Retinal ganglion cell type-specific expression of synuclein family members revealed by scRNA-sequencing.

Synuclein family members (Snca, Sncb, and Scng) are expressed in the retina, but their precise locations and roles are poorly understood. We performed an extensive analysis of the single-cell transcriptome in healthy and injured retinas to investigate their expression patterns and roles. We observed the expression of all synuclein family members in retinal ganglion cells (RGCs), which remained consistent across species (human, mouse, and chicken). We unveiled differential expression of Snca across distinct clusters (highly expressed in most), while Sncb and Sncg displayed uniform expression across all clusters. Further, we observed a decreased expression in RGCs following traumatic axonal injury. However, the proportion of α-Syn-positive RGCs in all RGCs and α-Syn-positive intrinsically photosensitive retinal ganglion cells (ipRGCs) in all ipRGCs remained unaltered. Lastly, we identified changes in communication patterns preceding cell death, with particular significance in the pleiotrophin-nucleolin (Ptn-Ncl) and neural cell adhesion molecule signaling pathways, where communication differences were pronounced between cells with varying expression levels of Snca. Our study employs an innovative approach using scRNA-seq to characterize synuclein expression in health retinal cells, specifically focusing on RGC subtypes, advances our knowledge of retinal physiology and pathology.

Understanding Genetic Risks: Computational Exploration of Human ß-Synuclein nsSNPs and their Potential Impact on Structural Alteration.

Synucleins are pivotal in neurodegenerative conditions. Beta-synuclein (ß-synuclein) is part of the synuclein protein family alongside alpha-synuclein (α-synuclein) and gamma-synuclein (γ-synuclein). These proteins, found mainly in brain tissue and cancers, are soluble and unstructured. ß-synuclein shares significant similarity with α-synuclein, especially in their N-terminus, with a 90% match. However, their aggregation tendencies differ significantly. While α-synuclein aggregation is believed to be counteracted by ß-synuclein, which occurs in conditions like Parkinson's disease, ß-synuclein may counteract α-synuclein's toxic effects on the nervous system, offering potential treatment for neurodegenerative diseases. Under normal circumstances, ß-synuclein may guard against disease by interacting with α-synuclein. Yet, in pathological environments with heightened levels or toxic substances, it might contribute to disease. Our research aims to explore potential harmful mutations in the ß-synuclein using computational tools to predict their destabilizing impact on protein structure. Consensus analysis revealed rs1207608813 (A63P), rs1340051870 (S72F), and rs1581178262 (G36C) as deleterious. These findings highlight the intricate relationship between nsSNPs and protein function, shedding light on their potential implications in disease pathways. Understanding the structural consequences of nsSNPs is crucial for elucidating their role in pathogenesis and developing targeted therapeutic interventions. Our results offer a robust computational framework for identifying neurodegenerative disorder-related mutations from SNP datasets, potentially reducing the costs associated with experimental characterization.

Differential impairment of cerebrospinal fluid synaptic biomarkers in the genetic forms of frontotemporal dementia.

BACKGROUND: Approximately a third of frontotemporal dementia (FTD) is genetic with mutations in three genes accounting for most of the inheritance: C9orf72, GRN, and MAPT. Impaired synaptic health is a common mechanism in all three genetic variants, so developing fluid biomarkers of this process could be useful as a readout of cellular dysfunction within therapeutic trials. METHODS: A total of 193 cerebrospinal fluid (CSF) samples from the GENetic FTD Initiative including 77 presymptomatic (31 C9orf72, 23 GRN, 23 MAPT) and 55 symptomatic (26 C9orf72, 17 GRN, 12 MAPT) mutation carriers as well as 61 mutation-negative controls were measured using a microflow LC PRM-MS set-up targeting 15 synaptic proteins: AP-2 complex subunit beta, complexin-2, beta-synuclein, gamma-synuclein, 14-3-3 proteins (eta, epsilon, zeta/delta), neurogranin, Rab GDP dissociation inhibitor alpha (Rab GDI alpha), syntaxin-1B, syntaxin-7, phosphatidylethanolamine-binding protein 1 (PEBP-1), neuronal pentraxin receptor (NPTXR), neuronal pentraxin 1 (NPTX1), and neuronal pentraxin 2 (NPTX2). Mutation carrier groups were compared to each other and to controls using a bootstrapped linear regression model, adjusting for age and sex. RESULTS: CSF levels of eight proteins were increased only in symptomatic MAPT mutation carriers (compared with controls) and not in symptomatic C9orf72 or GRN mutation carriers: beta-synuclein, gamma-synuclein, 14-3-3-eta, neurogranin, Rab GDI alpha, syntaxin-1B, syntaxin-7, and PEBP-1, with three other proteins increased in MAPT mutation carriers compared with the other genetic groups (AP-2 complex subunit beta, complexin-2, and 14-3-3 zeta/delta). In contrast, CSF NPTX1 and NPTX2 levels were affected in all three genetic groups (decreased compared with controls), with NPTXR concentrations being affected in C9orf72 and GRN mutation carriers only (decreased compared with controls). No changes were seen in the CSF levels of these proteins in presymptomatic mutation carriers. Concentrations of the neuronal pentraxins were correlated with brain volumes in the presymptomatic period for the C9orf72 and GRN groups, suggesting that they become abnormal in proximity to symptom onset. CONCLUSIONS: Differential synaptic impairment is seen in the genetic forms of FTD, with abnormalities in multiple measures in those with MAPT mutations, but only changes in neuronal pentraxins within the GRN and C9orf72 mutation groups. Such markers may be useful in future trials as measures of synaptic dysfunction, but further work is needed to understand how these markers change throughout the course of the disease.

Synuclein Analysis in Adult Xenopus laevis.

The α-, ß- and γ-synucleins are small soluble proteins expressed in the nervous system of mammals and evolutionary conserved in vertebrates. After being discovered in the cartilaginous fish Torpedo californica, synucleins have been sequenced in all vertebrates, showing differences in the number of genes and splicing isoforms in different taxa. Although α-, ß- and γ-synucleins share high homology in the N-terminal sequence, suggesting their evolution from a common ancestor, the three isoforms also differ in molecular characteristics, expression levels and tissue distribution. Moreover, their functions have yet to be fully understood. Great scientific interest on synucleins mainly derives from the involvement of α-synuclein in human neurodegenerative diseases, collectively named synucleinopathies, which involve the accumulation of amyloidogenic α-synuclein inclusions in neurons and glia cells. Studies on synucleinopathies can take advantage of the development of new vertebrate models other than mammals. Moreover, synuclein expression in non-mammalian vertebrates contribute to clarify the physiological role of these proteins in the evolutionary perspective. In this paper, gene expression levels of α-, ß- and γ-synucleins have been analysed in the main organs of adult Xenopus laevis by qRT-PCR. Moreover, recombinant α-, ß- and γ-synucleins were produced to test the specificity of commercial antibodies against α-synuclein used in Western blot and immunohistochemistry. Finally, the secondary structure of Xenopus synucleins was evaluated by circular dichroism analysis. Results indicate Xenopus as a good model for studying synucleinopathies, and provide a useful background for future studies on synuclein functions and their evolution in vertebrates.

ß-Synuclein: An Enigmatic Protein with Diverse Functionality.

α-Synuclein (αS) is a small, unstructured, presynaptic protein expressed in the brain. Its aggregated form is a major component of Lewy bodies, the large proteinaceous deposits in Parkinson's disease. The closely related protein, ß-Synuclein (ßS), is co-expressed with αS. In vitro, ßS acts as a molecular chaperone to inhibit αS aggregation. As a result of this assignation, ßS has been largely understudied in comparison to αS. However, recent reports suggest that ßS promotes neurotoxicity, implying that ßS is involved in other cellular pathways with functions independent of αS. Here, we review the current literature pertaining to human ßS in order to understand better the role of ßS in homeostasis and pathology. Firstly, the structure of ßS is discussed. Secondly, the ability of ßS to (i) act as a molecular chaperone; (ii) regulate synaptic function, lipid binding, and the nigrostriatal dopaminergic system; (iii) mediate apoptosis; (iv) participate in protein degradation pathways; (v) modulate intracellular metal levels; and (vi) promote cellular toxicity and protein aggregation is explored. Thirdly, the P123H and V70M mutations of ßS, which are associated with dementia with Lewy bodies, are discussed. Finally, the importance of post-translational modifications on the structure and function of ßS is reviewed. Overall, it is concluded that ßS has both synergistic and antagonistic interactions with αS, but it may also possess important cellular functions independent of αS.

Excess membrane binding of monomeric alpha-, beta- and gamma-synuclein is invariably associated with inclusion formation and toxicity.

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 ß-synuclein (ßS) 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-ß 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 ßS 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 ßS and γS could also become neurotoxic if they were more prone to membrane/lipid binding. Here, we increased ßS 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.

Dementia with Lewy bodies-associated ß-synuclein mutations V70M and P123H cause mutation-specific neuropathological lesions.

Beta (ß)-synuclein (ß-Syn) has long been considered to be an attenuator for the neuropathological effects caused by the Parkinson's disease-related alpha (α)-synuclein (α-Syn) protein. However, recent studies demonstrated that overabundant ß-Syn can form aggregates and induce neurodegeneration in central nervous system (CNS) neurons in vitro and in vivo, albeit at a slower pace as compared with α-Syn. Here, we demonstrate that ß-Syn mutants V70M, detected in a sporadic case of dementia with Lewy bodies (DLB), and P123H, detected in a familial case of DLB, robustly aggravate the neurotoxic potential of ß-Syn. Intriguingly, the two mutations trigger mutually exclusive pathways. ß-Syn V70M enhances morphological mitochondrial deterioration and degeneration of dopaminergic and non-dopaminergic neurons, but it has no influence on neuronal network activity. Conversely, ß-Syn P123H silences neuronal network activity, but it does not aggravate neurodegeneration. ß-Syn wild type (WT), V70M and P123H formed proteinase K-resistant intracellular fibrils within neurons, albeit with less stable C-termini as compared with α-Syn. Under cell-free conditions, ß-Syn V70M demonstrated a much slower pace of fibril formation as compared with WT ß-Syn, and P123H fibrils present with a unique phenotype characterized by large numbers of short, truncated fibrils. Thus, it is possible that V70M and P123H cause structural alterations in ß-Syn, which are linked to their distinct neuropathological profiles. The extent of the lesions caused by these neuropathological profiles is almost identical to that of overabundant α-Syn and is thus likely to be directly involved into the etiology of DLB. Overall, this study provides insights into distinct disease mechanisms caused by mutations of ß-Syn.

Temperature is a key determinant of alpha- and beta-synuclein membrane interactions in neurons.

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 ß-synuclein (ßS) under various conditions that differed in temperature and digitonin concentration (200-1200 µg/ml), we consistently found αS to be more membrane-associated than ßS. 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 ßS. 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.

Effect of Disease-Associated P123H and V70M Mutations on ß-Synuclein Fibrillation.

Synucleinopathies are a class of neurodegenerative diseases, including Parkinson's disease (PD), Dementia with Lewy bodies (DLB), and Multiple System Atrophy (MSA). The common pathological hallmark of synucleinopathies is the filamentous α-synuclein (α-Syn) aggregates along with membrane components in cytoplasmic inclusions in the brain. ß-Synuclein (ß-Syn), an isoform of α-Syn, inhibits α-Syn aggregation and prevents its neurotoxicity, suggesting the neuroprotective nature of ß-Syn. However, this notion changed with the discovery of disease-associated ß-Syn mutations, V70M and P123H, in patients with DLB. It is still unclear how these missense mutations alter the structural and amyloidogenic properties of ß-Syn, leading to neurodegeneration. Here, we characterized the biophysical properties and investigated the effect of mutations on ß-Syn fibrillation under different conditions. V70M and P123H show high membrane binding affinity compared to wild-type ß-Syn, suggesting their potential role in membrane interactions. ß-Syn and its mutants do not aggregate under normal physiological conditions; however, the proteins undergo self-polymerization in a slightly acidic microenvironment and/or in the presence of an inducer, forming long unbranched amyloid fibrils similar to α-Syn. Strikingly, V70M and P123H mutants exhibit accelerated fibrillation compared to native ß-Syn under these conditions. NMR study further revealed that these point mutations induce local perturbations at the site of mutation in ß-Syn. Overall, our data provide insight into the biophysical properties of disease-associated ß-Syn mutations and demonstrate that these mutants make the native protein more susceptible to aggregation in an altered microenvironment.

Possible Role of Amyloidogenic Evolvability in Dementia with Lewy Bodies: Insights from Transgenic Mice Expressing P123H ß-Synuclein.

Dementia with Lewy bodies (DLB) is the second most prevalent neurodegenerative dementia after Alzheimer's disease, and is pathologically characterized by formation of intracellular inclusions called Lewy bodies, the major constituent of which is aggregated α-synuclein (αS). Currently, neither a mechanistic etiology nor an effective disease-modifying therapy for DLB has been established. Although two missense mutations of ß-synuclein (ßS), V70M and P123H, were identified in sporadic and familial DLB, respectively, the precise mechanisms through which ßS mutations promote DLB pathogenesis remain elusive. To further clarify such mechanisms, we investigated transgenic (Tg) mice expressing P123H ßS, which develop progressive neurodegeneration in the form of axonal swelling and non-motor behaviors, such as memory dysfunction and depression, which are more prominent than motor deficits. Furthermore, cross-breeding of P123H ßS Tg mice with αS Tg mice worsened the neurodegenerative phenotype presumably through the pathological cross-seeding of P123H ßS with αS. Collectively, we predict that ßS misfolding due to gene mutations might be pathogenic. In this paper, we will discuss the possible involvement of amyloidogenic evolvability in the pathogenesis of DLB based on our previous papers regarding the P123H ßS Tg mice. Given that stimulation of αS evolvability by P123H ßS may underlie neuropathology in our mouse model, more radical disease-modifying therapy might be derived from the evolvability mechanism. Additionally, provided that altered ßS were involved in the pathogenesis of sporadic DLB, the P123H ßS Tg mice could be used for investigating the mechanism and therapy of DLB.

HLA DR2b-binding peptides from human endogenous retrovirus envelope, Epstein-Barr virus and brain proteins in the context of molecular mimicry in multiple sclerosis.

The aetiology of multiple sclerosis (MS) is as yet poorly understood. Multiple mechanisms in different disease stages are responsible for immunopathology in MS. HLA Class II DR2b (DRB1*1501 ß, DRA1*0101 α) is the strongest genetic risk factor for MS. Remnants of ancient retroviruses in the human genome, termed human endogenous retroviruses (HERV), and Epstein-Barr virus (EBV) infection are also associated with MS. In silico analyses of human endogenous retroviral envelope (HERV env) proteins and three myelin proteins that are principal targets of an autoimmune response in MS showed sequence similarities between potential TH epitopes within pairs of viral and myelin peptides predicted to bind HLA DR2b. This led to the proposal that such molecular mimicry may potentially trigger MS. HLA DR2b binding characteristics of previously identified peptides from the three myelin proteins and HERV env proteins as well as additional in silico predicted peptides from other encephalitogenic brain proteins and EBV proteins were studied to further investigate molecular mimicry. Peptides containing potential TH epitopes from the myelin oligodendrocyte glycoprotein and HERV env previously predicted to bind HLA DR2b as well as other pertinent potential HLA DR2b-restricted TH epitopes were confirmed to bind HLA DR2b molecules. Molecular modelling of HLA DR2b in complex with high affinity peptides derived from MOG and HERV env proteins showed that their binding could occur in a similar manner to a HLA DR2b-binding peptide containing a known TH epitope. A structurally related pair of peptides predicted to bind HLA DR2b from the EBV protein EBNA1 and ß synuclein, a brain protein implicated in MS, were also shown to similarly bind HLA DR2b. The findings justify investigating CD4+ T cell responses to the identified peptides.

ß-Synuclein-reactive T cells induce autoimmune CNS grey matter degeneration.

The grey matter is a central target of pathological processes in neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. The grey matter is often also affected in multiple sclerosis, an autoimmune disease of the central nervous system. The mechanisms that underlie grey matter inflammation and degeneration in multiple sclerosis are not well understood. Here we show that, in Lewis rats, T cells directed against the neuronal protein ß-synuclein specifically invade the grey matter and that this is accompanied by the presentation of multifaceted clinical disease. The expression pattern of ß-synuclein induces the local activation of these T cells and, therefore, determined inflammatory priming of the tissue and targeted recruitment of immune cells. The resulting inflammation led to significant changes in the grey matter, which ranged from gliosis and neuronal destruction to brain atrophy. In humans, ß-synuclein-specific T cells were enriched in patients with chronic-progressive multiple sclerosis. These findings reveal a previously unrecognized role of ß-synuclein in provoking T-cell-mediated pathology of the central nervous system.

Investigating the neuroprotective effect of AAV-mediated ß-synuclein overexpression in a transgenic model of synucleinopathy.

Parkinson's disease (PD) and multiple system atrophy (MSA) are neurodegenerative diseases characterized by inclusions mainly composed of α-synuclein (α-syn) aggregates. The objective of this study was to investigate if ß-synuclein (ß-syn) overexpression could have beneficial effects by inhibiting the aggregation of α-syn. The M83 transgenic mouse is a model of synucleinopathy, which develops severe motor symptoms associated with aggregation of α-syn. M83 neonate or adult mice were injected with adeno-associated virus vectors carrying the human ß-syn gene (AAVß-syn) or green fluorescent protein gene (AAVGFP) using different injection sites. The M83 disease was - or not - accelerated using extracts of M83 brains injected with brain extract from mouse (M83) or human (MSA) origins. AAV vectors expression was confirmed using Western blot and ELISA technics. AAV mediated ß-syn overexpression did not delay the disease onset or reduce the α-syn phosphorylated at serine 129 levels detected by ELISA, regardless of the AAV injection route and the inoculation of brain extracts. Instead, a proteinase-K resistant ß-syn staining was detected by immunohistochemistry, specifically in sick M83 mice overexpressing ß-syn after inoculation of AAVß-syn. This study indicated for the first time that viral vector-mediated ß-syn overexpression could form aggregates in a model of synucleinopathy.

Early manifestation of depressive-like behavior in transgenic mice that express dementia with Lewy body-linked mutant ß-synuclein.

AIM: We previously generated transgenic (Tg) mice that expressed P123H ß-synuclein (ßS), a dementia with Lewy body-linked mutant ßS. Notably, these mice recapitulated neurodegenerative features of Lewy body disease, reflected by motor dysfunction, greater protein aggregation, and memory impairment. Since recent studies suggested that non-motor symptoms, such as depression, might be manifested in the prodromal stage of Lewy body disease, the main objective of the present study was to investigate the early expression of behavior in P123H ßS Tg mice. METHODS: Nest building, locomotor activity, and depressive-like behavior were assessed using 6- to 10-month-old male and female P123H ßS Tg and wildtype mice. KEY RESULTS: P123H ßS Tg mice exhibited hyperlocomotor activity in a novel environment, a decrease in mobility time in the tail suspension test, and impairments in nest building. CONCLUSIONS: Importantly, these non-motor behaviors were manifested before the onset of motor dysfunction, suggesting that P123H ßS Tg mice could be a valid model for investigating the early phase of Lewy body disease.

INDEL Length and Haplotypes in the ß-Synuclein Gene: A Key to Differentiate Dementia with Lewy Bodies?

Lewy body diseases (LBD) include Parkinson's disease (PD) and dementia with Lewy bodies (DLB) and together with Alzheimer's disease (AD) they show an important neuropathological and clinical overlap. The human alpha- and beta-synuclein genes (SNCA and SNCB) are key factors for the development of Lewy body diseases. Here, we aimed to analyze the genotype distribution of potentially functional SNPs in SNCA and SNCB, perform haplotype analysis for SNCB, and to identify functional insertion and deletion (INDEL) variations within the regulatory region of SNCB which might be responsible for the drastically diminished beta-synuclein levels reported for pure DLB. Thus, we genotyped brain samples from AD, DLB, PD, and healthy controls for two SNCA and four SNCB SNPs. We also analyzed INDEL variations upstream of SNCB, determined SNCB expression levels, and correlated INDEL lengths with expression levels. Applying Fisher's exact, chi-square, ANOVA tests, and the ΔΔCt method, we found disease-specific genotype distribution of SNCA and SNCB SNPs. Additionally, we identified three INDEL variations upstream of SNCB and showed that the INDEL allele lengths were associated with SNCB expression levels. INDEL alleles associated with low SNCB expression were accumulated in pure DLB. Finally, one major and four minor DLB specific SNCB haplotypes were identified with Haploview and Arlequin. In summary, our study showed that different SNCA and SNCB genotypes are associated with the development of either PD or DLB, and that the frequencies of genotypes associated with low SNCB expression are elevated in DLB.

Immature-like molecular expression patterns in the hippocampus of a mouse model of dementia with Lewy body-linked mutant ß-synuclein.

AIM: Maturation abnormalities of the brain cells have been suggested in several neuropsychiatric disorders, including schizophrenia, bipolar disorder, autism spectrum disorders, and epilepsy. In this study, we examined the expression patterns of neuronal maturation markers in the brain of a mouse model of dementia with Lewy body-linked mutant ß-synuclein (ßS), especially in the hippocampus, to explore whether such brain abnormalities occur in neurodegenerative disorders as well. METHODS: Quantitative PCR (qPCR) and immunohistochemical analyses were performed using the hippocampus of 14-month-old P123H ßS transgenic (Tg) mice to evaluate the expression of molecular markers for maturation of dentate granule cells. RESULTS: Based on qPCR results, expression of Tdo2 and Dsp (markers of mature granule cells) was decreased and that of Drd1a (a marker of immature granule cells) was increased in the hippocampus of P123H ßS Tg mice compared to that in wild-type controls. Immunohistochemical analysis revealed decreased expression of mature granule cell markers Calb1 and Gria1, along with increased expression of the microglial marker Iba1, in the hippocampal dentate gyrus region of P123H ßS Tg mice. P123H ßS Tg mice exhibited immature-like neuronal molecular expression patterns and microgliosis in the hippocampus. Pseudo-immaturity of dentate granule cells, associated with neuroinflammation, may be a shared endophenotype in the brains of at least a subgroup of patients with neuropsychiatric disorders and neurodegenerative diseases.

Unveiling a Selective Mechanism for the Inhibition of α-Synuclein Aggregation by ß-Synuclein.

α-Synuclein (αS) is an intrinsically disordered protein that is associated with Parkinson's disease (PD) through its ability to self-assemble into oligomers and fibrils. Inhibition of this oligomerization cascade is an interesting approach to developing therapeutical strategies and ß-synuclein (ßS) has been described as a natural negative regulator of this process. However, the biological background and molecular mechanisms by which this inhibition occurs is unclear. Herein, we focused on assessing the effect of ßS on the aggregation of five αS pathological mutants linked to early-onset PD (A30P, E46K, H50Q, G51D and A53T). By coupling single molecule fluorescence spectroscopy to a cell-free protein expression system, we validated the ability of ßS to act as a chaperone of αS, effectively inhibiting its aggregation. Interestingly, we found that ßS does so in a selective manner, i.e., is a more effective inhibitor for certain αS pathological mutants-A30P and G51D-as compared to E46K, H50Q and A53T. Moreover, two-color coincidence experiments proved that this discrepancy is due to a preferential incorporation of ßS into smaller oligomers of αS. This was validated by showing that the chaperoning effect was lost when proteins were mixed after being expressed individually. This study highlights the potential of fluorescence spectroscopy to deconstruct αS aggregation cascade and its interplay with ßS.

Levels of α- and ß-synuclein regulate cellular susceptibility to toxicity from α-synuclein oligomers.

α-Synuclein (α-syn) is associated with a range of diseases, including Parkinson disease. In disease, α-syn is known to aggregate and has the potential to be neurotoxic. The association between copper and α-syn results in the formation of stellate toxic oligomers that are highly toxic to cultured neurons. We further investigated the mechanism of toxicity of α-syn oligomers. Cells that overexpress α-syn showed increased susceptibility to the toxicity of the oligomers, while those that overexpressed ß-syn showed increased resistance to the toxic oligomers. Elevated α-syn expression caused an increase in expression of the transcription factor Forkhead box O3a (FoxO3a). Inhibition of FoxO3a activity by the overexpression of DNA binding domain of FoxO3a resulted in significant protection from α-syn oligomer toxicity. Increased FoxO3a expression in cells was shown to be caused by increased ferrireductase activity and Fe(II) levels. These results suggest that α-syn increases FoxO3a expression as a result of its intrinsic ferrireductase activity. The results also suggest that FoxO3a plays a pivotal role in the toxicity of both Fe(II) and toxic α-syn species to neuronal cells.-Angelova, D. M., Jones, H. B. L., Brown, D. R. Levels of α- and ß-synuclein regulate cellular susceptibility to toxicity from α-synuclein oligomers.

A pH-dependent switch promotes ß-synuclein fibril formation via glutamate residues.

α-Synuclein (αS) is the primary protein associated with Parkinson's disease, and it undergoes aggregation from its intrinsically disordered monomeric form to a cross-ß fibrillar form. The closely related homolog ß-synuclein (ßS) is essentially fibril-resistant under cytoplasmic physiological conditions. Toxic gain-of-function by ßS has been linked to dysfunction, but the aggregation behavior of ßS under altered pH is not well-understood. In this work, we compare fibril formation of αS and ßS at pH 7.3 and mildly acidic pH 5.8, and we demonstrate that pH serves as an on/off switch for ßS fibrillation. Using αS/ßS domain-swapped chimera constructs and single residue substitutions in ßS, we localized the switch to acidic residues in the N-terminal and non-amyloid component domains of ßS. Computational models of ßS fibril structures indicate that key glutamate residues (Glu-31 and Glu-61) in these domains may be sites of pH-sensitive interactions, and variants E31A and E61A show dramatically altered pH sensitivity for fibril formation supporting the importance of these charged side chains in fibril formation of ßS. Our results demonstrate that relatively small changes in pH, which occur frequently in the cytoplasm and in secretory pathways, may induce the formation of ßS fibrils and suggest a complex role for ßS in synuclein cellular homeostasis and Parkinson's disease.

LRRK2 knockdown in zebrafish causes developmental defects, neuronal loss, and synuclein aggregation.

Although mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of genetic Parkinson's disease, their function is largely unknown. LRRK2 is pleiotropic in nature, shown to be involved in neurodegeneration and in more peripheral processes, including kidney functions, in rats and mice. Recent studies in zebrafish have shown conflicting evidence that removal of the LRRK2 WD40 domain may or may not affect dopaminergic neurons and/or locomotion. This study shows that ∼50% LRRK2 knockdown in zebrafish causes not only neuronal loss but also developmental perturbations such as axis curvature defects, ocular abnormalities, and edema in the eyes, lens, and otic vesicles. We further show that LRRK2 knockdown results in significant neuronal loss, including a reduction of dopaminergic neurons. Immunofluorescence demonstrates that endogenous LRRK2 is expressed in the lens, brain, heart, spinal cord, and kidney (pronephros), which mirror the LRRK2 morphant phenotypes observed. LRRK2 knockdown results further in the concomitant upregulation of ß-synuclein, PARK13, and SOD1 and causes ß-synuclein aggregation in the diencephalon, midbrain, hindbrain, and postoptic commissure. LRRK2 knockdown causes mislocalization of the Na(+) /K(+) ATPase protein in the pronephric ducts, suggesting that the edema might be linked to renal malfunction and that LRRK2 might be associated with pronephric duct epithelial cell differentiation. Combined, our study shows that LRRK2 has multifaceted roles in zebrafish and that zebrafish represent a complementary model to further our understanding of this central protein. © 2016 Wiley Periodicals, Inc.