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.

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.

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.

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.

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.

The loss of inhibitory C-terminal conformations in disease associated P123H ß-synuclein.

ß-synuclein (ßS) is a homologue of α-synuclein (αS), the major protein component of Lewy bodies in patients with Parkinson's disease. In contrast to αS, ßS does not form fibrils, mitigates αS toxicity in vivo and inhibits αS fibril formation in vitro. Previously a missense mutation of ßS, P123H, was identified in patients with Dementia with Lewy Body disease. The single P123H mutation at the C-terminus of ßS is able to convert ßS from a nontoxic to a toxic protein that is also able to accelerate formation of inclusions when it is in the presence of αS in vivo. To elucidate the molecular mechanisms of these processes, we compare the conformational properties of the monomer forms of αS, ßS and P123H-ßS, and the effects on fibril formation of coincubation of αS with ßS, and with P123H-ßS. NMR residual dipolar couplings and secondary structure propensities show that the P123H mutation of ßS renders it more flexible C-terminal to the mutation site and more αS-like. In vitro Thioflavin T fluorescence experiments show that P123H-ßS accelerates αS fibril formation upon coincubation, as opposed to wild type ßS that acts as an inhibitor of αS aggregation. When P123H-ßS becomes more αS-like it is unable to perform the protective function of ßS, which suggests that the extended polyproline II motif of ßS in the C-terminus is critical to its nontoxic nature and to inhibition of αS upon coincubation. These studies may provide a basis for understanding which regions to target for therapeutic intervention in Parkinson's disease.

Fish Synucleins: An Update.

Synucleins (syns) are a family of proteins involved in several human neurodegenerative diseases and tumors. Since the first syn discovery in the brain of the electric ray Torpedo californica, members of the same family have been identified in all vertebrates and comparative studies have indicated that syn proteins are evolutionary conserved. No counterparts of syns were found in invertebrates suggesting that they are vertebrate-specific proteins. Molecular studies showed that the number of syn members varies among vertebrates. Three genes encode for α-, ß- and γ-syn in mammals and birds. However, a variable number of syn genes and encoded proteins is expressed or predicted in fish depending on the species. Among biologically verified sequences, four syn genes were identified in fugu, encoding for α, ß and two γ (γ1 and γ2) isoforms, whereas only three genes are expressed in zebrafish, which lacks α-syn gene. The list of "non verified" sequences is much longer and is often found in sequence databases. In this review we provide an overview of published papers and known syn sequences in agnathans and fish that are likely to impact future studies in this field. Indeed, fish models may play a key role in elucidating some of the molecular mechanisms involved in physiological and pathological functions of syn proteins.

Influence of Lentiviral ß-Synuclein Overexpression in the Hippocampus of a Transgenic Mouse Model of Alzheimer's Disease on Amyloid Precursor Protein Metabolism and Pathology.

BACKGROUND: ß-Synuclein (ß-Syn) is a member of the highly homologous synuclein protein family. The most prominent family member, α-synuclein (α-Syn), abnormally accumulates in so-called Lewy bodies, one of the major pathological hallmarks of α-synucleinopathies. Notably, parts of the peptide backbone, called the nonamyloid component, are also found in amyloid plaques. However, ß-Syn seems to have beneficial effects by reducing α-Syn aggregation, and amyloid antiaggregatory activity has been described. OBJECTIVE: The aim of the study was to analyze if wild-type ß-Syn can counteract functional and pathological changes in a murine Alzheimer model over different time periods. METHODS: At the onset of pathology, lentiviral particles expressing human ß-Syn were injected into the hippocampus of transgenic mice overexpressing human amyloid precursor protein with Swedish and London mutations (APPSL). An empty vector served as the control. Behavioral analyses were performed 1, 3 and 6 months after injection followed by biochemical and histological examinations of brain samples. RESULTS: ß-Syn expression was locally concentrated and rather modest, but nevertheless changed its effect on APP expression and plaque load in a time- and concentration-dependent manner. Interestingly, the phosphorylation of glycogen synthase kinase 3 beta was enhanced in APPSL mice expressing human ß-Syn, but an inverse trend was observed in wild-type animals. CONCLUSION: The initially reported beneficial effects of ß-Syn could be partially reproduced, but locally elevated levels of ß-Syn might also cause neurodegeneration. To enlighten the controversial pathological mechanism of ß-Syn, further examinations considering the relationship between concentration and exposure time of ß-Syn are needed.

Differences in the binding of copper(I) to α- and ß-synuclein.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of abnormal α-synuclein (αS) deposits in the brain. Alterations in homeostasis and metal-induced oxidative stress may play a crucial role in the progression of αS amyloid assembly and pathogenesis of PD. Contrary to αS, ß-synuclein (ßS) is not involved in the PD etiology. However, it has been suggested that the ßS/αS ratio is altered in PD, indicating that a correct balance of these two proteins is implicated in the inhibition of αS aggregation. αS and ßS share similar abilities to coordinate Cu(II). In this study, we investigated and compared the interaction of Cu(I) with the N-terminal portion of ßS and αS by means of NMR, circular dichroism, and X-ray absorption spectroscopies. Our data show the importance of M10K mutation, which induces different Cu(I) chemical environments. Coordination modes 3S1O and 2S2O were identified for ßS and αS, respectively. These new insights into the bioinorganic chemistry of copper and synuclein proteins are a basis to understand the molecular mechanism by which ßS might inhibit αS aggregation.

Evolutionary aspects of the synuclein super-family and sub-families based on large-scale phylogenetic and group-discrimination analysis.

Over the last decade, many genetic studies have suggested that the synucleins, which are small, natively unfolded proteins, are closely related to Parkinson's disease and cancer. Less is known about the molecular basis of this role. A comprehensive analysis of the evolutionary path of the synuclein protein family may reveal the relationship between evolutionarily conserved residues and protein function or structure. The phylogeny of 252 unique synuclein sequences from 73 organisms suggests that gamma-synuclein is the common ancestor of alpha- and beta-synuclein. Although all three sub-families remain highly conserved, especially at the N-terminal, nearly 15% of the residues in each sub family clearly diverged during evolution, providing crucial guidance for investigations of the different properties of the members of the superfamily. His50 is found to be an alpha-specific conserved residue (91%) and, based on mutagenesis, evolutionarily developed a secondary copper binding site in the alpha synuclein family. Surprisingly, this site is located between two well-known polymorphisms of alpha-synuclein, E46K and A53T, which are linked to early-onset Parkinson's disease, suggesting that the mutation-induced impairment of copper binding could be a mechanism responsible for alpha-synuclein aggregation.

Counter-regulation of alpha- and beta-synuclein expression at the transcriptional level.

Alpha-synuclein is a cytosolic protein associated with a range of diseases including Parkinson's disease. In these diseases alpha-synuclein aggregates and this is believed to play a causative role in disease progression. Alpha-synuclein aggregation has been suggested to be caused by increased expression levels and has also been suggested to be countered by increased beta-synuclein expression. In this regard, strategies to counter-regulate the expression of the synucleins by increasing beta-synuclein expression relative to alpha-synuclein may be beneficial in preventing disease progression. We therefore studied the regulation of alpha-synuclein to try to identify pathways that might counter-regulate the synucleins. We identified members of the ZSCAN family of transcription factors as specific repressors of alpha-synuclein. In particular ZSCAN21 was found to both repress alpha-synuclein and increase beta-synuclein expression. These findings support the notion that a single pathway in the cell can counter-regulate the expression of the synucleins. Support for this came from experiments that showed that ZSCAN21 expression decreases alpha-synuclein aggregation in the cells.

Macula-less rat and macula-bearing monkey retinas exhibit common lifelong proteomic changes.

The visual consequences of age-related alterations in the neural retina have been well documented, but little is known about their molecular bases. We performed a comparative proteomic analysis of the retinas in marmosets and rats to identify proteins for which the expression profiles are altered with maturation and aging. Protein profiles were compared in the newborn, juvenile, middle-age, and aged retinas using 2-dimensional gel electrophoresis. Matrix-assisted desorption ionization-time-of-flight mass spectrometry revealed common proteins in rats and marmosets that exhibited changes in concentration throughout life. Western blot, quantitative reverse-transcriptase polymerase chain reaction, and immunohistochemistry analyses of selected proteins and their mRNA were used to determine whether the changes identified by proteomics were verifiable at the cellular and molecular levels. We found 4 proteins common to both species (Parkinson disease [autosomal recessive, early onset] 7/DJ-1, stathmin, peroxiredoxin, and ß-synuclein) whose concentrations were regulated with age. These findings were confirmed by Western blot, immunohistochemistry, and quantitative reverse-transcriptase polymerase chain reaction analyses. The proteins were localized in certain layers and subsets of cells within the retinas of both species. The expression of these proteins in the adult human retina was confirmed with immunohistochemistry. The present study is the first to provide evidence that the retina is physiologically characterized by specific lifelong changes in its proteome. These changes are independent of whether the retina bears a macula, occur in key functional pathways during the processing of visual signals, and might be involved in the development of age-related pathologic entities.

Molecular ageing of alpha- and Beta-synucleins: protein damage and repair mechanisms.

Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and ß-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and ß-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and ß-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[(3)H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated (3)H-methanol. All α-synuclein proteins accumulated isoaspartate at ∼1% of molecules/day, ∼20 times faster than for ß-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of (3)H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar ß-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and ß-synucleins, or α, or ß synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ∼57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with ß-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and ß-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of ß-synuclein.

ß-synuclein aggregates and induces neurodegeneration in dopaminergic neurons.

OBJECTIVE: Whereas the contribution of α-synuclein to neurodegeneration in Parkinson disease is well accepted, the putative impact of its close homologue, ß-synuclein, is enigmatic. ß-Synuclein is widely expressed throughout the central nervous system, as is α-synuclein, but the physiological functions of both proteins remain unknown. Recent findings have supported the view that ß-synuclein can act as an ameliorating regulator of α-synuclein-induced neurotoxicity, having neuroprotective rather than neurodegenerative capabilities, and being nonaggregating due to the absence of most of the aggregation-promoting NAC domain. However, a mutation of ß-synuclein linked to dementia with Lewy bodies rendered the protein neurotoxic in transgenic mice, and fibrillation of ß-synuclein has been demonstrated in vitro. METHODS: Neurotoxicity and aggregation properties of α-, ß-, and γ-synuclein were comparatively elucidated in the rat nigro-striatal projection and in cultured neurons. RESULTS: Supporting the hypothesis that ß-synuclein can act as a neurodegeneration-inducing factor, we demonstrated that wild-type ß-synuclein is neurotoxic for cultured primary neurons. Furthermore, ß-synuclein formed proteinase K-resistant aggregates in dopaminergic neurons in vivo, leading to pronounced and progressive neurodegeneration in rats. Expression of ß-synuclein caused mitochondrial fragmentation, but this fragmentation did not render mitochondria nonfunctional in terms of ion handling and respiration even at late stages of neurodegeneration. A comparison of the neurodegenerative effects induced by α-, ß-, and γ-synuclein revealed that ß-synuclein was eventually as neurotoxic as α-synuclein for nigral dopaminergic neurons, whereas γ-synuclein proved to be nontoxic and had very low aggregation propensity. INTERPRETATION: Our results suggest that the role of ß-synuclein as a putative modulator of neuropathology in aggregopathies like Parkinson disease and dementia with Lewy bodies needs to be revisited.

In vivo cross-linking reveals principally oligomeric forms of α-synuclein and ß-synuclein in neurons and non-neural cells.

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, ß-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.

Transcriptional profiling of striatal neurons in response to single or concurrent activation of dopamine D2, adenosine A(2A) and metabotropic glutamate type 5 receptors: focus on beta-synuclein expression.

G protein-coupled receptor oligomerization is a concept which is changing the understanding of classical pharmacology. Both, oligomerization and functional interaction between adenosine A(2A,) dopamine D(2) and metabotropic glutamate type 5 receptors have been demonstrated in the striatum. However, the transcriptional consequences of receptors co-activation are still unexplored. We aim here to determine the changes in gene expression of striatal primary cultured neurons upon isolated or simultaneous receptor activation. Interestingly, we found that 95 genes of the total analyzed (15,866 transcripts and variants) changed their expression in response to simultaneous stimulation of all three receptors. Among these genes, we focused on the ß-synuclein (ß-Syn) gene (SCNB). Quantitative PCR verified the magnitude and direction of change in expression of SCNB. Since ß-Syn belongs to the homologous synuclein family and may be considered a natural regulator of α-synuclein (α-Syn), it has been proposed that ß-Syn might act protectively against α-Syn neuropathology.

Strain-independent global effect of hippocampal proteins in mice trained in the Morris water maze.

A series of individual proteins have been linked to performance in the Morris water maze (MWM) but no global effects have been reported. It was therefore the aim of the study to show which proteins were strain-independent, global factors for training in the MWM. Strains C57BL/6J, apodemus sylvaticus and PWD/PhJ were used. MWM and gels from trained animals were from a previous own study and corresponding yoked groups were generated. Hippocampal proteins were extracted and run on two-dimensional gel electrophoresis. Spots with different expressional levels between trained and yoked groups were punched and identified by mass spectrometry (nano-LC-ESI-MS/MS, ion trap). Two-way ANOVA with two factors (strain and training) was carried out and a Bonferroni test was used to compare groups. 12 proteins from several pathways and cascades showed different levels in trained mice versus corresponding yoked animals in all strains tested. Four out of these proteins were verified by immunoblotting: beta-synuclein, profilin 2, nucleoside diphosphate kinase A (NME1) and isocitrate dehydrogenase 3. Four proteins verified by immunoblotting could be shown to be involved in training in the MWM as a global effect, independent of the strain tested.

[Role of genetics in the etiology of synucleinopathies]. / Papel de la genética en la etiología de las sinucleinopatías.

The protein family known as synucleins is composed of α-, ß- and γ-synuclein. The most widely studied is the α-synuclein protein due to its participation in essential processes of the central nervous system. Neurotoxicity of this protein is related to the presence of multiplications (duplications and triplications) and point mutations in the gene sequence of the α-synuclein gene (SNCA), differential expression of its isoforms and variations in post-transductional modifications. Neurotoxicity is also related to cytoplasmic inclusions known as Lewy bodies (LBs) and Lewy neurites (LNs), which are also present in α-synucleinopathies. In general, the ß-synuclein protein, codified by the SNCB gene, acts as a regulator of processes triggered by α-synuclein and its function is altered by variations in the gene sequence, while γ-synuclein, codified by the SNCG gene, seems to play a major role in certain tumoral processes.

Inhibiting α-synuclein oligomerization by stable cell-penetrating ß-synuclein fragments recovers phenotype of Parkinson's disease model flies.

The intracellular oligomerization of α-synuclein is associated with Parkinson's disease and appears to be an important target for disease-modifying treatment. Yet, to date, there is no specific inhibitor for this aggregation process. Using unbiased systematic peptide array analysis, we identified molecular interaction domains within the ß-synuclein polypeptide that specifically binds α-synuclein. Adding such peptide fragments to α-synuclein significantly reduced both amyloid fibrils and soluble oligomer formation in vitro. A retro-inverso analogue of the best peptide inhibitor was designed to develop the identified molecular recognition module into a drug candidate. While this peptide shows indistinguishable activity as compared to the native peptide, it is stable in mouse serum and penetrates α-synuclein over-expressing cells. The interaction interface between the D-amino acid peptide and α-synuclein was mapped by Nuclear Magnetic Resonance spectroscopy. Finally, administering the retro-inverso peptide to a Drosophila model expressing mutant A53T α-synuclein in the nervous system, resulted in a significant recovery of the behavioral abnormalities of the treated flies and in a significant reduction in α-synuclein accumulation in the brains of the flies. The engineered retro-inverso peptide can serve as a lead for developing a novel class of therapeutic agents to treat Parkinson's disease.