β-III Tubulin Fragments Inhibit α-Synuclein Accumulation in Models of Multiple System Atrophy

Multiple system atrophy (MSA) is a neurodegenerative disease caused by α-synuclein aggregation in oligodendrocytes and neurons. Using a transgenic mouse model overexpressing human α-synuclein in oligodendrocytes, we previously demonstrated that oligodendrocytic α-synuclein inclusions induce neuronal α-synuclein accumulation and progressive neuronal degeneration. α-Synuclein binds to β-III tubulin, leading to the neuronal accumulation of insoluble α-synuclein in an MSA mouse model. The present study demonstrates that α-synuclein co-localizes with β-III tubulin in the brain tissue from patients with MSA and MSA model transgenic mice as well as neurons cultured from these mice. Accumulation of insoluble α-synuclein in MSA mouse neurons was blocked by the peptide fragment β-III tubulin (residues 235–282). We have determined the α-synuclein-binding domain of β-III tubulin and demonstrated that a short fragment containing this domain can suppress α-synuclein accumulation in the primary cultured cells. Administration of a short α-synuclein-binding fragment of β-III tubulin may be a novel therapeutic strategy for MSA.     

FAS-Dependent Cell Death in α-Synuclein Transgenic Oligodendrocyte Models of Multiple System Atrophy

Multiple system atrophy is a parkinsonian neurodegenerative disorder. It is cytopathologically characterized by accumulation of the protein p25α in cell bodies of oligodendrocytes followed by accumulation of aggregated α-synuclein in so-called glial cytoplasmic inclusions. p25α is a stimulator of α-synuclein aggregation, and coexpression of α-synuclein and p25α in the oligodendroglial OLN-t40-AS cell line causes α-synuclein aggregate-dependent toxicity. In this study, we investigated whether the FAS system is involved in α-synuclein aggregate dependent degeneration in oligodendrocytes and may play a role in multiple system atrophy. Using rat oligodendroglial OLN-t40-AS cells we demonstrate that the cytotoxicity caused by coexpressing α-synuclein and p25α relies on stimulation of the death domain receptor FAS and caspase-8 activation. Using primary oligodendrocytes derived from PLP-α-synuclein transgenic mice we demonstrate that they exist in a sensitized state expressing pro-apoptotic FAS receptor, which makes them sensitive to FAS ligand-mediated apoptosis. Immunoblot analysis shows an increase in FAS in brain extracts from multiple system atrophy cases. Immunohistochemical analysis demonstrated enhanced FAS expression in multiple system atrophy brains notably in oligodendrocytes harboring the earliest stages of glial cytoplasmic inclusion formation. Oligodendroglial FAS expression is an early hallmark of oligodendroglial pathology in multiple system atrophy that mechanistically may be coupled to α-synuclein dependent degeneration and thus represent a potential target for protective intervention.     

Oral N-Acetyl-Cysteine Attenuates Loss of Dopaminergic Terminals in α-Synuclein Overexpressing Mice

Levels of glutathione are lower in the substantia nigra (SN) early in Parkinson''s disease (PD) and this may contribute to mitochondrial dysfunction and oxidative stress. Oxidative stress may increase the accumulation of toxic forms of α-synuclein (SNCA). We hypothesized that supplementation with n-acetylcysteine (NAC), a source of cysteine – the limiting amino acid in glutathione synthesis, would protect against α-synuclein toxicity. Transgenic mice overexpressing wild-type human α-synuclein drank water supplemented with NAC or control water supplemented with alanine from ages 6 weeks to 1 year. NAC increased SN levels of glutathione within 5–7 weeks of treatment; however, this increase was not sustained at 1 year. Despite the transient nature of the impact of NAC on brain glutathione, the loss of dopaminergic terminals at 1 year associated with SNCA overexpression was significantly attenuated by NAC supplementation, as measured by immunoreactivity for tyrosine hydroxylase in the striatum (p = 0.007; unpaired, two-tailed t-test), with a similar but nonsignificant trend for dopamine transporter (DAT) immunoreactivity. NAC significantly decreased the levels of human SNCA in the brains of PDGFb-SNCA transgenic mice compared to alanine treated transgenics. This was associated with a decrease in nuclear NFκB localization and an increase in cytoplasmic localization of NFκB in the NAC-treated transgenics. Overall, these results indicate that oral NAC supplementation decreases SNCA levels in brain and partially protects against loss of dopaminergic terminals associated with overexpression of α-synuclein in this model.     

Hyperphosphorylation and insolubility of alpha-synuclein in transgenic mouse oligodendrocytes

(Oligodendro)glial cytoplasmic inclusions composed of α-synuclein (αSYN) characterize multiple system atrophy (MSA). Mature oligodendrocytes (OLs) do not normally express αSYN, so MSA pathology may arise from aberrant expression of αSYN in OLs. To study pathological deposition of αSYN in OLs, transgenic mice were generated in which human wild-type αSYN was driven by a proteolipid protein promoter. Transgenic αSYN was detected in OLs but no other brain cell type. At the light microscopic level, the transgenic αSYN profiles resembled glial cytoplasmic inclusions. Strikingly, the diagnostic hyperphosphorylation at S129 of αSYN was reproduced in the transgenic mice. A significant proportion of the transgenic αSYN was detergent insoluble, as in MSA patients. The histological and biochemical abnormalities were specific for the disease-relevant αSYN because control green fluorescent protein was fully soluble and evenly distributed throughout OL cell bodies and processes. Thus, ectopic expression αSYN in OLs might initiate salient features of MSA pathology.     

Rasagiline Ameliorates Olfactory Deficits in an Alpha-Synuclein Mouse Model of Parkinson's Disease

Impaired olfaction is an early pre-motor symptom of Parkinson''s disease. The neuropathology underlying olfactory dysfunction in Parkinson''s disease is unknown, however α-synuclein accumulation/aggregation and altered neurogenesis might play a role. We characterized olfactory deficits in a transgenic mouse model of Parkinson''s disease expressing human wild-type α-synuclein under the control of the mouse α-synuclein promoter. Preliminary clinical observations suggest that rasagiline, a monoamine oxidase-B inhibitor, improves olfaction in Parkinson''s disease. We therefore examined whether rasagiline ameliorates olfactory deficits in this Parkinson''s disease model and investigated the role of olfactory bulb neurogenesis. α-Synuclein mice were progressively impaired in their ability to detect odors, to discriminate between odors, and exhibited alterations in short-term olfactory memory. Rasagiline treatment rescued odor detection and odor discrimination abilities. However, rasagiline did not affect short-term olfactory memory. Finally, olfactory changes were not coupled to alterations in olfactory bulb neurogenesis. We conclude that rasagiline reverses select olfactory deficits in a transgenic mouse model of Parkinson''s disease. The findings correlate with preliminary clinical observations suggesting that rasagiline ameliorates olfactory deficits in Parkinson''s disease.     

Increased Neuronal α-Synuclein Pathology Associates with Its Accumulation in Oligodendrocytes in Mice Modeling α-Synucleinopathies

Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by striatonigral degeneration and olivo-pontocerebellar atrophy. The histopathological hallmark of MSA is glial cytoplasmic inclusions (GCI) within oligodendrocytes, accompanied by neuronal degeneration. MSA is a synucleinopathy, and α-Synuclein (α-Syn) is the major protein constituent of the GCI. It is unclear how the neuronal α-Syn protein accumulates in oligodendrocytes. We tested the hypothesis that oligodendrocytes can take up neuronal-secreted α-Syn as part of the pathogenic mechanisms leading to MSA. We report that increases in the degree of α-Syn soluble oligomers or intracellular α-Syn levels, enhance its secretion from cultured MN9D dopaminergic cells, stably expressing the protein. In accord, we show that primary oligodendrocytes from rat brain and oligodendroglial cell lines take-up neuronal-secreted or exogenously added α-Syn from their conditioning medium. This uptake is concentration-, time-, and clathrin-dependent. Utilizing the demonstrated effect of polyunsaturated fatty acids (PUFA) to enhance α-Syn neuropathology, we show an in vivo effect for brain docosahexaenoic acid (DHA) levels on α-Syn localization to oligodendrocytes in brains of a mouse model for synucleinopathies, expressing human A53T α-Syn cDNA under the PrP promoter. Hence, pathogenic mechanisms leading to elevated levels of α-Syn in neurons underlie neuronal secretion and subsequent uptake of α-Syn by oligodendrocytes in MSA.     

Glucocerebrosidase deficits in sporadic Parkinson disease

Parkinson disease (PD) is a progressive neurodegenerative movement disorder characterized pathologically by abnormal SNCA/α-synuclein protein inclusions in neurons. Impaired lysosomal autophagic degradation of cellular proteins is implicated in PD pathogenesis and progression. Heterozygous GBA mutations, encoding lysosomal GBA/glucocerebrosidase (glucosidase, β, acid), are the greatest genetic risk factor for PD, and reduced GBA and SNCA accumulation are related in PD models. Here we review our recent human brain tissue study demonstrating that GBA deficits in sporadic PD are related to the early accumulation of SNCA, and dysregulation of chaperone-mediated autophagy (CMA) pathways and lipid metabolism.     

Involvement of Peripheral Nerves in the Transgenic PLP-α-Syn Model of Multiple System Atrophy: Extending the Phenotype

Multiple system atrophy (MSA) is a fatal, rapidly progressive neurodegenerative disease with (oligodendro-)glial cytoplasmic α-synuclein (α-syn) inclusions (GCIs). Peripheral neuropathies have been reported in up to 40% of MSA patients, the cause remaining unclear. In a transgenic MSA mouse model featuring GCI-like inclusion pathology based on PLP-promoter driven overexpression of human α-syn in oligodendroglia motor and non-motor deficits are associated with MSA-like neurodegeneration. Since α-syn is also expressed in Schwann cells we aimed to investigate whether peripheral nerves are anatomically and functionally affected in the PLP-α-syn MSA mouse model.

Results

To this end, heat/cold as well as mechanical sensitivity tests were performed. Furthermore, in vivo and ex vivo nerve conduction and the G-ratios of the sciatic nerve were analyzed, and thermosensitive ion channel mRNA expression in dorsal root ganglia (DRG) was assessed. The presence of human α-syn in Schwann cells was associated with subtle behavioral impairments. The G-ratio of the sciatic nerve, the conduction velocity of myelinated and unmyelinated primary afferents and the expression of thermosensitive ion channels in the sensory neurons, however, were similar to wildtype mice.

Conclusion

Our results suggest that the PNS appears to be affected by Schwann cell α-syn deposits in the PLP-α-syn MSA mouse model. However, there was no consistent evidence for functional PNS perturbations resulting from such α-syn aggregates suggesting a more central cause of the observed behavioral abnormalities. Nonetheless, our results do not exclude a causal role of α-syn in the pathogenesis of MSA associated peripheral neuropathy.     

Prion infection promotes extensive accumulation of α-synuclein in aged human α-synuclein transgenic mice

In neurodegenerative disorders of the aging population, misfolded proteins, such as PrP^Sc, α-synuclein, amyloid β protein and tau, can interact resulting in enhanced aggregation, cross seeding and accelerated disease progression. Previous reports have shown that in Creutzfeldt-Jakob disease and scrapie, α-synuclein accumulates near PrP^Sc deposits. However, it is unclear if pre-existing human α-synuclein aggregates modified prion disease pathogenesis, or if PrP^Sc exacerbates the α-synuclein pathology. Here, we inoculated infectious prions into aged α-synuclein transgenic (tg) and non-transgenic littermate control mice by the intracerebral route. Remarkably, inoculation of RML and mNS prions into α-synuclein tg mice resulted in more extensive and abundant intraneuronal and synaptic α-synuclein accumulation. In addition, infectious prions led to the formation of perineuronal α-synuclein deposits with a neuritic plaque-like appearance. Prion pathology was unmodified by the presence of α-synuclein. However, with the mNS prion strain there was a modest but significant acceleration in the time to terminal prion disease in mice having α-synuclein aggregates as compared with non-tg mice. Taken together, these studies support the notion that PrP^Sc directly or indirectly promotes α-synuclein pathology.     

Targeted Overexpression of α-Synuclein by rAAV2/1 Vectors Induces Progressive Nigrostriatal Degeneration and Increases Vulnerability to MPTP in Mouse

Mutations, duplication and triplication of α-synuclein genes are linked to familial Parkinson’s disease (PD), and aggregation of α-synuclein (α-syn) in Lewy bodies (LB) is involved in the pathogenesis of the disease. The targeted overexpression of α-syn in the substantia nigra (SN) mediated by viral vectors may provide a better alternative to recapitulate the neurodegenerative features of PD. Therefore, we overexpressed human wild-type α-syn using rAAV2/1 vectors in the bilateral SN of mouse and examined the effects for up to 12 weeks. Delivery of rAAV-2/1-α-syn caused significant nigrostriatal degeneration including appearance of dystrophic striatal neurites, loss of nigral dopaminergic (DA) neurons and dissolving nigral neuron bodies in a time-dependent manner. In addition, the α-syn overexpressed mice also developed significant deficits in motor function at 12 weeks when the loss of DA neurons exceeded a threshold of 50%. To investigate the sensitivity to neurotoxins in mice overexpressing α-syn, we performed an MPTP treatment with the subacute regimen 8 weeks after rAAV injection. The impact of the combined genetic and environmental insults on DA neuronal loss, striatal dopamine depletion, dopamine turnover and motor dysfunction was markedly greater than that of either alone. Moreover, we observed increased phosphorylation (S129), accumulation and nuclear distribution of α-syn after the combined insults. In summary, these results reveal that the overexpressed α-syn induces progressive nigrostriatal degeneration and increases the susceptibility of DA neurons to MPTP. Therefore, the targeted overexpression of α-syn and the combination with environmental toxins may provide valuable models for understanding PD pathogenesis and developing related therapies.     

Anti-Human α-Synuclein N-Terminal Peptide Antibody Protects against Dopaminergic Cell Death and Ameliorates Behavioral Deficits in an AAV-α-Synuclein Rat Model of Parkinson’s Disease

The protein α-synuclein (α-Syn) has a central role in the pathogenesis of Parkinson’s disease (PD) and immunotherapeutic approaches targeting this molecule have shown promising results. In this study, novel antibodies were generated against specific peptides from full length human α-Syn and evaluated for effectiveness in ameliorating α-Syn-induced cell death and behavioral deficits in an AAV-α-Syn expressing rat model of PD. Fisher 344 rats were injected with rAAV vector into the right substantia nigra (SN), while control rats received an AAV vector expressing green fluorescent protein (GFP). Beginning one week after injection of the AAV-α-Syn vectors, rats were treated intraperitoneally with either control IgG or antibodies against the N-terminal (AB1), or central region (AB2) of α-Syn. An unbiased stereological estimation of TH+, NeuN+, and OX6 (MHC-II) immunostaining revealed that the α-Syn peptide antibodies (AB1 and AB2) significantly inhibited α-Syn-induced dopaminergic cell (DA) and NeuN+ cell loss (one-way ANOVA (F (3, 30) = 5.8, p = 0.002 and (F (3, 29) = 7.92, p = 0.002 respectively), as well as decreasing the number of activated microglia in the ipsilateral SN (one-way ANOVA F = 14.09; p = 0.0003). Antibody treated animals also had lower levels of α-Syn in the ipsilateral SN (one-way ANOVA F (7, 37) = 9.786; p = 0.0001) and demonstrated a partial intermediate improvement of the behavioral deficits. Our data suggest that, in particular, an α-Syn peptide antibody against the N-terminal region of the protein can protect against DA neuron loss and, to some extent behavioral deficits. As such, these results may be a potential therapeutic strategy for halting the progression of PD.     

New Roles of Glycosaminoglycans in α-Synuclein Aggregation in a Cellular Model of Parkinson Disease

The causes of Parkinson disease (PD) remain mysterious, although some evidence supports mitochondrial dysfunctions and α-synuclein accumulation in Lewy bodies as major events. The abnormal accumulation of α-synuclein has been associated with a deficiency in the ubiquitin-proteasome system and the autophagy-lysosomal pathway. Cathepsin D (cathD), the major lysosomal protease responsible of α-synuclein degradation was described to be up-regulated in PD model. As glycosaminoglycans (GAGs) regulate cathD activity, and have been recently suggested to participate in PD physiopathology, we investigated their role in α-synuclein accumulation by their intracellular regulation of cathD activity. In a classical neuroblastoma cell model of PD induced by MPP⁺, the genetic expression of GAGs-biosynthetic enzymes was modified, leading to an increase of GAGs amounts whereas intracellular level of α-synuclein increased. The absence of sulfated GAGs increased intracellular cathD activity and limited α-synuclein accumulation. GAGs effects on cathD further suggested that specific sequences or sulfation patterns could be responsible for this regulation. The present study identifies, for the first time, GAGs as new regulators of the lysosome degradation pathway, regulating cathD activity and affecting two main biological processes, α-synuclein aggregation and apoptosis. Finally, this opens new insights into intracellular GAGs functions and new fields of investigation for glycobiological approaches in PD and neurobiology.     

Fibrillar α-synuclein toxicity depends on functional lysosomes

Neurodegeneration in Parkinson''s disease (PD) can be recapitulated in animals by administration of α-synuclein preformed fibrils (PFFs) into the brain. However, the mechanism by which these PFFs induce toxicity is unknown. Iron is implicated in PD pathophysiology, so we investigated whether α-synuclein PFFs induce ferroptosis, an iron-dependent cell death pathway. A range of ferroptosis inhibitors were added to a striatal neuron-derived cell line (STHdhQ7/7 cells), a dopaminergic neuron–derived cell line (SN4741 cells), and WT primary cortical neurons, all of which had been intoxicated with α-synuclein PFFs. Viability was not recovered by these inhibitors except for liproxstatin-1, a best-in-class ferroptosis inhibitor, when used at high doses. High-dose liproxstatin-1 visibly enlarged the area of a cell that contained acidic vesicles and elevated the expression of several proteins associated with the autophagy-lysosomal pathway similarly to the known lysosomal inhibitors, chloroquine and bafilomycin A1. Consistent with high-dose liproxstatin-1 protecting via a lysosomal mechanism, we further de-monstrated that loss of viability induced by α-synuclein PFFs was attenuated by chloroquine and bafilomycin A1 as well as the lysosomal cysteine protease inhibitors, leupeptin, E-64D, and Ca-074-Me, but not other autophagy or lysosomal enzyme inhibitors. We confirmed using immunofluorescence microscopy that heparin prevented uptake of α-synuclein PFFs into cells but that chloroquine did not stop α-synuclein uptake into lysosomes despite impairing lysosomal function and inhibiting α-synuclein toxicity. Together, these data suggested that α-synuclein PFFs are toxic in functional lysosomes in vitro. Therapeutic strategies that prevent α-synuclein fibril uptake into lysosomes may be of benefit in PD.     

α-Synuclein Oligomers Induced by Docosahexaenoic Acid Affect Membrane Integrity

A key feature of Parkinson disease is the aggregation of α-synuclein and its intracellular deposition in fibrillar form. Increasing evidence suggests that the pathogenicity of α-synuclein is correlated with the activity of oligomers formed in the early stages of its aggregation process. Oligomers toxicity seems to be associated with both their ability to bind and affect the integrity of lipid membranes. Previously, we demonstrated that α-synuclein forms oligomeric species in the presence of docosahexaenoic acid and that these species are toxic to cells. Here we studied how interaction of these oligomers with membranes results in cell toxicity, using cellular membrane-mimetic and cell model systems. We found that α-synuclein oligomers are able to interact with large and small unilamellar negatively charged vesicles acquiring an increased amount of α-helical structure, which induces small molecules release. We explored the possibility that oligomers effects on membranes could be due to pore formation, to a detergent-like effect or to fibril growth on the membrane. Our biophysical and cellular findings are consistent with a model where α-synuclein oligomers are embedded into the lipid bilayer causing transient alteration of membrane permeability.     

Nitrated α-Synuclein Induces the Loss of Dopaminergic Neurons in the Substantia Nigra of Rats

Background

The pathology of Parkinson''s disease (PD) is characterized by the degeneration of the nigrostriatal dopaminergic pathway, as well as the formation of intraneuronal inclusions known as Lewy bodies and Lewy neurites in the substantia nigra. Accumulations of nitrated α-synuclein are demonstrated in the signature inclusions of Parkinson''s disease. However, whether the nitration of α-synuclein is relevant to the pathogenesis of PD is unknown.

Methodology/Principal Findings

In this study, effect of nitrated α-synuclein to dopaminergic (DA) neurons was determined by delivering nitrated recombinant TAT-α-synuclein intracellular. We provide evidence to show that the nitrated α-synuclein was toxic to cultured dopaminergic SHSY-5Y neurons and primary mesencephalic DA neurons to a much greater degree than unnitrated α-synuclein. Moreover, we show that administration of nitrated α-synuclein to the substantia nigra pars compacta of rats caused severe reductions in the number of DA neurons therein, and led to the down-regulation of D₂R in the striatum in vivo. Furthermore, when administered to the substantia nigra of rats, nitrated α-synuclein caused PD-like motor dysfunctions, such as reduced locomotion and motor asymmetry, however unmodified α-synuclein had significantly less severe behavioral effects.

Conclusions/Significance

Our results provide evidence that α-synuclein, principally in its nitrated form, induce DA neuron death and may be a major factor in the etiology of PD.     

Glucocerebrosidase Deficiency in Drosophila Results in α-Synuclein-Independent Protein Aggregation and Neurodegeneration

Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher’s disease, and are the most common genetic risk factor for Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) excluding variants of low penetrance. Because α-synuclein-containing neuronal aggregates are a defining feature of PD and DLB, it is widely believed that mutations in GBA1 act by enhancing α-synuclein toxicity. To explore this hypothesis, we deleted the Drosophila GBA1 homolog, dGBA1b, and compared the phenotypes of dGBA1b mutants in the presence and absence of α-synuclein expression. Homozygous dGBA1b mutants exhibit shortened lifespan, locomotor and memory deficits, neurodegeneration, and dramatically increased accumulation of ubiquitinated protein aggregates that are normally degraded through an autophagic mechanism. Ectopic expression of human α-synuclein in dGBA1b mutants resulted in a mild enhancement of dopaminergic neuron loss and increased α-synuclein aggregation relative to controls. However, α-synuclein expression did not substantially enhance other dGBA1b mutant phenotypes. Our findings indicate that dGBA1b plays an important role in the metabolism of protein aggregates, but that the deleterious consequences of mutations in dGBA1b are largely independent of α-synuclein. Future work with dGBA1b mutants should reveal the mechanism by which mutations in dGBA1b lead to accumulation of protein aggregates, and the potential influence of this protein aggregation on neuronal integrity.     

The HSP70 Molecular Chaperone Is Not Beneficial in a Mouse Model of α-synucleinopathy

Background

Aggregation and misfolded α-synuclein is thought to be central in the pathogenesis of Parkinson''s disease (PD). Heat-shock proteins (HSPs) that are involved in refolding and degradation processes could lower the aggregate load of α-synuclein and thus be beneficial in α-synucleinopathies.

Methodology/Principal Findings

We co-overexpressed human A53T point-mutated α-synuclein and human HSP70 in mice, both under the control of Thy1 regulatory sequences. Behavior read-outs showed no beneficial effect of HSP70 expression in mice. In contrast, motor coordination, grip strength and weight were even worse in the α-synucleinopathy model in the presence of HSP70 overexpression. Biochemical analyses revealed no differences in α-synuclein oligomers/aggregates, truncations and phosphorylation levels and α-synuclein localization was unchanged in immunostainings.

Conclusion/Significance

Overexpressing HSP70 in a mouse model of α-synucleinopathy did not lower the toxic load of α-synuclein species and had no beneficial effect on α-synuclein-related motor deficits.     

Identification of Synaptosomal Proteins Binding to Monomeric and Oligomeric α-Synuclein

Monomeric α-synuclein (αSN) species are abundant in nerve terminals where they are hypothesized to play a physiological role related to synaptic vesicle turn-over. In Parkinson’s disease (PD) and dementia with Lewy body (DLB), αSN accumulates as aggregated soluble oligomers in terminals, axons and the somatodendritic compartment and insoluble filaments in Lewy inclusions and Lewy neurites. The autosomal dominant heritability associated to mutations in the αSN gene suggest a gain of function associated to aggregated αSN. We have conducted a proteomic screen to identify the αSN interactome in brain synaptosomes. Porcine brain synaptosomes were fractionated, solubilized in non-denaturing detergent and subjected to co-immunoprecipitation using purified recombinant human αSN monomers or oligomers as bait. The isolated αSN binding proteins were identified with LC-LTQ-orbitrap tandem mass spectrometry and quantified by peak area using Windows client application, Skyline Targeted Proteomic Environment. Data are available via ProteomeXchange with identifier PXD001462. To quantify the preferential binding an average fold increase was calculated by comparing binding to monomer and oligomer. We identified 10 proteins preferentially binding monomer, and 76 binding preferentially to oligomer and a group of 92 proteins not displaying any preferred conformation of αSN. The proteomic data were validated by immunoprecipitation in both human and porcine brain extracts using antibodies against monomer αSN interactors: Abl interactor 1, and myelin proteolipid protein, and oligomer interactors: glutamate decarboxylase 2, synapsin 1, glial fibrillary acidic protein, and VAMP-2. We demonstrate the existence of αSN conformation selective ligands and present lists of proteins, whose identity and functions will be useful for modeling normal and pathological αSN dependent processes.     

A Blood-Brain Barrier (BBB) Disrupter Is Also a Potent α-Synuclein (α-syn) Aggregation Inhibitor: A NOVEL DUAL MECHANISM OF MANNITOL FOR THE TREATMENT OF PARKINSON DISEASE (PD)*

The development of disease-modifying therapy for Parkinson disease has been a main drug development challenge, including the need to deliver the therapeutic agents to the brain. Here, we examined the ability of mannitol to interfere with the aggregation process of α-synuclein in vitro and in vivo in addition to its blood-brain barrier-disrupting properties. Using in vitro studies, we demonstrated the effect of mannitol on α-synuclein aggregation. Although low concentration of mannitol inhibited the formation of fibrils, high concentration significantly decreased the formation of tetramers and high molecular weight oligomers and shifted the secondary structure of α-synuclein from α-helical to a different structure, suggesting alternative potential pathways for aggregation. When administered to a Parkinson Drosophila model, mannitol dramatically corrected its behavioral defects and reduced the amount of α-synuclein aggregates in the brains of treated flies. In the mThy1-human α-synuclein transgenic mouse model, a decrease in α-synuclein accumulation was detected in several brain regions following treatment, suggesting that mannitol promotes α-synuclein clearance in the cell bodies. It appears that mannitol has a general neuroprotective effect in the transgenic treated mice, which includes the dopaminergic system. We therefore suggest mannitol as a basis for a dual mechanism therapeutic agent for the treatment of Parkinson disease.