Induce Α-synuclein Aggregation Research Articles

20S proteasome enhancers prevent cytotoxic tubulin polymerization-promoting protein induced α-synuclein aggregation

Synucleinopathies are a class of neurodegenerative diseases defined by the presence of α-synuclein inclusions. The location and composition of these α-synuclein inclusions directly correlate to the disease pattern. The inclusions in Multiple System Atrophy are located predominantly in oligodendrocytes and are rich in a second protein, p25α. P25α plays a key role in neuronal myelination by oligodendrocytes. In healthy oligodendrocytes, there is little to no α-synuclein present. If aberrant α-synuclein is present, p25α leaves the myelin sheaths and quickly co-aggregates with α-synuclein, resulting in the disruption of the cellular process and ultimately cell death. Herein, we report that p25α is susceptible for 20S proteasome-mediated degradation and that p25α induces α-synuclein aggregation, resulting in proteasome impairment and cell death. In addition, we identified small molecules 20S proteasome enhancers that prevent p25α induced α-synuclein fibrilization, restore proteasome impairment, and enhance cell viability.

SARS-CoV-2 Spike Protein 1 Causes Aggregation of α-Synuclein via Microglia-Induced Inflammation and Production of Mitochondrial ROS: Potential Therapeutic Applications of Metformin.

Abnormal aggregation of α-synuclein is the hallmark of neurodegenerative diseases, classified as α-synucleinopathies, primarily occurring sporadically. Their onset is associated with an interaction between genetic susceptibility and environmental factors such as neurotoxins, oxidative stress, inflammation, and viral infections. Recently, evidence has suggested an association between neurological complications in long COVID (sometimes referred to as 'post-acute sequelae of COVID-19') and α-synucleinopathies, but its underlying mechanisms are not completely understood. In this study, we first showed that SARS-CoV-2 Spike protein 1 (S1) induces α-synuclein aggregation associated with activation of microglial cells in the rodent model. In vitro, we demonstrated that S1 increases aggregation of α-synuclein in BE(2)M-17 dopaminergic neurons via BV-2 microglia-mediated inflammatory responses. We also identified that S1 directly affects aggregation of α-synuclein in dopaminergic neurons through increasing mitochondrial ROS, though only under conditions of sufficient α-Syn accumulation. In addition, we observed a synergistic effect between S1 and the neurotoxin MPP+ S1 treatment. Combined with a low dose of MPP+, it boosted α-synuclein aggregation and mitochondrial ROS production compared to S1 or the MPP+ treatment group. Furthermore, we evaluated the therapeutic effects of metformin. The treatment of metformin suppressed the S1-induced inflammatory response and α-synucleinopathy. Our findings demonstrate that S1 promotes α-synucleinopathy via both microglia-mediated inflammation and mitochondrial ROS, and they provide pathological insights, as well as a foundation for the clinical management of α-synucleinopathies and the onset of neurological symptoms after the COVID-19 outbreak.

Gut microbiota produces biofilm-associated amyloids with potential for neurodegeneration.

Age-related neurodegenerative diseases involving amyloid aggregation remain one of the biggest challenges of modern medicine. Alterations in the gastrointestinal microbiome play an active role in the aetiology of neurological disorders. Here, we dissect the amyloidogenic properties of biofilm-associated proteins (BAPs) of the gut microbiota and their implications for synucleinopathies. We demonstrate that BAPs are naturally assembled as amyloid-like fibrils in insoluble fractions isolated from the human gut microbiota. We show that BAP genes are part of the accessory genomes, revealing microbiome variability. Remarkably, the abundance of certain BAP genes in the gut microbiome is correlated with Parkinson's disease (PD) incidence. Using cultured dopaminergic neurons and Caenorhabditis elegans models, we report that BAP-derived amyloids induce α-synuclein aggregation. Our results show that the chaperone-mediated autophagy is compromised by BAP amyloids. Indeed, inoculation of BAP fibrils into the brains of wild-type mice promote key pathological features of PD. Therefore, our findings establish the use of BAP amyloids as potential targets and biomarkers of α-synucleinopathies.

ATM-mediated co-chaperone DNAJB11 phosphorylation facilitates α-synuclein folding upon DNA double-stranded breaks

Abstract Parkinson's disease (PD) is a prevalent neurodegenerative disorder marked by the pathological accumulation of α-synuclein aggregates in dopaminergic neurons. This α-synuclein dyshomeostasis is caused by an interplay between aging, genetic and environmental factors. Aging process-related DNA damage and impaired DNA repair have recently been observed in the PD process. However, the precise neuronal response to DNA damage in PD remains largely unknown. Here, we demonstrate that double-strand breaks (DSBs) induce α-synuclein aggregation. Analysis of a large-scale proteomic analysis of ATM and ATR substrates identified a potential candidate in the HSP70 folding system responding to DNA damage. ATM phosphorylates co-chaperone DNAJB11 at threonine 188 which specifically facilitates the delivery of misfolded α-synuclein, but not tau or transthyretin protein, to the HSP70 folding system upon DSBs. Alteration of this response impairs the neurite outgrowth. Remarkably, DNAJB11 threonine 188 phosphorylation correlates with disease severity in transgenic SNCA mutant PD mice and PD patients. These findings reveal a DNA damage-responded HSP70 folding mechanism through a J-domain co-chaperone, offering a potential therapeutic target for PD.

Therapeutic Potential of Fosgonimeton, a Small-Molecule Positive Modulator of the Neurotrophic HGF/MET Pathway, in Neurodegenerative Conditions (S14.008)

<h3>Objective:</h3> Evaluate the neurotrophic and neuroprotective effects of fosgonimeton, a small-molecule positive modulator of hepatocyte growth factor (HGF)/MET. <h3>Background:</h3> HGF signaling through the MET receptor promotes neural survival and function, making it a compelling target to combat neurodegeneration. We developed a series of small-molecule positive modulators of HGF/MET, including fosgonimeton, to target this neurotrophic system. <h3>Design/Methods:</h3> To demonstrate the neurotrophic effects of fosgonimeton, primary neurons were treated with the active metabolite of fosgonimeton (fosgo-AM) and assessed for synaptic count and neurite outgrowth. To evaluate neuroprotective effects, primary neurons were treated with fosgo-AM before exposure to relevant neurodegenerative insults, namely oxidative stress (hydrogen peroxide [H₂O₂]), neuroinflammation (lipopolysaccharide [LPS]), excitotoxicity (glutamate), and mitochondrial dysfunction (1-methyl-4-phenylpyridinium [MPP+]). The effects of fosgo-AM on protein aggregation in primary neuron cultures were evaluated via levels of phosphorylated tau (p-tau) induced by amyloid β (Aβ) toxicity in cortical neurons and α-synuclein aggregation induced by 6-hydroxydopamine (6-OHDA) in dopaminergic neurons. In vivo, the therapeutic usefulness of fosgonimeton was assessed for LPS-induced cognitive impairment in mice and 6-OHDA–induced motor deficits in rats. <h3>Results:</h3> In vitro, fosgo-AM significantly increased the number of synapses and neurite length in hippocampal and cortical neurons, respectively. Fosgo-AM was neuroprotective of cortical neurons subjected to oxidative, neuroinflammatory, excitotoxic, and mitochondrial challenges. Significant reduction in the number of disease biomarkers was observed with fosgo-AM treatment, including Aβ-induced p-tau accumulation in cortical neurons and 6-OHDA–induced α-synuclein aggregation in dopaminergic neurons. In vivo, fosgonimeton administration significantly attenuated LPS-induced cognitive impairment and 6-OHDA–induced motor deficits in rodent models. <h3>Conclusions:</h3> These results, from in vitro and in vivo systems, consistently support the broad application of fosgonimeton to neurodegenerative conditions. Collectively, our observations highlight the therapeutic potential of HGF/MET modulation in the management of neurodegenerative diseases that include AD and PD, supporting further clinical exploration of this approach. <b>Disclosure:</b> Dr. Reda has received personal compensation for serving as an employee of Athira Pharma, Inc. . Dr. Reda has stock in Athira Pharma, Inc. . Dr. Taylor has received personal compensation for serving as an employee of Athira Pharma, Inc. Dr. Taylor has stock in Athira Pharma, Inc.. Dr. Taylor has received intellectual property interests from a discovery or technology relating to health care. Dr. Johnston has received personal compensation for serving as an employee of Athira Pharma, Inc. Dr. Johnston has stock in Athira Pharma, Inc. An immediate family member of Dr. Johnston has stock in Athira Pharma, Inc. Dr. Johnston has received intellectual property interests from a discovery or technology relating to health care. Dr. Berthiaume has received personal compensation for serving as an employee of Athira Pharma. Dr. Berthiaume has stock in Athira Pharma. An immediate family member of Dr. Berthiaume has stock in Athira Pharma. Dr. Setti has received personal compensation for serving as an employee of Athira Pharma. Dr. Setti has stock in Athira Pharma. Dr. Church has received personal compensation for serving as an employee of Athira Pharma. Dr. Church has stock in Athira Pharma. Dr. Church has received intellectual property interests from a discovery or technology relating to health care.

Infections of Cryptococcus species induce degeneration of dopaminergic neurons and accumulation of α-Synuclein in Caenorhabditis elegans.

Cryptococcosis in the central nervous system (CNS) can present with motor declines described as Parkinsonism. Although several lines of evidence indicate that dopaminergic (DA) neuron degeneration and α-synuclein accumulation contribute to the hallmark of Parkinsonism and Parkinson's disease (PD), little is known about cryptococcal infections associated with neuronal degeneration. In this study, the effects of Cryptococcus neoformans and C. gattii infections on dopaminergic neuron degeneration, α-synuclein accumulation, and lifespan in Caenorhabditis elegans were investigated. The results showed that cryptococcal infections significantly (P<0.05) induced DA neuron degeneration similar to a selective cathecholamine neurotoxin 6-hydroxydopamine (6-OHDA) in C. elegans (BZ555 strain) when compared to mock infected controls. Cryptococcal infections also significantly (P< 0.05) induced α-synuclein aggregation in C. elegans (NL5901 strain). Moreover, lifespan of the infected worms was significantly decreased (P<0.0001). In conclusion, DA neurodegeneration and α-synuclein accumulation are associated with lifespan reduction during cryptococcal infection in C elegans.

High-Glucose Diet Attenuates the Dopaminergic Neuronal Function in C. elegans, Leading to the Acceleration of the Aging Process.

Parkinson’s disease (PD) is a neurodegenerative disease characterized by the selective degeneration of neurons, primarily in the substantia nigra. Environmental or exogenous factors that cause Parkinson’s disease have not been sufficiently elucidated. Our study aims to investigate the causative effect of a high-glucose diet on Parkinson’s disease-relevant dopaminergic neuronal system in Caenorhabditis elegans. Aging parameters were first observed by measuring the lifespan, body movement, and body sizes with and without the background of high glucose. The toxic effect of a high-glucose diet was further explored by observing the dopaminergic neurons using transgenic Pdat-1::gfp strains, BZ555, under a Zeiss microscope, and the experiments were extended by assessing dopamine-related behavioral analysis including basal slowing response and alcohol avoidance. The aggregation of the α-synucleins was also assessed by observing the NL5901 mutants. Worms fed with 250 mM glucose showed daf-2-independent regulation of aging, displaying a short lifespan (≤15 days), long body size (max. 140%), and slow movement (min. 30%, 10 bends/min). Anterior dopaminergic neurons were rapidly inactivated (70%) by a glucose-rich diet from 12 h of exposure, suggesting specific degeneration in ADE neurons. The dysregulation of neurons led to deteriorations in dopaminergic behaviors including basal slowing response (BSR). A high-glucose diet decreased dopamine synthesis (40 pg/mg vs 15 pg/mg protein) and induced α-synuclein aggregation in the muscles. Results demonstrate the potential of a high-glucose diet as a trigger of dopaminergic neuronal dysregulation conjugating aging acceleration.

Shear Stress Induces α-Synuclein Aggregation Due to a Less Strained Protein Backbone and Protein Tyrosyl Groups Do Not Intervene in the Aggregation

Parkinson’s disease (PD) is an increasingly prevalent and currently incurable neurodegenerative disorder. The aggregation of the amyloid disordered protein α-synuclein (Syn) has been implicated in the development of PD. In the literature, it has been suggested that tyrosine residues of Syn play an important role in the interactions established during the fibrillation process. Herein, the prevalence of the referred interactions under shear stress conditions of Nα-acetyl-L-tyrosinamide (NAYA) and of Syn solutions by using membrane centrifugal filters with different cut-off of 200 nm, 100 kDa, 50 kDa and 30 kDa, under centrifugation conditions, were investigated. In order to determine the nature of the interactions involving the protein tyrosine residues the NAYA compound, which mimics the peptide bonds in protein and also possesses a tyrosyl group similar to the tyrosyl groups found in the Syn protein molecular structure, was used. It is expected that for a small molecule, such as NAYA, no molecular association occurs, contrary to what exists in the Syn protein solutions, which can more adequately retrieve the type of interactions formed, involving the tyrosyl group. Therefore, sensing the tyrosyl group absorption, spectroscopic techniques, in particular, were used. For NAYA, an intramolecular interaction between the tyrosyl group and the peptide bond was evidenced. For NAYA and Syn, it was observed that decreasing the membrane centrifugal filters pore size, under centrifugation conditions, was concomitant with the minimization of the intramolecular interactions between the tyrosyl group and the peptide bond. With this, it is likely to assume that shear stress conditions in the Syn solutions propel protein aggregation by a less strained protein backbone. Contrary to the centrifugation of NAYA solutions, centrifuging Syn solutions revealed molecular association and a progressive exposure of protein tyrosyl groups to water. Thus, we can also infer that shear stress conditions in the Syn solutions cause the protein tyrosyl groups to not intervene in the protein aggregation.

Construction of model strain of yeast Saccharomyces cerevisiae with regulated expression of recombinant human alpha-synuclein

Background. Improper folding and accumulation of a-synuclein aggregates are among the causes of Parkinson’s disease. The most important factor influencing the process of α-synuclein aggregation is the level of this protein in neurons which depends on the balance between its synthesis, degradation and secretion. Under certain conditions, when α-synuclein is synthesized at a high level, monomers of this protein can aggregate on the lipid membrane, which leads to the formation of amyloids, fibrils and protofibrils unable to perform their physiological functions. Since it is virtually impossible to study the properties of α-synuclein in vivo, researchers are actively using model biological systems (single-celled microorganisms, human cell lines, animal models etc.). The aim of this study was to construct a recombinant strain of Saccharomyces cerevisiae with controlled expression of human α-synuclein to study the regulation and properties of this protein and for screening for new low molecular weight chemi­cal compounds which can induce α-synuclein aggregation and/or degradation. Materials and methods. A recombinant strain of S. cerevisiae with controlled expression of α-synuclein conjugated to a green fluorescent protein was isolated. Western blotting with specific anti-α-synuclein antibodies was used to detect recombinant α-synuclein in yeast cells. Intracellular localization of heterologous chimeric green fluorescent protein conjugated to α-synuclein was also examined by fluorescence microscopy. Results. To construct a recombinant strain of S. cerevisiae, the coding sequence of the human wild-type α-synuclein gene was expressed under the regulated promoter of the ScMET25 gene. Analysis of the effect of different concentrations of exogenous methionine as a factor regulating the expression of the ScMET25 promoter on the content of recombinant protein showed that the expression of the human α-synuclein gene in S. cerevisiae is repressed in the presence of methionine at a concentration of 10 mg/L and higher. During long-term cultivation of yeast cells, this effect decreased due to the depletion of methionine in the growth medium. As a result, recombinant protein synthesis was restored, and α-synuclein content in such cells approached that of cells grown in a medium with a low concentration of (5 mg/L), or without methionine. It was also found that overproduction of recombinant α-synuclein in S. cerevisiae cells had virtually no effect on culture growth, indicating the absence or a very weak toxic effect of human α-synuclein on yeast physiology. Conclusions. The obtained data indicate a concentration-dependent effect of methionine on the level of recombinant α-synuclein synthesis in S. cerevisiae yeast cells. Such controlled expression of the studied protein can be used to screen for compounds capable of promoting dose-dependent aggregation or degradation of α-synuclein in yeast cells and potentially in human cells as well.

Glucosylceramide Associated with Gaucher Disease Forms Amyloid-like Twisted Ribbon Fibrils That Induce α-Synuclein Aggregation.

A major risk factor for Gaucher’s disease is loss of function mutations in the GBA1 gene that encodes lysosomal β-glucocerebrosidase, resulting in accumulation of glucosylceramide (GlcCer), a key lysosomal sphingolipid. GBA1 mutations also enhance the risk for Parkinson’s disease, whose hallmark is the aggregation of α-synuclein (αSyn). However, the role of accumulated GlcCer in αSyn aggregation is not completely understood. Using various biophysical assays, we demonstrate that GlcCer self-assembles to form amyloid-like fibrillar aggregates in vitro. The GlcCer assemblies are stable in aqueous media of different pH and exhibit a twisted ribbon-like structure. Near lysosomal pH GlcCer aggregates induced αSyn aggregation and stabilized its nascent oligomers. We found that several bona fide inhibitors of proteinaceous amyloids effectively inhibited aggregation of GlcCer. This study contributes to the growing evidence of cross-talk between proteinaceous amyloids and amyloid-like aggregates of metabolites accumulated in diseases and suggests these aggregates as therapeutic targets.

Phenotypic manifestation of \u03b1-synuclein strains derived from Parkinson\u2019s disease and multiple system atrophy in human dopaminergic neurons

α-Synuclein is critical in the pathogenesis of Parkinson’s disease and related disorders, yet it remains unclear how its aggregation causes degeneration of human dopaminergic neurons. In this study, we induced α-synuclein aggregation in human iPSC-derived dopaminergic neurons using fibrils generated de novo or amplified in the presence of brain homogenates from Parkinson’s disease or multiple system atrophy. Increased α-synuclein monomer levels promote seeded aggregation in a dose and time-dependent manner, which is associated with a further increase in α-synuclein gene expression. Progressive neuronal death is observed with brain-amplified fibrils and reversed by reduction of intraneuronal α-synuclein abundance. We identified 56 proteins differentially interacting with aggregates triggered by brain-amplified fibrils, including evasion of Parkinson’s disease-associated deglycase DJ-1. Knockout of DJ-1 in iPSC-derived dopaminergic neurons enhance fibril-induced aggregation and neuronal death. Taken together, our results show that the toxicity of α-synuclein strains depends on aggregate burden, which is determined by monomer levels and conformation which dictates differential interactomes. Our study demonstrates how Parkinson’s disease-associated genes influence the phenotypic manifestation of strains in human neurons.

α-synuclein aggregates induce c-Abl activation and dopaminergic neuronal loss by a feed-forward redox stress mechanism.

Oxidative stress and α-synuclein aggregation both drive neurodegeneration in Parkinson's disease, and the protein kinase c-Abl provides a potential amplifying link between these pathogenic factors. Suppressing interactions between these factors may thus be a viable therapeutic approach for this disorder. To evaluate this possibility, pre-formed α-synuclein fibrils (PFFs) were used to induce α-synuclein aggregation in neuronal cultures. Exposure to PFFs induced oxidative stress and c-Abl activation in wild-type neurons. By contrast, α-synuclein - deficient neurons, which cannot form α-synuclein aggregates, failed to exhibit either oxidative stress or c-Abl activation. N-acetyl cysteine, a thiol repletion agent that supports neuronal glutathione metabolism, suppressed the PFF - induced redox stress and c-Abl activation in the wild-type neurons, and likewise suppressed α-synuclein aggregation. Parallel findings were observed in mouse brain: PFF-induced α-synuclein aggregation in the substantia nigra was associated with redox stress, c-Abl activation, and dopaminergic neuronal loss, along with microglial activation and motor impairment, all of which were attenuated with oral N-acetyl cysteine. Similar results were obtained using AAV-mediated α-synuclein overexpression as an alternative means of driving α-synuclein aggregation in vivo. These findings show that α-synuclein aggregates induce c-Abl activation by a redox stress mechanism. c-Abl activation in turn promotes α-synuclein aggregation, in a feed-forward interaction. The capacity of N-acetyl cysteine to interrupt this interaction adds mechanistic support its consideration as a therapeutic in Parkinson's disease.

Brain Endothelial P-Glycoprotein Level Is Reduced in Parkinson's Disease via a Vitamin D Receptor-Dependent Pathway.

The progressive neurodegeneration in Parkinson’s disease (PD) is accompanied by neuroinflammation and endothelial vascular impairment. Although the vitamin D receptor (VDR) is expressed in both dopamine neurons and brain endothelial cells, its role in the regulation of endothelial biology has not been explored in the context of PD. In a 6-hydroxydopamine (6-OHDA)-induced PD mouse model, we observed reduced transcription of the VDR and its downstream target genes, CYP24 and MDR1a. The 6-OHDA-induced transcriptional repression of these genes were recovered after the VDR ligand—1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) treatment. Similarly, reduced vascular protein expression of P-glycoprotein (P-gp), encoded by MDR1a, after 6-OHDA administration was reversed by 1,25(OH)2D3. Moreover, marked reduction of endothelial P-gp expression with concomitant α-synuclein aggregation was found in a combinatorial AAV-αSyn/αSyn preformed fibril (PFF) injection mouse model and postmortem PD brains. Supporting the direct effect of α-synuclein aggregation on endothelial biology, PFF treatment of human umbilical vein endothelial cells (HUVECs) was sufficient to induce α-synuclein aggregation and repress transcription of the VDR. PFF-induced P-gp downregulation and impaired functional activity in HUVECs completely recovered after 1,25(OH)2D3 treatment. Taken together, our results suggest that a dysfunctional VDR-P-gp pathway could be a potential target for the maintenance of vascular homeostasis in PD pathological conditions.

α-Synuclein filaments from transgenic mouse and human synucleinopathy-containing brains are major seed-competent species

Assembled α-synuclein in nerve cells and glial cells is the defining pathological feature of neurodegenerative diseases called synucleinopathies. Seeds of α-synuclein can induce the assembly of monomeric protein. Here, we used sucrose gradient centrifugation and transiently transfected HEK 293T cells to identify the species of α-synuclein from the brains of homozygous, symptomatic mice transgenic for human mutant A53T α-synuclein (line M83) that seed aggregation. The most potent fractions contained Sarkosyl-insoluble assemblies enriched in filaments. We also analyzed six cases of idiopathic Parkinson's disease (PD), one case of familial PD, and six cases of multiple system atrophy (MSA) for their ability to induce α-synuclein aggregation. The MSA samples were more potent than those of idiopathic PD in seeding aggregation. We found that following sucrose gradient centrifugation, the most seed-competent fractions from PD and MSA brains are those that contain Sarkosyl-insoluble α-synuclein. The fractions differed between PD and MSA, consistent with the presence of distinct conformers of assembled α-synuclein in these different samples. We conclude that α-synuclein filaments are the main driving force for amplification and propagation of pathology in synucleinopathies.

Microglial exosomes facilitate α-synuclein transmission in Parkinson's disease.

Accumulation of neuronal α-synuclein is a prominent feature in Parkinson's disease. More recently, such abnormal protein aggregation has been reported to spread from cell to cell and exosomes are considered as important mediators. The focus of such research, however, has been primarily in neurons. Given the increasing recognition of the importance of non-cell autonomous-mediated neurotoxicity, it is critical to investigate the contribution of glia to α-synuclein aggregation and spread. Microglia are the primary phagocytes in the brain and have been well-documented as inducers of neuroinflammation. How and to what extent microglia and their exosomes impact α-synuclein pathology has not been well delineated. We report here that when treated with human α-synuclein preformed fibrils, exosomes containing α-synuclein released by microglia are fully capable of inducing protein aggregation in the recipient neurons. Additionally, when combined with microglial proinflammatory cytokines, these exosomes further increased protein aggregation in neurons. Inhibition of exosome synthesis in microglia reduced α-synuclein transmission. The in vivo significance of these exosomes was demonstrated by stereotaxic injection of exosomes isolated from α-synuclein preformed fibrils treated microglia into the mouse striatum. Phosphorylated α-synuclein was observed in multiple brain regions consistent with their neuronal connectivity. These animals also exhibited neurodegeneration in the nigrostriatal pathway in a time-dependent manner. Depleting microglia in vivo dramatically suppressed the transmission of α-synuclein after stereotaxic injection of preformed fibrils. Mechanistically, we report here that α-synuclein preformed fibrils impaired autophagy flux by upregulating PELI1, which in turn, resulted in degradation of LAMP2 in activated microglia. More importantly, by purifying microglia/macrophage derived exosomes in the CSF of Parkinson's disease patients, we confirmed the presence of α-synuclein oligomer in CD11b+ exosomes, which were able to induce α-synuclein aggregation in neurons, further supporting the translational aspect of this study. Taken together, our study supports the view that microglial exosomes contribute to the progression of α-synuclein pathology and therefore, they may serve as a promising therapeutic target for Parkinson's disease.

Neuron-autonomous susceptibility to induced synuclein aggregation is exacerbated by endogenous Lrrk2 mutations and ameliorated by Lrrk2 genetic knock-out.

Neuronal aggregates containing α-synuclein are a pathological hallmark of several degenerative diseases; including Parkinson’s disease, Parkinson’s disease with dementia and dementia with Lewy bodies. Understanding the process of α-synuclein aggregation, and discovering means of preventing it, may help guide therapeutic strategy and drug design. Recent advances provide tools to induce α-synuclein aggregation in neuronal cultures. Application of exogenous pre-formed fibrillar α-synuclein induces pathological phosphorylation and accumulation of endogenous α-synuclein, typical of that seen in disease. Genomic variability and mutations in α-synuclein and leucine-rich repeat kinase 2 proteins are the major genetic risk factors for Parkinson’s disease. Reports demonstrate fibril-induced α-synuclein aggregation is increased in cells from leucine-rich repeat kinase 2 pathogenic mutant (G2019S) overexpressing mice, and variously decreased by leucine-rich repeat kinase 2 inhibitors. Elsewhere in vivo antisense knock-down of leucine-rich repeat kinase 2 protein has been shown to protect mice from fibril-induced α-synuclein aggregation, whereas kinase inhibition did not. To help bring clarity to this issue, we took a purely genetic approach in a standardized neuron-enriched culture, lacking glia. We compared fibril treatment of leucine-rich repeat kinase 2 germ-line knock-out, and G2019S germ-line knock-in, mouse cortical neuron cultures with those from littermates. We found leucine-rich repeat kinase 2 knock-out neurons are resistant to α-synuclein aggregation, which predominantly forms within axons, and may cause axonal fragmentation. Conversely, leucine-rich repeat kinase 2 knock-in neurons are more vulnerable to fibril-induced α-synuclein accumulation. Protection and resistance correlated with basal increases in a lysosome marker in knock-out, and an autophagy marker in knock-in cultures. The data add to a growing number of studies that argue leucine-rich repeat kinase 2 silencing, and potentially kinase inhibition, may be a useful therapeutic strategy against synucleinopathy.

Iron-induced oxidative stress contributes to α-synuclein phosphorylation and up-regulation via polo-like kinase 2 and casein kinase 2

α-Synuclein plays a central role in synucleinopathies pathogenesis such as Parkinson's disease (PD). Phosphorylation is the most common and important protein modification linked to α-synuclein pathologies. There is mounting evidence suggested iron and α-synuclein are closely related in PD. We previously reported iron up-regulated α-synuclein mRNA levels and induced α-synuclein aggregation. In the present study, we aimed to investigate whether and how phosphorylation was involved in iron-induced α-synuclein regulations. The results showed that iron could induce pS129 α-synuclein (phosphorylation at Ser129) and α-synuclein upregulation in the substantia nigra of iron-overloaded rats and iron-treated SH-SY5Y cells, accompanied by the elevated levels of polo-like kinase 2 (PLK2) and casein kinase 2 (CK2). Over-expression of CK2 or PLK2 induced pS129 α-synuclein up-regulation and inhibitors of CK2 or PLK2 could suppress iron-induced α-synuclein phosphorylation. Antioxidant NAC could fully block iron-induced upregulation of CK2, PLK2 and pS129 α-synuclein levels, indicating oxidative stress plays a critical role in iron-induced α-synuclein phosphorylation. However, iron-induced α-synuclein up-regulation could only be partially blocked by CK2/PLK2 inhibitor or NAC. These findings demonstrate that iron-induced oxidative stress is largely responsible for α-synuclein phosphorylation and upregulation via CK2 and PLK2, and α-synuclein upregulation is not fully phosphorylation-dependent.

Acrolein acts as a neurotoxin in the nigrostriatal dopaminergic system of rat: involvement of \u03b1-synuclein aggregation and programmed cell death

Clinical studies report significant increases in acrolein (an α,β-unsaturated aldehyde) in the substantia nigra (SN) of patients with Parkinson’s disease (PD). In the present study, acrolein-induced neurotoxicity in the nigrostriatal dopaminergic system was investigated by local infusion of acrolein (15, 50, 150 nmoles/0.5 μl) in the SN of Sprague-Dawley rats. Acrolein-induced neurodegeneration of nigrostriatal dopaminergic system was delineated by reductions in tyrosine hydroxylase (TH) levels, dopamine transporter levels and TH-positive neurons in the infused SN as well as in striatal dopamine content. At the same time, apomorphine-induced turning behavior was evident in rats subjected to a unilateral infusion of acrolein in SN. Acrolein was pro-oxidative by increasing 4-hydroxy-2-nonenal and heme oxygenase-1 levels. Furthermore, acrolein conjugated with proteins at lysine residue and induced α-synuclein aggregation in the infused SN. Acrolein was pro-inflammatory by activating astrocytes and microglia. In addition, acrolein activated caspase 1 in the infused SN, suggesting acrolein-induced inflammasome formation. The neurotoxic mechanisms underlying acrolein-induced neurotoxicity involved programmed cell death, including apoptosis and necroptosis. Compared with well-known Parkinsonian neurotoxins, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and rotenone which do not exist in the SN of PD patients, our in vivo study shows that acrolein acts as a Parkinsonian neurotoxin in the nigrostriatal dopaminergic system of rat brain.

Apocyanin, NADPH oxidase inhibitor prevents lipopolysaccharide induced α-synuclein aggregation and ameliorates motor function deficits in rats: Possible role of biochemical and inflammatory alterations

Parkinson’s disease (PD), is an age-related, progressive neurodegenerative disorder that affects movement and is characterized by the loss of dopaminergic neurons in the nigrostriatal region. Although the clinical and pathological features of PD are complex, recent studies have indicated that microglial NADPH oxidase play a key role in its pathology. A little information is available regarding the role of apocyanin, an NADPH oxidase inhibitor, in ameliorating α-synuclein aggregation and neurobehavioral consequences of PD. Therefore, the present study evaluated its therapeutic potentials for the treatment of neurobehavioral consequences in lipolysaccharide (LPS) induced PD model. For the establishment of PD model LPS (5μg/5μl PBS) was injected into the Substantia nigra (SN) of rats. Apocyanin (10mg/kgb.wt) was injected intraperitoneal. Statistical analysis revealed that apocynin significantly ameliorated LPS induced inflammatory response characterized by NFkB, TNF-α and IL-1β upregulation as assessed by ELISA. It also prevented dopaminergic neurons from toxic insult of LPS as indicated by inhibition of apoptotic markers i.e., caspase 3 and caspase 9 as depicted from RT-PCR and ELISA studies. This was further supported by TUNEL assay for DNA fragmentation. Effectiveness of apocyanin in protecting dopaminergic neuronal degeneration was further confirmed by assessment of α-synuclein deposition as depicted by IHC analysis. Consequently, an improvement in the behavioral outcome was observed following apocyanin treatment as depicted from various behavioral tests performed. Hence the data suggests that specific NADPH oxidase inhibitors, such as apocynin, may provide a new therapeutic approach to the control of neurological disabilities induced by LPS induced PD.

Ellagic acid mitigates SNO-PDI induced aggregation of Parkinsonian biomarkers.

Nitrosative stress mediated S-nitrosylation (SNO) of protein disulfide isomerase (PDI), a housekeeping oxidoreductase, has been implicated in the pathogenesis of sporadic Parkinson's (PD) and Alzheimer's (AD) diseases. Previous cell line studies have indicated that SNO-PDI formation provokes synphilin-1 aggregation, the minor Parkinsonian biomarker protein. Yet no work exists investigating whether SNO-PDI induces α-synuclein aggregation, the major Lewy body constituent associated with Parkinson's pathogenesis. Here, we report that SNO-PDI formation is linked to the aggregation of α-synuclein and also provokes α-synuclein:synphilin-1 deposits (Lewy-body-like debris) normally found in the PD brain. Furthermore, we have examined the ability of a small molecule, 2,3,7,8-tetrahydroxy-chromeno[5,4,3-cde]chromene-5,10-dione (ellagic acid; EA) to scavenge NOx radicals and to protect cells from SNO-PDI formation via rotenone insult both, cell-based and cell-independent in vitro experiments. Furthermore, EA not only mitigates nitrosative-stress-induced aggregation of synphilin-1 but also α-synuclein and α-synuclein:synphilin-1 composites (Lewy-like neurites) in PC12 cells. Mechanistic analyses of the neuroprotective phenomena revealed that EA lowered rotenone-instigated reactive oxygen species (ROS) and reactive nitrogen species (RNS) in PC12 cells, imparted antiapoptotic tributes, and directly interfered with SNO-PDI formation. Lastly, we demonstrate that EA can bind human serum albumin (HSA). These results collectively indicate that small molecules can provide a therapeutic foothold for overcoming Parkinson's through a prophylactic approach.