unclear. iPSC models, while promising for disease modeling, do not form inclusions in a reasonable timeframe and suffer from limited tractability. Here, we developed an iPSC toolbox utilizing piggyBac-based or targeted transgenes to rapidly induce CNS cells with concomitant expression of aggregation-prone proteins. This system is amenable to screening and longitudinal tracking at single-cell and single-inclusion resolution. For proof-of-principle, cortical neuron α-synuclein “inclusionopathy” models were engineered to form inclusions through exogenous seeding or α-synuclein mutation. These models recapitulated known fibril- and lipid-rich inclusion subtypes, uncovering dynamic interactions between them, and refined the classification of inclusions in postmortem brain. Genetic-modifier and protein-interaction screens pinpointed proteins like RhoA whose sequestration into specific inclusion subtypes is likely to be toxic. This iPSC platform should enhance our understanding of proteinaceous pathologies in neurodegeneration and facilitate therapeutics development.

In contrast to Aβ plaques, the spatiotemporal distribution of neurofibrillary tangles of hyperphosphorylated tau (p-tau) predicts cognitive impairment in Alzheimer’s disease (AD), underscoring the key pathological role of p-tau and the utmost need to develop AD therapeutics centering upon the control of p-tau aggregation and cytotoxicity. Our drug discovery program is focused on compounds that prevent the aggregation and cytotoxicity of p-tau moieties of the tau isoform 1N4R due to its prevalence (1 N) and long-distance trans-synaptic propagation (4R). We prepared and tested twenty-four newly synthesized small molecules representing the urea (1, 2, 3), sulfonylurea (4), and sulfonamide (5–24) series and evaluated their anti-aggregation effects with biophysical methods (thioflavin T and S fluorescence assays, transmission electron microscopy) and intracellular inclusion cell-based assays. Pre-evaluation was performed on alpha-synuclein (α-syn) to identify molecules to be challenged with p-tau. The sulfonamide derivatives 18 and 20 exhibited an anti-fribrillization activity on α-syn and p-tau. Sulfonamide compounds 18 and 20 reduced inclusion formation in M17D neuroblastoma cells that express inclusion-prone αSynuclein3K::YFP. This project advances new concepts in targeting prone-to-aggregate proteins such as α-syn and p-tau, and provides a molecular scaffold for further optimization and pre-clinical studies focused on AD drug development.

α-Synuclein (αS) has been well-documented to play a role in human synucleinopathies such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). First, the lesions found in PD/DLB brains—Lewy bodies and Lewy neurites—are rich in aggregated αS. Second, genetic evidence links missense mutations and increased αS expression to familial forms of PD/DLB. Third, toxicity and cellular stress can be caused by αS under certain experimental conditions. In contrast, the homologs β-synuclein (βS) and γ-synuclein (γS) are not typically found in Lewy bodies/neurites, have not been clearly linked to brain diseases and have been largely non-toxic in experimental settings. In αS, the so-called non-amyloid-β component of plaques (NAC) domain, constituting amino acids 61–95, has been identified to be critical for aggregation in vitro. This domain is partially absent in βS and only incompletely conserved in γS, which could explain why both homologs do not cause disease. However, αS in vitro aggregation and cellular toxicity have not been firmly linked experimentally, and it has been proposed that excess αS membrane binding is sufficient to induce neurotoxicity. Indeed, recent characterizations of Lewy bodies have highlighted the accumulation of lipids and membranous organelles, raising the possibility that βS and γS could also become neurotoxic if they were more prone to membrane/lipid binding. Here, we increased βS and γS membrane affinity by strategic point mutations and demonstrate that these proteins behave like membrane-associated monomers, are cytotoxic and form round cytoplasmic inclusions that can be prevented by inhibiting stearoyl-CoA desaturase.

Objective Parkinson disease (PD) has useful symptomatic treatments that do not slow the neurodegenerative process, and no significant disease-modifying treatments are approved. A key therapeutic target in PD is α-synuclein (αS), which is both genetically implicated and accumulates in Lewy bodies rich in vesicles and other lipid membranes. Reestablishing αS homeostasis is a central goal in PD. Based on previous lipidomic analyses, we conducted a mouse trial of a stearoyl-coenzyme A desaturase (SCD) inhibitor ("5b") that prevented αS-positive vesicular inclusions and cytotoxicity in cultured human neurons. Methods Oral dosing and brain activity of 5b were established in nontransgenic mice. 5b in drinking water was given to mice expressing wild-type human αS (WT) or an amplified familial PD αS mutation (E35K + E46K + E61K ["3K"]) beginning near the onset of nigral and cortical neurodegeneration and the robust PD-like motor syndrome in 3K. Motor phenotypes, brain cytopathology, and SCD-related lipid changes were quantified in 5b- versus placebo-treated mice. Outcomes were compared to effects of crossing 3K to SCD1-/- mice. Results 5b treatment reduced αS hyperphosphorylation in E46K-expressing human neurons, in 3K neural cultures, and in both WT and 3K αS mice. 5b prevented subtle gait deficits in WT αS mice and the PD-like resting tremor and progressive motor decline of 3K αS mice. 5b also increased αS tetramers and reduced proteinase K-resistant lipid-rich aggregates. Similar benefits accrued from genetically deleting 1 SCD allele, providing target validation. Interpretation Prolonged reduction of brain SCD activity prevented PD-like neuropathology in multiple PD models. Thus, an orally available SCD inhibitor potently ameliorates PD phenotypes, positioning this approach to treat human α-synucleinopathies. ANN NEUROL 2021;89:74-90.

There is growing recognition in the field of neurodegenerative diseases that mixed proteinopathies are occurring at greater frequency than originally thought. This is particularly true for three amyloid proteins defining most of these neurological disorders, amyloid-beta (Aβ), tau, and alpha-synuclein (αSyn). The co-existence and often co-localization of aggregated forms of these proteins has led to the emergence of concepts positing molecular interactions and cross-seeding between Aβ, tau, and αSyn aggregates. Amongst this trio, αSyn has received particular attention in this context during recent years due to its ability to modulate Aβ and tau aggregation in vivo, to interact at a molecular level with Aβ and tau in vivo and to cross-seed tau in mice. Here we provide a comprehensive, critical, and accessible review about the expression, role and nature of endogenous soluble αSyn oligomers because of recent developments in the understanding of αSyn multimerization, misfolding, aggregation, cross-talk, spreading and cross-seeding in neurodegenerative disorders, including Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, Alzheimer's disease, and Huntington's disease. We will also discuss our current understanding about the relative toxicity of endogenous αSyn oligomers in vivo and in vitro, and introduce potential opportunities to counter their deleterious effects.

Increasing evidence has shown that Parkinson’s disease (PD) impairs midbrain dopaminergic, cortical and other neuronal subtypes in large part due to the build-up of lipid- and vesicle-rich α-synuclein (αSyn) cytotoxic inclusions. We previously identified stearoyl-CoA desaturase (SCD) as a potential therapeutic target for synucleinopathies. A brain-penetrant SCD inhibitor, YTX-7739, was developed and has entered Phase 1 clinical trials. Here, we report the efficacy of YTX-7739 in reversing pathological αSyn phenotypes in various in vitro and in vivo PD models. In cell-based assays, YTX-7739 decreased αSyn-mediated neuronal death, reversed the abnormal membrane interaction of amplified E46K (“3K”) αSyn, and prevented pathological phenotypes in A53T and αSyn triplication patient-derived neurospheres, including dysregulated fatty acid profiles and pS129 αSyn accumulation. In 3K PD-like mice, YTX-7739 crossed the blood–brain barrier, decreased unsaturated fatty acids, and prevented progressive motor deficits. Both YTX-7739 treatment and decreasing SCD activity through deletion of one copy of the SCD1 gene (SKO) restored the physiological αSyn tetramer-to-monomer ratio, dopaminergic integrity, and neuronal survival in 3K αSyn mice. YTX-7739 efficiently reduced pS129 + and PK-resistant αSyn in both human wild-type αSyn and 3K mutant mice similar to the level of 3K-SKO. Together, these data provide further validation of SCD as a PD therapeutic target and YTX-7739 as a clinical candidate for treating human α-synucleinopathies.

The protein α-synuclein, a key player in Parkinson’s disease (PD) and other synucleinopathies, exists in different physiological conformations: cytosolic unfolded aggregation-prone monomers and helical aggregation-resistant multimers. It has been shown that familial PD-associated missense mutations within the α-synuclein gene destabilize the conformer equilibrium of physiologic α-synuclein in favor of unfolded monomers. Here, we characterized the relative levels of unfolded and helical forms of cytosolic α-synuclein in post-mortem human brain tissue and showed that the equilibrium of α-synuclein conformations is destabilized in sporadic PD and DLB patients. This disturbed equilibrium is decreased in a brain region-specific manner in patient samples pointing toward a possible “prion-like” propagation of the underlying pathology and forms distinct disease-specific patterns in the two different synucleinopathies. We are also able to show that a destabilization of multimers mechanistically leads to increased levels of insoluble, pathological α-synuclein, while pharmacological stabilization of multimers leads to a “prion-like” aggregation resistance. Together, our findings suggest that these disease-specific patterns of α-synuclein multimer destabilization in sporadic PD and DLB are caused by both regional neuronal vulnerability and “prion-like” aggregation transmission enabled by the destabilization of local endogenous α-synuclein protein.

Significance The mechanisms responsible for brain α-synuclein (αS) dyshomeostasis, caused by Gaucher’s GBA1 mutations that increase Parkinson’s disease (PD) risk, are largely unknown. We previously showed that abrogating physiological αS tetramers by a familial PD-E46K–amplified 3K mutation produces PD-like syndrome in mice and that treatment with stearoyl-CoA desaturase inhibitors increased a portion of the αS tetramers, benefitting the motor phenotypes. Here, we show that—similar to previous findings in GBA1-mutant PD culture—GCase elevation prolonged the stabilization of wild-type and 3K mutant αS tetramers in wtGBA1–transduced mouse brains, improving lysosomal integrity and motor and cognitive phenotypes. These data help elucidating lipid modulators that impact the αS physiological state in vivo and the development of PD therapeutic approaches., Loss-of-function mutations in acid beta-glucosidase 1 (GBA1) are among the strongest genetic risk factors for Lewy body disorders such as Parkinson’s disease (PD) and Lewy body dementia (DLB). Altered lipid metabolism in PD patient–derived neurons, carrying either GBA1 or PD αS mutations, can shift the physiological α-synuclein (αS) tetramer–monomer (T:M) equilibrium toward aggregation-prone monomers. A resultant increase in pSer129+ αS monomers provides a likely building block for αS aggregates. 3K αS mice, representing a neuropathological amplification of the E46K PD–causing mutation, have decreased αS T:M ratios and vesicle-rich αS+ aggregates in neurons, accompanied by a striking PD-like motor syndrome. We asked whether enhancing glucocerebrosidase (GCase) expression could benefit αS dyshomeostasis by delivering an adeno-associated virus (AAV)–human wild-type (wt) GBA1 vector into the brains of 3K neonates. Intracerebroventricular AAV-wtGBA1 at postnatal day 1 resulted in prominent forebrain neuronal GCase expression, sustained through 6 mo. GBA1 attenuated behavioral deficits both in working memory and fine motor performance tasks. Furthermore, wtGBA1 increased αS solubility and the T:M ratio in both 3K-GBA mice and control littermates and reduced pS129+ and lipid-rich aggregates in 3K-GBA. We observed GCase distribution in more finely dispersed lysosomes, in which there was increased GCase activity, lysosomal cathepsin D and B maturation, decreased perilipin-stabilized lipid droplets, and a normalized TFEB translocation to the nucleus, all indicative of improved lysosomal function and lipid turnover. Therefore, a prolonged increase of the αS T:M ratio by elevating GCase activity reduced the lipid- and vesicle-rich aggregates and ameliorated PD-like phenotypes in mice, further supporting lipid modulating therapies in PD.

Significance Misfolding and accumulation of the protein α-synuclein (αS) inside nerve cells characterize Parkinson’s disease (PD) and related brain diseases, for which no disease-modifying therapies exist. Robust cell models are needed that recapitulate abnormal αS folding/accumulation in real time, especially for compound screening. We took advantage of the engineered αS “3K” (E35K+E46K+E61K) mutation, which amplifies the familial PD-causing E46K and readily forms round inclusions. We rescued the inclusions with known αS modulators and then screened a ∼2,000-compound library. We identified the potential therapeutic target stearoyl-CoA desaturase; its inhibition cleared αS inclusions, in accord with the effects of conditioning in saturated fatty acids. We propose a model for how fatty acids serve as key modulators of cellular αS homeostasis., Microscopy of Lewy bodies in Parkinson’s disease (PD) suggests they are not solely filamentous deposits of α-synuclein (αS) but also contain vesicles and other membranous material. We previously reported the existence of native αS tetramers/multimers and described engineered mutations of the αS KTKEGV repeat motifs that abrogate the multimers. The resultant excess monomers accumulate in lipid membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial PD mutants, such as E46K. Here, we use the αS “3K” (E35K+E46K+E61K) engineered mutation to probe the mechanisms of reported small-molecule modifiers of αS biochemistry and then identify compounds via a medium-throughput automated screen. αS 3K, which forms round, vesicle-rich inclusions in cultured neurons and causes a PD-like, l-DOPA–responsive motor phenotype in transgenic mice, was fused to YFP, and fluorescent inclusions were quantified. Live-cell microscopy revealed the highly dynamic nature of the αS inclusions: for example, their rapid clearance by certain known modulators of αS toxicity, including tacrolimus (FK506), isradipine, nilotinib, nortriptyline, and trifluoperazine. Our automated 3K cellular screen identified inhibitors of stearoyl-CoA desaturase (SCD) that robustly prevent the αS inclusions, reduce αS 3K neurotoxicity, and prevent abnormal phosphorylation and insolubility of αS E46K. SCD inhibition restores the E46K αS multimer:monomer ratio in human neurons, and it actually increases this ratio for overexpressed wild-type αS. In accord, conditioning 3K cells in saturated fatty acids rescued, whereas unsaturated fatty acids worsened, the αS phenotypes. Our cellular screen allows probing the mechanisms of synucleinopathy and refining drug candidates, including SCD inhibitors and other lipid modulators.

The α-synuclein protein (αS) is the major constituent of pathological neuronal inclusions both in Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB) with differential brain region-specific pathology patterns and clinical presentations. Two hypotheses that were recently put forward were either that of brain-region specific vulnerability or that of different αS aggregates, “strains”, that would affect different brain regions via “prion-like” spread. What governs these patterns, their hypothesized “prion-like” progression and region-specific vulnerability to αS aggregation in different synucleinopathies is still largely unknown. Data collected in the last decade suggests that αS can exhibit in different conformations under physiological and/or pathological conditions, which in turn help govern aggregation propensity. The cytosolic unfolded, monomeric form of αS (αSCU) is aggregation-prone and can misfold into soluble, toxic oligomers, protofilaments, and amyloid fibrils with self-templating properties, while the cytosolic helically folded, multimeric form (αSCH) resists disease-associated changes.

Previous work from our group showed that certain engineered missense mutations to the α-synuclein (αS) KTKEGV repeat motifs abrogate the protein’s ability to form native multimers. The resultant excess monomers accumulate in lipid-membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial Parkinson’s disease mutants such as E46K. We presented an initial characterization of the lipid-rich inclusions and found similarities to the αS- and vesicle-rich inclusions that form in baker’s yeast when αS is expressed. We also discussed, with some caution, a possible role of membrane-rich inclusions as precursors to filamentous Lewy bodies, the widely accepted hallmark pathology of Parkinson’s disease and other synucleinopathies. In the meantime, advances in the microscopic characterization of Lewy bodies have highlighted the presence of crowded organelles and lipid membranes in addition to αS accumulation. This prompted us to revisit the αS inclusions caused by our repeat motif variants in neuroblastoma cells. In addition to our previous characterization, we found that these inclusions can often be seen by brightfield microscopy, overlap with endogenous vesicle markers in immunofluorescence experiments, stain positive for lipid dyes, and can be found to be closely associated with mitochondria. We also observed abnormal tubulation of membranes, which was subtle in inducible lines and pronounced in cells that transiently expressed high amounts of the highly disruptive KTKEGV motif mutant “KLKEGV”. Membrane tubulation had been reported before as an αS activity in reductionist systems. Our in-cellulo demonstration now suggests that this mechanism could possibly be a relevant aspect of aberrant αS behavior in cells.

α-Synuclein aggregation in Lewy bodies/neurites is the defining feature of familial and ‘sporadic’ Parkinson’s disease. We previously identified helical αSyn tetramers in normal cells. PD-causing αSyn mutations lower the tetramer:monomer (T:M) ratio, associated with αSyn hyperphosphorylation and cytotoxicity in cultured neurons and a progressive motor syndrome of resting tremor and gait deficits in transgenic mice. We asked whether LRRK2 mutations, the most common genetic cause of what were previously considered sporadic PD cases, also alter αSyn tetramer homeostasis. iPSC-derived patient neurons carrying the most prevalent LRRK2 mutation (G2019S) had decreased T:M ratios and pSer129 hyperphosphorylation of their endogenous αSyn. A LRRK2 kinase inhibitor restored the T:M ratio and prevented hyperphosphorylation of αSyn and the LRRK2-substrate Rab10. With the discovery that PD-causing mutations of glucocerebrosidase in Gaucher’s carriers also decrease T:M ratios, our findings indicate that three distinct genetic forms of PD involve life-long destabilization of αSyn tetramers as a common pathogenic mechanism in PD.

The neuronal protein α-synuclein (αS) is central to the pathogenesis of Parkinson's disease and other progressive brain diseases such as Lewy body dementia and multiple system atrophy. These diseases, collectively referred to as 'synucleinopathies', have long been considered purely proteinopathies: diseases characterized by the misfolding of a protein into small and large aggregates mainly consisting of that protein (in this case: α-synuclein). However, recent morphological insights into Lewy bodies, the hallmark neuropathology of human synucleinopathies, suggests these lesions are also rich in vesicles and other membranous organelles. Moreover, αS physiology and pathology are both strongly associated with various aspects of intracellular vesicle trafficking and lipid biology. αS physiologically binds to synaptic and other small vesicles, and several functions of αS in regulating vesicle biology have been proposed. Familial PD-linked αS excess and missense mutations have been shown to impair vesicle trafficking and alter lipid homeostasis. On the other hand, vesicle trafficking and lipid-related genes have emerged as Parkinson's risk factors, suggesting a bidirectional relationship. The answer to the question "Does abnormal αS accumulation cause impaired vesicle trafficking and lipid dyshomeostasis or is αS aggregation the consequence of such impairments?" may be "Both". Here, we review current knowledge of the αS-lipid and αS-vesicle trafficking interplay, with a special focus on Parkinson's disease and Lewy body dementia.

Amyloidosis, a group of diseases caused by the fibrillation of prone-to-aggregate proteins, remains one of the most difficult ailments to treat human medicine. This study focuses on the amyloid protein, alpha-synuclein (α-syn), the major pathogenetic contributor to well-known diseases such as Parkinson's Disease (PD) and multiple system atrophy (MSA). We examine the effectiveness of our novel family of molecules, diaryl derivatives of urea, on the inhibition of early- and late-stage α-syn aggregation. Screening with Thioflavin-T (ThT) fluorescence assay has identified one compound 12 as a promising inhibitor for fibril formation (late-stage aggregation). Utilizing the Photo-induced Cross-linking of Unmodified Proteins (PICUP) assays, compound 12 impeded α-syn oligomerization (early-stage aggregation). In contrast to resveratrol, a polyphenol used as positive control, compound 12 delayed the lag time and did not generate globular structures of 5 nm per transmission electron microscopy (TEM). EC50 of compound 12 is 27.6 ± 1.3 µM. Compound 12 reduced cell inclusions and cytotoxicity in a dose-dependent manner using the inclusion-forming neuroblastoma cell-based assays. This family of compound has the potential to become an invaluable tool to abrogate, at the early-stage, α-syn aggregation.

α-Synuclein (αS) and tau have a lot in common. Dyshomeostasis and aggregation of both proteins are central in the pathogenesis of neurodegenerative diseases: Parkinson's disease, dementia with Lewy bodies, multi-system atrophy and other 'synucleinopathies' in the case of αS; Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy and other 'tauopathies' in the case of tau. The aggregated states of αS and tau are found to be (hyper)phosphorylated, but the relevance of the phosphorylation in health or disease is not well understood. Both tau and αS are typically characterized as 'intrinsically disordered' proteins, while both engage in transient interactions with cellular components, thereby undergoing structural changes and context-specific folding. αS transiently binds to (synaptic) vesicles forming a membrane-induced amphipathic helix; tau transiently interacts with microtubules forming an 'extended structure'. The regulation and exact nature of the interactions are not fully understood. Here we review recent and previous insights into the dynamic, transient nature of αS and tau with regard to the mode of interaction with their targets, the dwell-time while bound, and the cis and trans factors underlying the frequent switching between bound and unbound states. These aspects are intimately linked to hypotheses on how subtle changes in the transient behaviors may trigger the earliest steps in the pathogenesis of the respective brain diseases. Based on a deeper understanding of transient αS and tau conformations in the cellular context, new therapeutic strategies may emerge, and it may become clearer why existing approaches have failed or how they could be optimized.

The pathology of Parkinson's disease and other synucleinopathies is characterized by the formation of intracellular inclusions comprised primarily of misfolded, fibrillar α-synuclein (α-syn). One strategy to slow disease progression is to prevent the misfolding and aggregation of its native monomeric form. Here we present findings that support the contention that the tricyclic antidepressant compound nortriptyline (NOR) has disease-modifying potential for synucleinopathies. Findings from in vitro aggregation and kinetics assays support the view that NOR inhibits aggregation of α-syn by directly binding to the soluble, monomeric form, and by enhancing reconfiguration of the monomer, inhibits formation of toxic conformations of the protein. We go on to demonstrate that NOR inhibits the accumulation, aggregation and neurotoxicity of α-syn in multiple cell and animal models. These findings suggest that NOR, a compound with established safety and efficacy for treatment of depression, may slow progression of α-syn pathology by directly binding to soluble, native, α-syn, thereby inhibiting pathological aggregation and preserving its normal functions.

Lipids play a more significant role in Parkinson’s disease and its related brain disorders than is currently recognized, supporting a “lipid cascade”. The 14 kDa protein α-synuclein (αS) is strongly associated with Parkinson’s disease (PD), dementia with Lewy bodies (DLB), other synucleinopathies such as multiple system atrophy, and even certain forms of Alzheimer’s disease. Rigorously deciphering the biochemistry of αS in native systems is the key to developing treatments. αS is highly expressed in the brain, the second most lipid-rich organ, and has been proposed to be a lipid-binding protein that physiologically interacts with phospholipids and fatty acids (FAs). αS-rich cytoplasmic inclusions called Lewy bodies and Lewy neurites are the hallmark lesions of synucleinopathies. Excess αS–membrane interactions may trigger proteinaceous αS aggregation by stimulating its primary nucleation. However, αS may also exert its toxicity prior to or independent of its self-aggregation, e.g., via excessive membrane interactions, which may be promoted by certain lipids and FAs. A complex αS-lipid landscape exists, which comprises both physiological and pathological states of αS. As novel insights about the composition of Lewy lesions occur, new lipid-related PD drug candidates emerge, and genome-wide association studies (GWAS) increasingly validate new hits in lipid-associated pathways, it seems timely to review our current knowledge of lipids in PD and consider the roles for these pathways in synucleinopathies.Fig. 1αS ↔ lipid interplay: aspects of cellular αS homeostasis (blue oval), aspects of lipid homeostasis (green oval), and overlapping aspects.Pathological states are labeled in red. Simplified schematic of both select αS and select lipid species. Several existing publications suggest αS effects on lipids and vice versa, as indicated by arrows. DG diglyceride, ER endoplasmic reticulum, FA fatty acid, LD, lipid droplet, TG triglyceride.

β-Sheet-rich aggregates of α-synuclein (αS) are the hallmark neuropathology of Parkinson's disease (PD) and related synucleinopathies, whereas the native conformations of αS in healthy cells are under debate. Cross-linking analyses in intact cells detect a large portion of endogenous αS in apparent multimeric states, most notably as putative tetramers (αS60) that run around 60 kDa on SDS-PAGE, but also point at the dynamic nature of cellular αS states. Standardization of αS cross-linking methods will facilitate efforts to study the effects of genetic, pharmacological, and environmental factors on αS conformation. Here, we present detailed protocols for cross-linking cellular αS multimers in cultured cells and brain tissues. These protocols will benefit future studies aimed at characterizing αS conformation in its cellular environment, both at steady state and upon perturbation, be it chronic or acute.

β-Sheet-rich aggregates of α-synuclein (αS) are the hallmark neuropathology of Parkinson's disease (PD) and related synucleinopathies, whereas the native conformations of αS in healthy cells are under debate. Cross-linking analyses in intact cells detect a large portion of endogenous αS in apparent multimeric states, most notably as putative tetramers (αS60) that run around 60 kDa on SDS-PAGE, but also point at the dynamic nature of cellular αS states. Standardization of αS cross-linking methods will facilitate efforts to study the effects of genetic, pharmacological, and environmental factors on αS conformation. Here, we present detailed protocols for cross-linking cellular αS multimers in cultured cells and brain tissues. These protocols will benefit future studies aimed at characterizing αS conformation in its cellular environment, both at steady state and upon perturbation, be it chronic or acute.

Aggregation of α-synuclein (αS) leads to the hallmark neuropathology of Parkinson’s disease (PD) and related synucleinopathies. αS has been described to exist in both cytosolic and membrane-associated forms, the relative abundance of which has remained unsettled. To study αS under the most relevant conditions by a quantitative method, we cultured and matured rodent primary cortical neurons for >17 days and determined αS cytosol:membrane distribution via centrifugation-free sequential extractions based on the weak ionic detergent digitonin. We noticed that at lower temperatures (4 °C or room temperature), αS was largely membrane-associated. At 37 °C, however, αS solubility was markedly increased. In contrast, the extraction of control proteins (GAPDH, cytosolic; calnexin, membrane) was not affected by temperature. When we compared the relative distribution of the synuclein homologs αS and β-synuclein (βS) under various conditions that differed in temperature and digitonin concentration (200–1200 μg/ml), we consistently found αS to be more membrane-associated than βS. Both proteins, however, exhibited temperature-dependent membrane binding. Under the most relevant conditions (37 °C and 800 μg/ml digitonin, i.e., the lowest digitonin concentration that extracted cytosolic GAPDH to near completion), cytosolic distribution was 49.8% ± 9.0% for αS and 63.6% ± 6.6% for βS. PD-linked αS A30P was found to be largely cytosolic, confirming previous studies that had used different methods. Our work highlights the dynamic nature of cellular synuclein behavior and has important implications for protein-biochemical and cell-biological studies of αS proteostasis, such as testing the effects of genetic and pharmacological manipulations.

In Parkinson's disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in αS or lipid/fatty acid homeostasis affect each other. Lipidomic profiling of human αS-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of αS dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased αS yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in αS-overexpressing rat neurons. In a C. elegans model, SCD knockout prevented αS-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on αS homeostasis: in human neural cells, excess OA caused αS inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for αS-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach. Keywords: Parkinson’s disease; synucleinopathy; alpha-synuclein; stearoyl-CoA-desaturase; unsaturated fatty acid; oleic acid; lipid droplets; diglyceride; triglyceride; tetramer; inclusions

α-Synuclein (αS) is a highly abundant neuronal protein that aggregates into β-sheet-rich inclusions in Parkinson's disease (PD). αS was long thought to occur as a natively unfolded monomer, but recent work suggests it also occurs normally in α-helix-rich tetramers and related multimers. To elucidate the fundamental relationship between αS multimers and monomers in living neurons, we performed systematic mutagenesis to abolish self-interactions and learn which structural determinants underlie native multimerization. Unexpectedly, tetramers/multimers still formed in cells expressing each of 14 sequential 10-residue deletions across the 140-residue polypeptide. We postulated compensatory effects among the six highly conserved and one to three additional αS repeat motifs (consensus: KTKEGV), consistent with αS and its homologs β- and γ-synuclein all forming tetramers while sharing only the repeats. Upon inserting in-register missense mutations into six or more αS repeats, certain mutations abolished tetramer formation, shown by intact-cell cross-linking and independently by fluorescent-protein complementation. For example, altered repeat motifs KLKEGV, KTKKGV, KTKEIV, or KTKEGW did not support tetramerization, indicating the importance of charged or small residues. When we expressed numerous different in-register repeat mutants in human neural cells, all multimer-abolishing but no multimer-neutral mutants caused frank neurotoxicity akin to the proapoptotic protein Bax. The multimer-abolishing variants became enriched in buffer-insoluble cell fractions and formed round cytoplasmic inclusions in primary cortical neurons. We conclude that the αS repeat motifs mediate physiological tetramerization, and perturbing them causes PD-like neurotoxicity. Moreover, the mutants we describe are valuable tools for studying normal and pathological properties of αS and screening for tetramer-stabilizing therapeutics.

α-Synuclein (αS) forms round cytoplasmic inclusions in Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Evidence suggests a physiological function of αS in vesicle trafficking and release. In contrast to earlier tenets, recent work indicates that αS normally exists in cells in a dynamic equilibrium between monomers and tetramers/multimers. We engineered αS mutants incapable of multimerization, leading to excess monomers at vesicle membranes. By EM, such mutants induced prominent vesicle clustering, leading to round cytoplasmic inclusions. Immunogold labeling revealed abundant αS intimately associated with vesicles of varied size. Fluorescence microscopy with marker proteins showed that the αS-associated vesicles were of diverse endocytic and secretory origin. An αS ‘3K’ mutant (E35K + E46K + E61K) that amplifies the PD/DLB-causing E46K mutation induced αS-rich vesicle clusters resembling the vesicle-rich areas of Lewy bodies, supporting pathogenic relevance. Mechanistically, E46K can increase αS vesicle binding via membrane-induced amphipathic helix formation, and ‘3K’ further enhances this effect. Another engineered αS variant added hydrophobicity to the hydrophobic half of αS helices, thereby stabilizing αS-membrane interactions. Importantly, substituting charged for uncharged residues within the hydrophobic half of the stabilized helix not only reversed the strong membrane interaction of the multimer-abolishing αS variant but also restored multimerization and prevented the aberrant vesicle interactions. Thus, reversible αS amphipathic helix formation and dynamic multimerization regulate a normal function of αS at vesicles, and abrogating multimers has pathogenic consequences.

This scientific commentary refers to ‘Direct visualization of alpha-synuclein oligomers reveals previously undetected pathology in Parkinson’s disease brain’, by Roberts et al. (doi: 10.1093/brain/awv040). Parkinson’s disease is the commonest movement disorder, with an estimated life-time risk of 1.5% (de Rijk et al., 1995). It is characterized by severe cell loss in the substantia nigra and neuronal/neuritic accumulation of aggregated α-synuclein (αSyn) (Spillantini et al., 1997), a 140-amino acid protein of unclear function. Point mutations lead to early-onset Parkinson’s disease (Polymeropoulos, 1997), and importantly, total αSyn levels—which can be increased following duplication or triplication of the alpha-synuclein gene SNCA—help to determine time of onset and rate of progression (Fuchs et al., 2007). The causes of the ∼90% of Parkinson’s disease cases that are sporadic are not well understood. Overall, there is abundant evidence linking αSyn and its aggregation to the pathogenic mechanism of Parkinson’s disease, dementia with Lewy bodies and other neurodegenerative diseases collectively termed ‘synucleinopathies’. However, which forms of αSyn are cytotoxic and how these are distributed within the human brain are largely unknown, due in no small part to the lack of sensitive techniques for their specific detection. Classical immunohistochemistry cannot differentiate between physiological and pathological αSyn assemblies, and differentiation based on morphology of the αSyn signal—diffuse or punctate—is not helpful in detecting pathological oligomers that are dispersed inside or even outside of cells. Aggregate-binding dyes such as thioflavin, on the other hand, detect only very large β-sheet-rich inclusions of αSyn and are not protein-specific, simultaneously detecting other aggregation-prone proteins such as tau and amyloid-β in Alzheimer’s disease. Detecting intermediate-sized and potentially diffusible aggregates of αSyn in human tissue has therefore been a major challenge in the field. The study by Roberts et al. published in this issue of Brain provides a significant step towards solving this problem by establishing a method that promises to make small and intermediate αSyn aggregates visible (Roberts et al., 2015). Currently it is believed that neither the soluble monomeric nor the soluble tetrameric forms of αSyn (both presumed to be physiological) are cytotoxic. The monomeric form, however, is known to readily aggregate in vitro into soluble higher oligomeric forms and thence into amyloid-like fibrils, with the latter found in the pathological hallmarks of synucleinopathies, Lewy bodies and Lewy neurites (Spillantini et al., 1997). The fibrils appear to be devoid of neurotoxicity themselves, but they are thought to work as a temporarily protective reservoir of putatively active intermediates that can be released into the tissue, thereby contributing to the pathological mechanism (Fig. 1). While a variety of studies have focused on which toxic αSyn aggregates can form in vitro on the way from physiological monomers to the biologically inactive amyloid fibril, no evidence has emerged so far as to which of these intermediate forms are present in human disease-associated tissue. Roberts et al. now use an elegant technique to answer this question directly in frozen brain slices from patients with Parkinson’s disease. The authors adapted a method originally developed to detect interaction between two proteins, the proximity ligation assay (PLA) (Soderberg et al., 2006). The assay utilizes two antibodies coupled to specific short nucleotide sequences that, when in close proximity, undergo complementation and are ligated to form complete DNA circles, which in turn are amplified by ‘rolling circle’ DNA replication. The amplified DNA then binds fluorescent probes, hence enabling the detection of protein-protein complexes. Here, Roberts et al. chose to use two identical monoclonal anti-αSyn antibodies, thereby visualizing any protein aggregates composed of two or more αSyn molecules in tissue sections, and were thus poised to detect the earliest aggregation forms and give us a glimpse of the first aggregation events in the αSyn mechanism of Parkinson’s disease. Figure 1 Possible pathways of αSyn aggregation in synucleinopathies. Destabilization of the physiological forms of αSyn initiates aggregation and ultimately Lewy body formation. Detection of the respective intermediates is important for understanding ... This new method appears to preferentially detect intermediate sized αSyn oligomers while having much lower sensitivity for fibrillar material and monomeric αSyn in vitro. In tissue sections, large aggregates are only very occasionally (and then only peripherally) stained by this method, and physiological tetramers/multimers—for the existence of which there is growing evidence (Bartels et al., 2011; Dettmer et al., 2013; Burre et al., 2014; Wang et al., 2014)—also appear to be spared. In their Fig. 1C, for example, Roberts et al. show how wild-type αSyn-mediated YFP-complementation occurs diffusely in the cytosol, suggesting the presence of physiological tetramers/multimers, whereas αSyn-PLA in the same cells detects only local punctate staining indicative of an abnormally aggregated subpopulation, perhaps caused by overexpression/tagging of αSyn. It is tempting to speculate that this unexpected specificity may turn out to be one of the method’s biggest strengths: using other αSyn antibodies besides syn211 in the same assay could lead to a preferential detection of alternative pathological oligomers, potentially deepening our understanding of different αSyn aggregation species and possibly identifying species that are highly typical of the disease and completely absent in healthy brains. Combining different antibodies in this new method might lead to an even bigger tool set, which could indeed turn out to be very useful for the αSyn field. This, however, will require detailed controls that, for example, exclude signals caused by two antibodies binding to the same αSyn molecule. Roberts et al. provide such controls and appropriately discuss certain ambiguities that they observed. The abovementioned detection, limited to early and not mature aggregates, is useful for addressing several open questions in the αSyn field. First, is disease correlated with the accumulation of oligomers rather than mature Lewy bodies? Second, which brain regions are affected in the earlier, probably soluble oligomer-driven stage of the disease? Third, how common are these oligomers in the advanced stage of the disease? Robert et al.’s use of their new method to compare eight patients with Parkinson’s disease to an equal number of age- and sex-matched healthy controls revealed the presence of extensive, disease-associated oligomer staining in the medulla, midbrain and cingulate cortex. These brain regions are of particular interest, given their hypothesized involvement in the early stages of Parkinson’s disease and the presumed ‘spreading’ of cytopathology from the dorsal motor nucleus through the substantia nigra to the neocortex, as first proposed by Braak et al. (2004). Analogous to the aggregation time course of synthetic αSyn in vitro, one would expect a variety of aggregates among the different regions that are affected in a post-mortem Parkinson’s brain, with more mature aggregates in the regions that had been affected earliest, and more diffuse staining in areas of pathology emerging closer to the time of the patient’s death. While Roberts et al. focus only on regions that should be heavily affected according to the Braak hypothesis, the distinct staining with conventional immunohistochemistry (which detects mature Lewy bodies) versus PLA (which apparently detects early intermediates) is intriguing. In the study, mature Lewy bodies in the brainstem, thought to be the ‘oldest’ aggregates in patients according to Braak staging, are picked up by standard immunohistochemistry but rarely by PLA. Other aggregates, like extrasomal Lewy bodies and pale bodies on the other hand, are indeed detected with high sensitivity by PLA, indicating that they represent the less dense aggregates presumably characteristic of early pathology. Interestingly, Lewy bodies in areas that are putatively affected later in Parkinson’s disease, like the cingulate cortex, are detected more strongly by PLA than by conventional immunohistochemistry. This result further strengthens the claim that these regions have a later temporal involvement in the apparent pathological spread of αSyn aggregation. Excitingly, the authors also find diffuse cytoplasmic staining by PLA in neurons of patients but not healthy controls, which may give us a glimpse into the earliest events in disease pathogenesis. This diffuse staining extended to the neuropil and the white matter of the medulla and cingulate cortex. One could speculate that this points towards the involvement of the axons of the respective neurons, or perhaps even glial cells, in the earliest pathogenic events. For future studies, it would be very interesting to analyse a wider array of different brain regions to search for lesions detected with PLA. This might sketch out a timeline of αSyn aggregation in Parkinson’s disease and might even offer clues as to why certain neuronal populations, i.e. in the substantia nigra, are more severely affected than other αSyn-expressing regions in the brain. Finally, the authors show the general applicability of the PLA method in brains from other synucleinopathies, including dementia with Lewy bodies and multiple system atrophy. The technique used by Roberts et al. therefore holds great promise for elucidating the temporal hierarchy behind a variety of neurodegenerative diseases associated with αSyn aggregation.

Misfolding and pathogenic aggregation of α-synuclein (αSyn) is a hallmark of familial and sporadic Parkinson's disease, but the physiological state of the protein in cells remains unsettled. We have further examined our hypothesis that endogenous αSyn can occur in normal cells as a metastable, helically folded tetramer, not solely as the unfolded monomer long thought to be its native form. At this meeting, we reviewed our recent approaches for trapping αSyn in intact cells via in vivo crosslinking, a 5-step purification of αSyn from normal human brain, and the generation of new monoclonal antibodies to αSyn that enable general and oligomer-selective ELISAs. Crosslinking in intact living cells confirmed that αSyn occurs in the cytosol of neurons and non-neural cells in substantial part as metastable tetramers and related oligomers, plus varying amounts of free monomers. The non-pathogenic homolog, β-synuclein, forms closely similar oligomeric assemblies, suggesting that the oligomers we observe for αSyn are also physiological. In contrast to other normal oligomeric proteins (e.g., DJ-1), αSyn tetramers dissociate rapidly to monomers upon conventional cell lysis but are retained partially as tetramers if cells are lysed at high protein concentrations (‘molecular crowding'). Thus, αSyn exists natively as helical tetramers that are in dynamic equilibrium with unfolded monomers. The tetramers appear relatively resistant to aggregation, in contrast to monomers, which may give rise to fibrillar inclusions.

Aggregation of α-synuclein (αSyn) in neurons produces the hallmark cytopathology of Parkinson disease and related synucleinopathies. Since its discovery, αSyn has been thought to exist normally in cells as an unfolded monomer. We recently reported that αSyn can instead exist in cells as a helically folded tetramer that resists aggregation and binds lipid vesicles more avidly than unfolded recombinant monomers (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107–110). However, a subsequent study again concluded that cellular αSyn is an unfolded monomer (Fauvet, B., Mbefo, M. K., Fares, M. B., Desobry, C., Michael, S., Ardah, M. T., Tsika, E., Coune, P., Prudent, M., Lion, N., Eliezer, D., Moore, D. J., Schneider, B., Aebischer, P., El-Agnaf, O. M., Masliah, E., and Lashuel, H. A. (2012) J. Biol. Chem. 287, 15345–15364). Here we describe a simple in vivo cross-linking method that reveals a major ∼60-kDa form of endogenous αSyn (monomer, 14.5 kDa) in intact cells and smaller amounts of ∼80- and ∼100-kDa forms with the same isoelectric point as the 60-kDa species. Controls indicate that the apparent 60-kDa tetramer exists normally and does not arise from pathological aggregation. The pattern of a major 60-kDa and minor 80- and 100-kDa species plus variable amounts of free monomers occurs endogenously in primary neurons and erythroid cells as well as neuroblastoma cells overexpressing αSyn. A similar pattern occurs for the homologue, β-synuclein, which does not undergo pathogenic aggregation. Cell lysis destabilizes the apparent 60-kDa tetramer, leaving mostly free monomers and some 80-kDa oligomer. However, lysis at high protein concentrations allows partial recovery of the 60-kDa tetramer. Together with our prior findings, these data suggest that endogenous αSyn exists principally as a 60-kDa tetramer in living cells but is lysis-sensitive, making the study of natural αSyn challenging outside of intact cells. Background: αSyn is central to Parkinsonism, but its native state is unsettled. Results: A new, facile method for cross-linking αSyn in living cells, including neurons, reveals a major 60-kDa form consistent with a tetramer. Cell lysis destabilizes it, yielding mostly monomers. Conclusion: αSyn exists principally as a metastable tetramer in vivo. Significance: Models of native αSyn as an unfolded monomer should be reconsidered.

Summary α-Synuclein (αS) regulates vesicle exocytosis but forms insoluble deposits in Parkinson’s disease (PD). Developing disease-modifying therapies requires animal models that reproduce cardinal features of PD. We recently described a previously unrecognized physiological form of αS, α-helical tetramers, and showed that familial PD-causing missense mutations shift tetramers to aggregation-prone monomers. Here, we generated mice expressing the fPD E46K mutation plus 2 homologous E→K mutations in adjacent KTK E GV motifs. This tetramer-abrogating mutant causes phenotypes similar to PD. αS monomers accumulate at membranes and form vesicle-rich inclusions. αS becomes insoluble, proteinase K-resistant, Ser129-phosphorylated, and C-terminally truncated, as in PD. These changes affect regions controlling motor behavior, including a decrease in nigrostriatal dopaminergic neurons. The outcome is a progressive motor syndrome including tremor and gait and limb deficits partially responsive to L-DOPA. This fully penetrant phenotype indicates that tetramers are required for normal αS homeostasis and that chronically shifting tetramers to monomers may result in PD, with attendant therapeutic implications.

β-Sheet-rich α-synuclein (αS) aggregates characterize Parkinson's disease (PD). αS was long believed to be a natively unfolded monomer, but recent work suggests it also occurs in α-helix-rich tetramers. Crosslinking traps principally tetrameric αS in intact normal neurons, but not after cell lysis, suggesting a dynamic equilibrium. Here we show that freshly biopsied normal human brain contains abundant αS tetramers. The PD-causing mutation A53T decreases tetramers in mouse brain. Neurons derived from an A53T patient have decreased tetramers. Neurons expressing E46K do also, and adding 1-2 E46K-like mutations into the canonical αS repeat motifs (KTKEGV) further reduces tetramers, decreases αS solubility and induces neurotoxicity and round inclusions. The other three fPD missense mutations likewise decrease tetramer:monomer ratios. The destabilization of physiological tetramers by PD-causing missense mutations and the neurotoxicity and inclusions induced by markedly decreasing tetramers suggest that decreased α-helical tetramers and increased unfolded monomers initiate pathogenesis. Tetramer-stabilizing compounds should prevent this.

α-Synuclein (αSyn) is a highly abundant neuronal protein whose exact structure and function are under debate. Misfolding and aggregation of this normally soluble, 140-residue polypeptide underlies a group of neurodegenerative disorders called synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). The αSyn field has focused increasing attention on the hypotheses that certain aggregates of αSyn may be directly toxic to the neurons in which they arise and/or that aggregates can be released from some neurons and diffuse by undefined mechanisms to other neurons to seed αSyn in the recipient cells, thus propagating neuropathology by a non-cell autonomous process ('pathogenic spread'). While intense interest in these hypotheses has led to new approaches and tools to model aspects of the disorders, it is important to analyze which molecular events initiate αSyn aggregation inside neurons in the first place. Here, we review new insights into how neuronal αSyn homeostasis may be maintained under physiological conditions but perturbed by pathological factors.