Mutant Alpha-synuclein Research Articles

Evidence for prodromal changes in neuronal excitability and neuroinflammation in the hippocampus in young alpha-synuclein (A30P) transgenic mice.

Neuronal hyperexcitability and neuroinflammation are thought to occur at early stages in a range of neurodegenerative diseases. Neuroinflammation, notably activation of microglia, has been identified as a potential prodromal marker of dementia with Lewy bodies (DLB). Using a transgenic mouse model of DLB that over-expresses human mutant (A30P) alpha-synuclein (hα-syn) we have investigated whether early neuroinflammation is evident in the hippocampus in young pre-symptomatic animals. Previous studies have shown early hyperexcitability in the hippocampal CA3 region in male A30P mice at 2-4 months of age, therefore, in the current study we have immunostained this region for markers of neuronal activity (c-Fos), reactive astrocytes (glial fibrillary acidic protein, GFAP), microglia (ionizing calcium binding adapter protein 1, Iba-1) and reactive microglia (inducible nitric oxide synthase, iNOS). We found an interesting biphasic change in the expression of c-Fos in A30P mice with high expression at 1 month, consistent with early onset of hyperexcitability, but lower expression from 2-4 months in male A30P mice compared to wild-type (WT) controls, possibly indicating chronic hyperexcitability. Neuroinflammation was indicated by significant increases in the % area of GFAP and the number of Iba-1+ cells that expressed iNOS immunoreactivity in the CA3 region in 2-4 months A30P male mice compared to WT controls. A similar increase in % area of GFAP was observed in female A30P mice, however, the Iba-1 count was not different between female WT and A30P mice. In WT mice aged 2-4 months only 4.6% of Iba-1+ cells co-expressed iNOS. In contrast, in age matched A30P mice 87% of cells co-expressed Iba-1 and iNOS. Although there was no difference in GFAP immunoreactivity at 1 month, Iba-1/iNOS co-expression was also increased in a cohort of 1 month old A30P mice. Abnormal hα-syn expression in A30P mice caused early changes in network excitability, as indicated by c-Fos expression, and neuroinflammation which might contribute to disease progression.

Mice Expressing A53T/A30P Mutant Alpha-Synuclein in Dopamine Neurons Do Not Display Behavioral Deficits.

Alpha-synuclein has been implicated in neurodegenerative diseases such as Parkinson's disease and dementia with Lewy bodies, with A53T and A30P mutations shown to be disease causing. It has been reported that hemizygous transgenic mice with tyrosine hydroxylase promotor-driven expression of A53T/A30P mutant alpha-synuclein in dopamine neurons provide a useful preclinical model of these conditions by virtue of developing behavioral deficits. Here, we report a lack of replication of this finding. Despite detecting robust overexpression of A53T/A30P mutant alpha-synuclein in dopamine neurons, we did not observe decreased tyrosine hydroxylase immunofluorescence or behavioral deficits in these mice. Our results demonstrate that preclinical models of synucleinopathy need careful validation in the field.

Modulation of TRPV1 and TRPA1 Channels Function by Sea Anemones' Peptides Enhances the Viability of SH-SY5Y Cell Model of Parkinson's Disease.

Cellular dysfunction during Parkinson's disease leads to neuroinflammation in various brain regions, inducing neuronal death and contributing to the progression of the disease. Different ion channels may influence the process of neurodegeneration. The peptides Ms 9a-1 and APHC3 can modulate the function of TRPA1 and TRPV1 channels, and we evaluated their cytoprotective effects in differentiated to dopaminergic neuron-like SH-SY5Y cells. We used the stable neuroblastoma cell lines SH-SY5Y, producing wild-type alpha-synuclein and its mutant A53T, which are prone to accumulation of thioflavin-S-positive aggregates. We analyzed the viability of cells, as well as the mRNA expression levels of TRPA1, TRPV1, ASIC1a channels, alpha-synuclein, and tyrosine hydroxylase after differentiation of these cell lines using RT-PCR. Overexpression of alpha-synuclein showed a neuroprotective effect and was accompanied by a reduction of tyrosine hydroxylase expression. A mutant alpha-synuclein A53T significantly increased the expression of the pro-apoptotic protein BAX and made cells more susceptible to apoptosis. Generally, overexpression of alpha-synuclein could be a model for the early stages of PD, while expression of mutant alpha-synuclein A53T mimics a genetic variant of PD. The peptides Ms 9a-1 and APHC3 significantly reduced the susceptibility to apoptosis of all cell lines but differentially influenced the expression of the genes of interest. Therefore, these modulators of TRPA1 and TRPV1 have the potential for the development of new therapeutic agents for neurodegenerative disease treatment.

Significance of Cell Membrane Repair in Parkinson's Disease

Objective: Parkinson’s disease (PD) is the second most common neurodegenerative disorder and affects an estimated 10 million people worldwide. PD pathogenesis involves the death of neuronal cells due to the breakdown of their cell membranes. Membrane repair is a mechanism that appears in nearly every eukaryotic cell that repairs disruptions in the plasma membrane to maintain cell health and function. The aim of the present study was to determine the if compromised membrane repair can contribute to the progression of PD. Materials and Methods: Using a glass bead mechanical damage assay, we injured N2A neuroblastoma cells with three different conditions: GFP transfected control cells, cells expressing alpha synuclein, and cells expressing A53T, an alpha synuclein mutant linked to PD. We then measured the amount of an intracellular enzyme, lactase dehydrogenase (LDH), that was released from the cells after injury with glass beads to determine how well the membrane repaired. (n=16) We also injured the membrane of these cells with a laser injury heat damage assay. In this mechanism, a laser is used to injure the cells, and influx of FM4-64 dye into the cell is measured to determine the rate of membrane repair. (n=60) Results: In the mechanical damage assay, cells expressing alpha synuclein released more LDH, which suggests that membrane repair may be compromised in PD. In the laser damage assay, cells expressing alpha synuclein showed significantly more FM4-64 dye influx. We observed increased dye influx in WT Alpha Synuclein cells. This shows that Alpha Synuclein transfected cells do not repair as effectively as control cells when exposed to the same conditions. These findings support that membrane repair may be compromised in PD. Elucidating that membrane repair is impaired in PD leads us to investigate if existing membrane repair enhancing agents could be potential therapeutics for the disease. American Physiological Society Summer Undergraduate Research Fellowship (APS SURF) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Absence of Gem1 (mammalian Miro/Rhot) mitigates alpha-synuclein toxicity in a yeast model of Parkinson's disease

Alpha-synuclein aggregation is a hallmark of Parkinson's disease (PD). Mutants A30P and A53T alpha-synuclein are known to exacerbate the toxicity of alpha-synuclein, which includes oxidative stress, mitochondrial and endoplasmic reticulum (ER) dysfunction. Saccharomyces cerevisiae (budding yeast) is a cellular model widely used to investigate mechanisms underlying neurodegenerative disorders, such as PD. In yeast, Gem1 (Miro/Rhot mammalian orthologue) coordinates mitochondrial dynamics and ER homeostasis, which is impaired in the presence of mutant alpha-synuclein and can lead to cell death. In this study, A30P or A53T alpha-synuclein were expressed in wild type or ΔGem (deletion of Gem1 gene) yeast strains. ΔGem cells presented decreased viability and increased mitochondrial H2O2 production and ER stress compared to wild type cells. However, in the presence of mutant alpha-synuclein, ΔGem cells showed increased growth compared to cells that do not express mutant alpha-synuclein. ΔGem cells expressing A53T alpha-synuclein also presented reduced ER stress and increased ability to deal with oxidative stress. Together, our results suggest that deletion of Gem1 activates pathways that strengthen cells against other stressful agents such as the presence of mutant alpha-synuclein.

Somatic copy number variant mutations in alpha-synuclein and genome-wide in brains of synucleinopathy cases

Synucleinopathies are mostly sporadic neurodegenerative disorders, and include Parkinson’s disease (PD), and multiple system atrophy (MSA). Inherited copy number variants (CNVs) ofSNCA(α-synuclein) are rare causes of familial disease. There is increasing evidence for somatic mutations in the human brain, and we have hypothesized a role for these in synucleinopathies.We further investigated somatic CNVs in brains from synucleinopathy cases, using Fluorescent in-situ Hybri- disation forSNCAgains, and single-cell whole genome sequencing for the first time in a synucleinopathy. In the cingulate cortex, mosaicism forSNCAgains was higher in MSA and PD than controls in neurons (> 2% in both diseases), and for MSA also in non-neurons. In MSA substantia nigra (SN), we notedSNCAgains in> 3% of cells. Cells with CNVs were more likely to have α-synuclein inclusions, in a pattern corresponding to cell categories mostly relevant to the disease: dopaminergic neurons in Lewy-body cases, and other cells in striatonigral degeneration-dominant MSA (MSA-SND). HigherSNCACNV mosaicism in typical MSA-SND SN non-dopaminergic cells may correlate with younger onset, and in PD cingulate neurons with younger death. Whole genome sequencing of 169 single cells from two MSA cases showed somatic CNVs in ~ 30%. Neurons had gains and losses, and other cells almost exclusively gains.We propose that somaticSNCACNVs contribute to the aetiology and pathogenesis of synucleinopathies, and that genome-wide somatic CNVs merit further studyc.proukakis@ucl.ac.uk

A NAC domain mutation (E83Q) unlocks the pathogenicity of human alpha-synuclein and recapitulates its pathological diversity.

The alpha-synuclein mutation E83Q, the first in the NAC domain of the protein, was recently identified in a patient with dementia with Lewy bodies. We investigated the effects of this mutation on the aggregation of aSyn monomers and the structure, morphology, dynamic, and seeding activity of the aSyn fibrils in neurons. We found that it markedly accelerates aSyn fibrillization and results in the formation of fibrils with distinct structural and dynamic properties. In cells, this mutation is associated with higher levels of aSyn, accumulation of pS129, and increased toxicity. In a neuronal seeding model of Lewy body (LB) formation, the E83Q mutation significantly enhances the internalization of fibrils into neurons, induces higher seeding activity, and results in the formation of diverse aSyn pathologies, including the formation of LB-like inclusions that recapitulate the immunohistochemical and morphological features of brainstem LBs observed in brains of patients with Parkinson’s disease.

Lipid level alteration in human and cellular models of alpha synuclein mutations

Lipid profiles in biological fluids from patients with Parkinson’s disease (PD) are increasingly investigated in search of biomarkers. However, the lipid profiles in genetic PD remain to be determined, a gap of knowledge of particular interest in PD associated with mutant α-synuclein (SNCA), given the known relationship between this protein and lipids. The objective of this research is to identify serum lipid composition from SNCA A53T mutation carriers and to compare these alterations to those found in cells and transgenic mice carrying the same genetic mutation. We conducted an unbiased lipidomic analysis of 530 lipid species from 34 lipid classes in serum of 30 participants with SNCA mutation with and without PD and 30 healthy controls. The primary analysis was done between 22 PD patients with SNCA+ (SNCA+/PD+) and 30 controls using machine-learning algorithms and traditional statistics. We also analyzed the lipid composition of human clonal-cell lines and tissue from transgenic mice overexpressing the same SNCA mutation. We identified specific lipid classes that best discriminate between SNCA+/PD+ patients and healthy controls and found certain lipid species, mainly from the glycerophosphatidylcholine and triradylglycerol classes, that are most contributory to this discrimination. Most of these alterations were also present in human derived cells and transgenic mice carrying the same mutation. Our combination of lipidomic and machine learning analyses revealed alterations in glycerophosphatidylcholine and triradylglycerol in sera from PD patients as well as cells and tissues expressing mutant α-Syn. Further investigations are needed to establish the pathogenic significance of these α-Syn-associated lipid changes.

Novel Ubiquitin Specific Protease-13 Inhibitors Alleviate Neurodegenerative Pathology.

Ubiquitin Specific Protease-13 (USP13) promotes protein de-ubiquitination and is poorly understood in neurodegeneration. USP13 is upregulated in Alzheimer’s disease (AD) and Parkinson’s disease (PD), and USP13 knockdown via shRNA reduces neurotoxic proteins and increases proteasome activity in models of neurodegeneration. We synthesized novel analogues of spautin-1 which is a non-specific USP13 inhibitor but unable to penetrate the brain. Our synthesized small molecule compounds are able to enter the brain, more potently inhibit USP13, and significantly reduce alpha-synuclein levels in vivo and in vitro. USP13 inhibition in transgenic mutant alpha-synuclein (A53T) mice increased the ubiquitination of alpha-synuclein and reduced its protein levels. The data suggest that novel USP13 inhibitors improve neurodegenerative pathology via antagonism of de-ubiquitination, thus alleviating neurotoxic protein burden in neurodegenerative diseases.

Studying the effect of alpha-synuclein and Parkinson\u2019s disease linked mutants on inter pathway connectivities

Parkinson’s disease is a common neurodegenerative disease. The differential expression of alpha-synuclein within Lewy Bodies leads to this disease. Some missense mutations of alpha-synuclein may resultant in functional aberrations. In this study, our objective is to verify the functional adaptation due to early and late-onset mutation which can trigger or control the rate of alpha-synuclein aggregation. In this regard, we have proposed a computational model to study the difference and similarities among the Wild type alpha-synuclein and mutants i.e., A30P, A53T, G51D, E46K, and H50Q. Evolutionary sequence space analysis is also performed in this experiment. Subsequently, a comparative study has been performed between structural information and sequence space outcomes. The study shows the structural variability among the selected subtypes. This information assists inter pathway modeling due to mutational aberrations. Based on the structural variability, we have identified the protein–protein interaction partners for each protein that helps to increase the robustness of the inter-pathway connectivity. Finally, few pathways have been identified from 12 semantic networks based on their association with mitochondrial dysfunction and dopaminergic pathways.

Retraction Note to: Differential regulation of wild-type and mutant alpha-synuclein binding to synaptic membranes by cytosolic factors

This article has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1186/s12868-021-00644-1

Development of a dopaminergic neurodegeneration assay using laser cytometry of Caenorhabditis elegans models of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons resulting in bradykinesia, tremor, gait abnormalities, and numerous non-motor complications. Knowledge of the molecular mechanisms underlying dopaminergic neuron death remains limited, partially due to the lack of accurate disease models. Here, we report the development of a high-throughput assay for monitoring dopaminergic neurodegeneration in Caenorhabditis elegans (C. elegans). Two transgenic strains of C. elegans containing human PD-linked genes were used, one expressing mutant (A53T) alpha-synuclein, and the other expressing mutant (G2019S) leucine-rich repeat kinase 2 (LRRK2). Both strains express GFP in their dopaminergic neurons and were crossed into a neuronal-RNAi sensitive background expressing mCherry under a pharyngeal promoter. This allows for the accurate measurement of neuronal viability (via GFP) and a normalization and sorting control of the total number of worms plated (via mCherry). Daily laser cytometry readings of GFP/mCherry fluorescence intensity revealed that robust temporal degeneration occurs within the first eight days of adulthood in both PD models, but not in a wild-type control strain. We determined a signal (cell loss) to baseline ratio of approximately 4-fold, sufficient for screening applications. Particularly in the case of the mutant LRRK2 model, neurodegeneration is severe: between day 3 and day 8 of adulthood, total GFP fluorescence intensity drops by 75-85%. In the LRRK2 mutants, administration of selective LRRK2 inhibitors (LRRK2-IN-1 and CZC 25146) confers neuroprotection in a dose-dependent manner. Similarly, using high content imaging, we found that the LRRK2 mutant-expressing worms displayed susceptibility to RNA interference. Administration of RNAi targeting LRRK2 slowed the course of the neurodegeneration in these worms. Similarly, administration of RNAi targeting GFP resulted in about 50% reduction of the mean GFP intensity at day 5. Our results indicate that this assay provides a reproducible, high-throughput measurement of dopaminergic neurodegeneration using an in vivo model. Future studies may exploit this model to conduct quantitative high-throughput screens to identify small molecules that inhibit neurodegeneration or use RNAi libraries to identify genes mediating the neurodegenerative response, and hence new drug targets for PD treatment.

Early Disruption of Cortical Sleep-Related Oscillations in a Mouse Model of Dementia With Lewy Bodies (DLB) Expressing Human Mutant (A30P) Alpha-Synuclein.

Changes in sleep behavior and sleep-related cortical activity have been reported in conditions associated with abnormal alpha-synuclein (α-syn) expression, in particular Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Notably, changes can occur in patients years before the onset of cognitive decline. Sleep-related network oscillations play a key role in memory function, but how abnormal α-syn impacts the generation of such activity is currently unclear. To determine whether early changes in sleep-related network activity could also be observed, prior to any previously reported cognitive dysfunction, we used mice that over-express human mutant α-syn (A30P). Recordings in vivo were performed under urethane anesthesia in the medial prefrontal cortex (mPFC) and CA1 region of the hippocampus in young male (2.5 – 4 months old) A30P and age-matched wild type (WT) mice. We found that the slow oscillation (SO) < 1 Hz frequency was significantly faster in both the mPFC and hippocampus in A30P mice, and Up-state-associated fast oscillations at beta (20 – 30 Hz) and gamma (30 – 80 Hz) frequencies were delayed relative to the onset of the Up-state. Spindle (8 – 15 Hz) activity in the mPFC was also altered in A30P mice, as spindles were shorter in duration and had reduced density compared to WT. These changes demonstrate that dysregulation of sleep-related oscillations occurs in young A30P mice long before the onset of cognitive dysfunction. Our data suggest that, as seen in patients, changes in sleep-related oscillations are an early consequence of abnormal α-syn aggregation in A30P mice.

Distinct residual and disordered structures of alpha-synuclein analyzed by amide-proton exchange and NMR signal intensity

The residual solution structures of two alpha-synuclein mutants, A30P and A53T, observed in family members of patients with Parkinson's disease were compared with that of wild-type by NMR. The A53T substitution had been shown to accelerate fibril formation of alpha-synuclein, whereas the A30P mutation has the negative and positive effects on the formation of the fibril and spherical oligomer, respectively. The remaining structure was analyzed via amide-proton exchange and signal intensity measurements using NMR. Amide-proton exchange was used for both the calculation of kex values and ratio of kex at different temperatures. Effects of the A30P (N-terminal region) mutation were observed at the C-terminal region as a more flexible structure, suggesting that long-range interactions exist between the N- and C-terminal regions in alpha-synuclein. In addition, the N-terminal region adopted a more rigid structure in the A53T and A30P mutants than in the wild-type. It was concluded that the structural change caused by the mutations is related to the formation of a beta-hairpin at the initiation site of the N-terminal core structure. Furthermore, the signal intensity was used to estimate the rigidity of the structure. Higher signal intensities were observed for A30P at the 112, 113, and 116 C-terminal residues, suggesting that this region adopts more flexible structure. The ratio of the intensities at different temperatures indicated more flexible or rigid structures in the N-terminal region of A30P than in that of wild-type. Thus, using different approaches and temperatures is a good method to analyze residual structure in intrinsically disordered proteins.

Mitochondria and Parkinson's Disease: Clinical, Molecular, and Translational Aspects.

Mitochondrial dysfunction represents a well-established player in the pathogenesis of both monogenic and idiopathic Parkinson’s disease (PD). Initially originating from the observation that mitochondrial toxins cause PD, findings from genetic PD supported a contribution of mitochondrial dysfunction to the disease. Here, proteins encoded by the autosomal recessively inherited PD genes Parkin, PTEN-induced kinase 1 (PINK1), and DJ-1 are involved in mitochondrial pathways. Additional evidence for mitochondrial dysfunction stems from models of autosomal-dominant PD due to mutations in alpha-synuclein (SNCA) and leucine-rich repeat kinase 2 (LRRK2). Moreover, patients harboring alterations in mitochondrial polymerase gamma (POLG) often exhibit signs of parkinsonism. While some molecular studies suggest that mitochondrial dysfunction is a primary event in PD, others speculate that it is the result of impaired mitochondrial clearance. Most recent research even implicated damage-associated molecular patterns released from non-degraded mitochondria in neuroinflammatory processes in PD. Here, we summarize the manifold literature dealing with mitochondria in the context of PD. Moreover, in light of recent advances in the field of personalized medicine, patient stratification according to the degree of mitochondrial impairment followed by mitochondrial enhancement therapy may hold potential for at least a subset of genetic and idiopathic PD cases. Thus, in the second part of this review, we discuss therapeutic approaches targeting mitochondrial dysfunction with the aim to prevent or delay neurodegeneration in PD.

Bioluminescence Imaging of Neuroinflammation in a Mouse Model of Parkinson's Disease.

In Parkinson's disease (PD) and related disorders pathological alpha-synuclein has been discussed to propagate via a prion-like mechanism in the CNS. The application of exogenous alpha-synuclein fibrils via injection to animal models of PD has been shown to be a useful method to study prion-like propagation of pathological alpha-synuclein and of transmission pathways that play a critical role in recapitulating characteristics of synucleinopathies. Using bigenic mice expressing mutant human alpha-synuclein in neurons and firefly luciferase in astrocytes we showed that transmission via the tongue and the peritoneum represent entrance points for pathological alpha-synuclein to invade the CNS. Here we present a method to quantify astrogliosis by bioluminescence imaging in an animal model of PD. This method allows noninvasive tracking of the neuroinflammatory process that often precedes neurological signs of disease and represents an alternative to behavioral or histological and biochemical analysis to detect disease.

A split-GFP tool reveals differences in the sub-mitochondrial distribution of wt and mutant alpha-synuclein

Parkinson’s disease (PD), the second most common neurodegenerative disorder, is characterized by dopaminergic neuronal loss that initiates in the substantia nigra pars compacta and by the formation of intracellular inclusions mainly constituted by aberrant α-synuclein (α-syn) deposits known as Lewy bodies. Most cases of PD are sporadic, but about 10% are familial, among them those caused by mutations in SNCA gene have an autosomal dominant transmission. SNCA encodes α-syn, a small 140-amino acids protein that, under physiological conditions, is mainly localized at the presynaptic terminals. It is prevalently cytosolic, but its presence has been reported in the nucleus, in the mitochondria and, more recently, in the mitochondria-associated ER membranes (MAMs). Whether different cellular localizations may reflect specific α-syn activities is presently unclear and its action at mitochondrial level is still a matter of debate. Mounting evidence supports a role for α-syn in several mitochondria-derived activities, among which maintenance of mitochondrial morphology and modulation of complex I and ATP synthase activity. α-syn has been proposed to localize at the outer membrane (OMM), in the intermembrane space (IMS), at the inner membrane (IMM) and in the mitochondrial matrix, but a clear and comparative analysis of the sub-mitochondrial localization of WT and mutant α-syn is missing. Furthermore, the reasons for this spread sub-mitochondrial localization under physiological and pathological circumstances remain elusive. In this context, we decided to selectively monitor the sub-mitochondrial distribution of the WT and PD-related α-syn mutants A53T and A30P by taking advantage from a bimolecular fluorescence complementation (BiFC) approach. We also investigated whether cell stress could trigger α-syn translocation within the different mitochondrial sub-compartments and whether PD-related mutations could impinge on it. Interestingly, the artificial targeting of α-syn WT (but not of the mutants) to the mitochondrial matrix impacts on ATP production, suggesting a potential role within this compartment.

Biochemical and morphological classification of disease-associated alpha-synuclein mutants aggregates

Alpha-synuclein (a-syn) aggregation in brain is implicated in several synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Until date, at least six disease-associated mutations in a-syn (namely A30P, E46K, H50Q, G51D, A53T, and A53E) are known to cause dominantly inherited familial forms of synucleinopathies. Previous studies using recombinant proteins have reported that a subset of disease-associated mutants show higher aggregation propensities and form spectroscopically distinguishable aggregates compared to wild-type (WT). However, morphological and biochemical comparison of the aggregates for all disease-associated a-syn mutants have not yet been performed. In this study, we performed electron microscopic examination, guanidinium hydrochloride (GdnHCl) denaturation, and protease digestion to classify the aggregates from their respective point mutations. Using electron microscopy we observed variations of amyloid fibrillar morphologies among the aggregates of a-syn mutants, mainly categorized into two groups: twisted fibrils observed for both WT and E46K while straight fibrils for the other mutants. GdnHCl denaturation experiments revealed the a-syn mutants except for E46K were more resistant than WT against the denaturation. Mass spectrometry analysis of protease-treated aggregates showed a variety of protease-resistant cores, which may correspond to their morphological properties. The difference of their properties could be implicated in the clinicopathological difference of synucleinopathies with those mutations.

RETRACTED ARTICLE: Alpha-synuclein overexpression in the olfactory bulb initiates prodromal symptoms and pathology of Parkinson’s disease

BackgroundParkinson’s disease (PD) is a neurodegenerative disease characterized by intraneuronal Lewy Body (LB) aggregates composed of misfolded alpha-synuclein (α-syn). The spread of misfolded α-syn follows a typical pattern: starting in the olfactory bulb (OB) and the gut, this pathology is followed by the progressive invasion of misfolded α-syn to the posterior part of the brain. It is unknown whether the administration of human mutant alpha-synuclein (hm-α-syn, a human mutation which occurs in familial PD) into the OB of rats would trigger similar α-syn propagation and subsequently cause pathological changes in broader brain fields associated to PD and establish an animal model of prodromal PD.Methodshm-α-syn was overexpressed in the OB of rats with an AAV injection. Then motor and non-motor symptoms of the SD rats were tested in different behavioral tasks following the AAV injection. In follow-up studies, pathological mechanisms of α-syn spread were explored at the histological, biochemical and micro-structure levels.ResultsThe experimental results indicated that hm-α-syn was overexpressed in the OB 3 weeks after the AAV injection. 1) overexpression of the Hm-α-syn in the OB by the AAV injection could transfer to wider adjacent fields beyond the monosynaptic scope. 2) The number of tyrosine hydroxylase positive cells body and fibers was decreased in the substantia nigra (SN) 12 weeks after AAV injection. This was consistent with decreased levels of the DA neurotransmitter. Importantly, behavioral dysfunctions were found that included olfactory impairment after 3 weeks, motor ability impairment and decreased muscular coordination on a rotarod 6 weeks after the AAV injection.3) The morphological level studies found that the Golgi staining revealed the number of neuronal branches and synapses in the OB, prefrontal cortex (PFC), hippocampus (Hip) and striatum caudate putamen (CPU) were decreased. 4) phosphorylated α-syn, at Ser-129 (pSer129), was found to be increased in hm-α-syn injected animals in comparison to controls that overexpressed GFP alone, which was also found in the most of LB stained by the thioflavine S (ThS) in the SN field. 5) A marker of autophagy (LC3B) was increased in serval fields, which was colacolizated with a marker of apoptosis in the SN field.ConclusionsThese results demonstrate that expression of exogenous mutant α-syn in the OB induces pathological changes in the sensitive brain fields by transferring pathogenic α-syn to adjacent fields. This method may be useful for establishing an animal model of prodromal PD.

A53T Mutant Alpha-Synuclein Induces Tau-Dependent Postsynaptic Impairment Independently of Neurodegenerative Changes

Abnormalities in α-synuclein are implicated in the pathogenesis of Parkinson's disease (PD). Because α-synuclein is highly concentrated within presynaptic terminals, presynaptic dysfunction has been proposed as a potential pathogenic mechanism. Here, we report novel, tau-dependent, postsynaptic deficits caused by A53T mutant α-synuclein, which is linked to familial PD. We analyzed synaptic activity in hippocampal slices and cultured hippocampal neurons from transgenic mice of either sex expressing human WT, A53T, and A30P α-synuclein. Increased α-synuclein expression leads to decreased spontaneous synaptic vesicle release regardless of genotype. However, only those neurons expressing A53T α-synuclein exhibit postsynaptic dysfunction, including decreased miniature postsynaptic current amplitude and decreased AMPA to NMDA receptor current ratio. We also found that long-term potentiation and spatial learning were impaired by A53T α-synuclein expression. Mechanistically, postsynaptic dysfunction requires glycogen synthase kinase 3β-mediated tau phosphorylation, tau mislocalization to dendritic spines, and calcineurin-dependent AMPA receptor internalization. Previous studies reveal that human A53T α-synuclein has a high aggregation potential, which may explain the mutation's unique capacity to induce postsynaptic deficits. However, patients with sporadic PD with severe tau pathology are also more likely to have early onset cognitive decline. Our results here show a novel, functional role for tau: mediating the effects of α-synuclein on postsynaptic signaling. Therefore, the unraveled tau-mediated signaling cascade may contribute to the pathogenesis of dementia in A53T α-synuclein-linked familial PD cases, as well as some subgroups of PD cases with extensive tau pathology.SIGNIFICANCE STATEMENT Here, we report mutation-specific postsynaptic deficits that are caused by A53T mutant α-synuclein, which is linked to familial Parkinson's disease (PD). The overexpression of WT, A53T, or A30P human α-synuclein leads to decreased spontaneous synaptic vesicle release. However, only those neurons expressing A53T α-synuclein exhibit tau phosphorylation-dependent postsynaptic dysfunction, which is characterized by decreased miniature postsynaptic current amplitude and decreased AMPA to NMDA receptor current ratio. The mutation-specific postsynaptic effects caused by human A53T α-synuclein will help us better understand the neurobiological basis of this specific form of familial PD. The differential effects of exogenous human WT, A53T, A30P, and E46K α-synuclein on glutamatergic synaptic responses will help to explain the clinical heterogeneity of sporadic and familial PD.