T-Type Ca2+ Enhancer SAK3 Activates CaMKII and Proteasome Activities in Lewy Body Dementia Mice Model

Lewy bodies are pathological characteristics of Lewy body dementia (LBD) and are composed of α-synuclein (α-Syn), which is mostly degraded via the ubiquitin–proteasome system. More importantly, 26S proteasomal activity decreases in the brain of LBD patients. We recently introduced a T-type calcium channel enhancer SAK3 (ethyl-8-methyl-2,4-dioxo-2-(piperidin-1-yl)- 2H-spiro[cyclopentane-1,3-imidazo [1,2-a]pyridin]-2-ene-3-carboxylate) for Alzheimer’s disease therapeutics. SAK3 enhanced the proteasome activity via CaMKII activation in amyloid precursor protein knock-in mice, promoting the degradation of amyloid-β plaques to improve cognition. At this point, we addressed whether SAK3 promotes the degradation of misfolded α-Syn and the aggregates in α-Syn preformed fibril (PFF)-injected mice. The mice were injected with α-Syn PFF in the dorsal striatum, and SAK3 (0.5 or 1.0 mg/kg) was administered orally for three months, either immediately or during the last month after injection. SAK3 significantly inhibited the accumulation of fibrilized phosphorylated-α-Syn in the substantia nigra. Accordingly, SAK3 significantly recovered mesencephalic dopamine neurons from cell death. Decreased α-Syn accumulation was closely associated with increased proteasome activity. Elevated CaMKII/Rpt-6 signaling possibly mediates the enhanced proteasome activity after SAK3 administration in the cortex and hippocampus. CaMKII/Rpt-6 activation also accounted for improved memory and cognition in α-Syn PFF-injected mice. These findings indicate that CaMKII/Rpt-6-dependent proteasomal activation by SAK3 recovers from α-Syn pathology in LBD.     

Intracerebral Administration of a Ligand-ASO Conjugate Selectively Reduces α-Synuclein Accumulation in Monoamine Neurons of Double Mutant Human A30P*A53T*α-Synuclein Transgenic Mice

α-Synuclein (α-Syn) protein is involved in the pathogenesis of Parkinson’s disease (PD). Point mutations and multiplications of the α-Syn, which encodes the SNCA gene, are correlated with early-onset PD, therefore the reduction in a-Syn synthesis could be a potential therapy for PD if delivered to the key affected neurons. Several experimental strategies for PD have been developed in recent years using oligonucleotide therapeutics. However, some of them have failed or even caused neuronal toxicity. One limiting step in the success of oligonucleotide-based therapeutics is their delivery to the brain compartment, and once there, to selected neuronal populations. Previously, we developed an indatraline-conjugated antisense oligonucleotide (IND-1233-ASO), that selectively reduces α-Syn synthesis in midbrain monoamine neurons of mice, and nonhuman primates. Here, we extended these observations using a transgenic male mouse strain carrying both A30P and A53T mutant human α-Syn (A30P*A53T*α-Syn). We found that A30P*A53T*α-Syn mice at 4–5 months of age showed 3.5-fold increases in human α-Syn expression in dopamine (DA) and norepinephrine (NE) neurons of the substantia nigra pars compacta (SNc) and locus coeruleus (LC), respectively, compared with mouse α-Syn levels. In parallel, transgenic mice exhibited altered nigrostriatal DA neurotransmission, motor alterations, and an anxiety-like phenotype. Intracerebroventricular IND-1233-ASO administration (100 µg/day, 28 days) prevented the α-Syn synthesis and accumulation in the SNc and LC, and recovered DA neurotransmission, although it did not reverse the behavioral phenotype. Therefore, the present therapeutic strategy based on a conjugated ASO could be used for the selective inhibition of α-Syn expression in PD-vulnerable monoamine neurons, showing the benefit of the optimization of ASO molecules as a disease modifying therapy for PD and related α-synucleinopathies.     

The Effects of Aβ1-42 Binding to the SARS-CoV-2 Spike Protein S1 Subunit and Angiotensin-Converting Enzyme 2

Increasing evidence suggests that elderly people with dementia are vulnerable to the development of severe coronavirus disease 2019 (COVID-19). In Alzheimer’s disease (AD), the major form of dementia, β-amyloid (Aβ) levels in the blood are increased; however, the impact of elevated Aβ levels on the progression of COVID-19 remains largely unknown. Here, our findings demonstrate that Aβ_1-42, but not Aβ_1-40, bound to various viral proteins with a preferentially high affinity for the spike protein S1 subunit (S1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the viral receptor, angiotensin-converting enzyme 2 (ACE2). These bindings were mainly through the C-terminal residues of Aβ_1-42. Furthermore, Aβ_1-42 strengthened the binding of the S1 of SARS-CoV-2 to ACE2 and increased the viral entry and production of IL-6 in a SARS-CoV-2 pseudovirus infection model. Intriguingly, data from a surrogate mouse model with intravenous inoculation of Aβ_1-42 show that the clearance of Aβ_1-42 in the blood was dampened in the presence of the extracellular domain of the spike protein trimers of SARS-CoV-2, whose effects can be prevented by a novel anti-Aβ antibody. In conclusion, these findings suggest that the binding of Aβ_1-42 to the S1 of SARS-CoV-2 and ACE2 may have a negative impact on the course and severity of SARS-CoV-2 infection. Further investigations are warranted to elucidate the underlying mechanisms and examine whether reducing the level of Aβ_1-42 in the blood is beneficial to the fight against COVID-19 and AD.     

(De)stabilization of Alpha-Synuclein Fibrillary Aggregation by Charged and Uncharged Surfactants

Parkinson’s disease (PD) is the second most common neurodegenerative disorder. An important hallmark of PD involves the pathological aggregation of proteins in structures known as Lewy bodies. The major component of these proteinaceous inclusions is alpha (α)-synuclein. In different conditions, α-synuclein can assume conformations rich in either α-helix or β-sheets. The mechanisms of α-synuclein misfolding, aggregation, and fibrillation remain unknown, but it is thought that β-sheet conformation of α-synuclein is responsible for its associated toxic mechanisms. To gain fundamental insights into the process of α-synuclein misfolding and aggregation, the secondary structure of this protein in the presence of charged and non-charged surfactant solutions was characterized. The selected surfactants were (anionic) sodium dodecyl sulphate (SDS), (cationic) cetyltrimethylammonium chloride (CTAC), and (uncharged) octyl β-D-glucopyranoside (OG). The effect of surfactants in α-synuclein misfolding was assessed by ultra-structural analyses, in vitro aggregation assays, and secondary structure analyses. The α-synuclein aggregation in the presence of negatively charged SDS suggests that SDS-monomer complexes stimulate the aggregation process. A reduction in the electrostatic repulsion between N- and C-terminal and in the hydrophobic interactions between the NAC (non-amyloid beta component) region and the C-terminal seems to be important to undergo aggregation. Fourier transform infrared spectroscopy (FTIR) measurements show that β-sheet structures comprise the assembly of the fibrils.     

Interferon Beta Activity Is Modulated via Binding of Specific S100 Proteins

Interferon-β (IFN-β) is a pleiotropic cytokine used for therapy of multiple sclerosis, which is also effective in suppression of viral and bacterial infections and cancer. Recently, we reported a highly specific interaction between IFN-β and S100P lowering IFN-β cytotoxicity to cancer cells (Int J Biol Macromol. 2020; 143: 633–639). S100P is a member of large family of multifunctional Ca²⁺-binding proteins with cytokine-like activities. To probe selectivity of IFN-β—S100 interaction with respect to S100 proteins, we used surface plasmon resonance spectroscopy, chemical crosslinking, and crystal violet assay. Among the thirteen S100 proteins studied S100A1, S100A4, and S100A6 proteins exhibit strictly Ca²⁺-dependent binding to IFN-β with equilibrium dissociation constants, K_d, of 0.04–1.5 µM for their Ca²⁺-bound homodimeric forms. Calcium depletion abolishes the S100—IFN-β interactions. Monomerization of S100A1/A4/A6 decreases K_d values down to 0.11–1.0 nM. Interferon-α is unable of binding to the S100 proteins studied. S100A1/A4 proteins inhibit IFN-β-induced suppression of MCF-7 cells viability. The revealed direct influence of specific S100 proteins on IFN-β activity uncovers a novel regulatory role of particular S100 proteins, and opens up novel approaches to enhancement of therapeutic efficacy of IFN-β.     

In Vitro Phosphorylation Does not Influence the Aggregation Kinetics of WT α-Synuclein in Contrast to Its Phosphorylation Mutants

The aggregation of alpha-synuclein (α-SYN) into fibrils is characteristic for several neurodegenerative diseases, including Parkinson’s disease (PD). Ninety percent of α-SYN deposited in Lewy Bodies, a pathological hallmark of PD, is phosphorylated on serine129. α-SYN can also be phosphorylated on tyrosine125, which is believed to regulate the membrane binding capacity and thus possibly its normal function. A better understanding of the effect of phosphorylation on the aggregation of α-SYN might shed light on its role in the pathogenesis of PD. In this study we compare the aggregation properties of WT α-SYN with the phospho-dead and phospho-mimic mutants S129A, S129D, Y125F and Y125E and in vitro phosphorylated α-SYN using turbidity, thioflavin T and circular dichroism measurements as well as transmission electron microscopy. We show that the mutants S129A and S129D behave similarly compared to wild type (WT) α-SYN, while the mutants Y125F and Y125E fibrillate significantly slower, although all mutants form fibrillar structures similar to the WT protein. In contrast, in vitro phosphorylation of α-SYN on either S129 or Y125 does not significantly affect the fibrillization kinetics. Moreover, FK506 binding proteins (FKBPs), enzymes with peptidyl-prolyl cis-trans isomerase activity, still accelerate the aggregation of phosphorylated α-SYN in vitro, as was shown previously for WT α-SYN. In conclusion, our results illustrate that phosphorylation mutants can display different aggregation properties compared to the more biologically relevant phosphorylated form of α-SYN.     

Srpk3 Decrease Associated with Alpha-Synuclein Increase in Muscles of MPTP-Induced Parkinson’s Disease Mice

Parkinson’s disease (PD) is characterized by a loss of dopaminergic cells in the substantia nigra, and its histopathological features include the presence of fibrillar aggregates of α-synuclein (α-syn), which are called Lewy bodies and Lewy neurites. Lewy pathology has been identified not only in the brain but also in various tissues, including muscles. This study aimed to investigate the link between serine/arginine-rich protein specific kinase 3 (srpk3) and α-syn in muscles in PD. We conducted experiments on the quadriceps femoris of a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model and the C2C12 cell line after treatment with 1-methyl-4-phenylpyridinium (MPP+) and srpk3 short interfering RNA (siRNA). Compared to the control group, the MPTP group showed significantly reduced expression of srpk3, but increased expression of α-syn. In MPP+-treated C2C12 cells, srpk3 expression gradually decreased and α-syn expression increased with the increasing MPP+ concentration. Moreover, experiments in C2C12 cells using srpk3 siRNA showed increased expressions of α-syn and phosphorylated α-syn. Our results showed that srpk3 expression could be altered by MPTP intoxication in muscles, and this change may be related to changes in α-syn expression. Furthermore, this study could contribute to advancement of research on the mechanism by which srpk3 plays a role in PD.     

Interferon-β Activity Is Affected by S100B Protein

Interferon-β (IFN-β) is a pleiotropic cytokine secreted in response to various pathological conditions and is clinically used for therapy of multiple sclerosis. Its application for treatment of cancer, infections and pulmonary diseases is limited by incomplete understanding of regulatory mechanisms of its functioning. Recently, we reported that IFN-β activity is affected by interactions with S100A1, S100A4, S100A6, and S100P proteins, which are members of the S100 protein family of multifunctional Ca²⁺-binding proteins possessing cytokine-like activities (Int J Mol Sci. 2020;21(24):9473). Here we show that IFN-β interacts with one more representative of the S100 protein family, the S100B protein, involved in numerous oncological and neurological diseases. The use of chemical crosslinking, intrinsic fluorescence, and surface plasmon resonance spectroscopy revealed IFN-β binding to Ca²⁺-loaded dimeric and monomeric forms of the S100B protein. Calcium depletion blocks the S100B–IFN-β interaction. S100B monomerization increases its affinity to IFN-β by 2.7 orders of magnitude (equilibrium dissociation constant of the complex reaches 47 pM). Crystal violet assay demonstrated that combined application of IFN-β and S100B (5–25 nM) eliminates their inhibitory effects on MCF-7 cell viability. Bioinformatics analysis showed that the direct modulation of IFN-β activity by the S100B protein described here could be relevant to progression of multiple oncological and neurological diseases.     

The Effect of Oxidized Dopamine on the Structure and Molecular Chaperone Function of the Small Heat-Shock Proteins, αB-Crystallin and Hsp27

Oxidation of the neurotransmitter, dopamine (DA), is a pathological hallmark of Parkinson’s disease (PD). Oxidized DA forms adducts with proteins which can alter their functionality. αB-crystallin and Hsp27 are intracellular, small heat-shock molecular chaperone proteins (sHsps) which form the first line of defense to prevent protein aggregation under conditions of cellular stress. In vitro, the effects of oxidized DA on the structure and function of αB-crystallin and Hsp27 were investigated. Oxidized DA promoted the cross-linking of αB-crystallin and Hsp27 to form well-defined dimer, trimer, tetramer, etc., species, as monitored by SDS-PAGE. Lysine residues were involved in the cross-links. The secondary structure of the sHsps was not altered significantly upon cross-linking with oxidized DA but their oligomeric size was increased. When modified with a molar equivalent of DA, sHsp chaperone functionality was largely retained in preventing both amorphous and amyloid fibrillar aggregation, including fibril formation of mutant (A53T) α-synuclein, a protein whose aggregation is associated with autosomal PD. In the main, higher levels of sHsp modification with DA led to a reduction in chaperone effectiveness. In vivo, DA is sequestered into acidic vesicles to prevent its oxidation and, intracellularly, oxidation is minimized by mM levels of the antioxidant, glutathione. In vitro, acidic pH and glutathione prevented the formation of oxidized DA-induced cross-linking of the sHsps. Oxidized DA-modified αB-crystallin and Hsp27 were not cytotoxic. In a cellular context, retention of significant chaperone functionality by mildly oxidized DA-modified sHsps would contribute to proteostasis by preventing protein aggregation (particularly of α-synuclein) that is associated with PD.     

The Role of α-Synuclein Oligomers in Parkinson’s Disease

α-synuclein (α-syn) is a protein associated with the pathogenesis of Parkinson’s disease (PD), the second most common neurodegeneration disease with no effective treatment. However, how α-syn drives the pathology of PD remains elusive. Recent studies suggest that α-syn oligomers are the primary cause of neurotoxicity and play a critical role in PD. In this review, we discuss the process of α-syn oligomers formation and the current understanding of the structures of oligomers. We also describe seed and propagation effects of oligomeric forms of α-syn. Then, we summarize the mechanism by which α-syn oligomers exert neurotoxicity and promote neurodegeneration, including mitochondrial dysfunction, endoplasmic reticulum stress, proteostasis dysregulation, synaptic impairment, cell apoptosis and neuroinflammation. Finally, we investigate treatment regimens targeting α-syn oligomers at present. Further research is needed to understand the structure and toxicity mechanism of different types of oligomers, so as to provide theoretical basis for the treatment of PD.     

Ellagic Acid Prevents α-Synuclein Aggregation and Protects SH-SY5Y Cells from Aggregated α-Synuclein-Induced Toxicity via Suppression of Apoptosis and Activation of Autophagy

Parkinson’s disease (PD) is a neurodegenerative disease characterized by the loss of dopamine neurons and the deposition of misfolded proteins known as Lewy bodies (LBs), which contain α-synuclein (α-syn). The causes and molecular mechanisms of PD are not clearly understood to date. However, misfolded proteins, oxidative stress, and impaired autophagy are believed to play important roles in the pathogenesis of PD. Importantly, α-syn is considered a key player in the development of PD. The present study aimed to assess the role of Ellagic acid (EA), a polyphenol found in many fruits, on α-syn aggregation and toxicity. Using thioflavin and seeding polymerization assays, in addition to electron microscopy, we found that EA could dramatically reduce α-syn aggregation. Moreover, EA significantly mitigated the aggregated α-syn-induced toxicity in SH-SY5Y cells and thus enhanced their viability. Mechanistically, these cytoprotective effects of EA are mediated by the suppression of apoptotic proteins BAX and p53 and a concomitant increase in the anti-apoptotic protein, BCL-2. Interestingly, EA was able to activate autophagy in SH-SY5Y cells, as evidenced by normalized/enhanced expression of LC3-II, p62, and pAKT. Together, our findings suggest that EA may attenuate α-syn toxicity by preventing aggregation and improving viability by restoring autophagy and suppressing apoptosis.     

The Prion-Like Spreading of Alpha-Synuclein in Parkinson’s Disease: Update on Models and Hypotheses

The pathological aggregation of the presynaptic protein α-synuclein (α-syn) and propagation through synaptically coupled neuroanatomical tracts is increasingly thought to underlie the pathophysiological progression of Parkinson’s disease (PD) and related synucleinopathies. Although the precise molecular mechanisms responsible for the spreading of pathological α-syn accumulation in the CNS are not fully understood, growing evidence suggests that de novo α-syn misfolding and/or neuronal internalization of aggregated α-syn facilitates conformational templating of endogenous α-syn monomers in a mechanism reminiscent of prions. A refined understanding of the biochemical and cellular factors mediating the pathological neuron-to-neuron propagation of misfolded α-syn will potentially elucidate the etiology of PD and unravel novel targets for therapeutic intervention. Here, we discuss recent developments on the hypothesis regarding trans-synaptic propagation of α-syn pathology in the context of neuronal vulnerability and highlight the potential utility of novel experimental models of synucleinopathies.     

Astragalus membranaceus Inhibits Peritoneal Fibrosis via Monocyte Chemoattractant Protein (MCP)-1 and the Transforming Growth Factor-β1 (TGF-β1) Pathway in Rats Submitted to Peritoneal Dialysis

Inflammation and transforming growth factor-β1 (TGF-β1) contribute to the development of peritoneal fibrosis (PF), which is associated with peritoneal dialysis (PD). Astragalus membranaceus (Astragalus) has anti-inflammatory and anti-fibrotic effects in many diseases. The goal of this study was to determine the anti-fibrotic effects of Astragalus on the PF response to PD. A rat model of PD was induced using standard PD fluid, and PF was verified by HE and Masson’s staining, as well as through the expression of fibroblast surface protein (FSP) and collagen III. The expression levels of monocyte chemoattractant protein (MCP)-1, F4/80 (macrophage/monocyte marker in rat), TGF-β1 and the downstream proteins phospho-SMAD 2/3 in dialyzed peritoneal tissue treated with or without Astragalus was evaluated using immunohistochemistry analysis. Overall correlations between MCP-1 and TGF-β1 staining were analyzed using both the Spearman and Pearson methods. The results showed that Astragalus could inhibit the recruitment and activation of monocytes/macrophages, thereby reducing the production of TGF-β1 in the dialyzed peritoneal membrane. PF was also significantly decreased following treatment with Astragalus. MCP-1 expression had a strong positive correlation with TGF-β1 sensitivity, suggesting that the anti-fibrotic function of Astragalus was mediated by MCP-1 and the TGF-β1 pathway. Our results indicate that Astragalus could be a useful agent against PD-induced PF.     

Cell-Free Hemoglobin Does Not Attenuate the Effects of SARS-CoV-2 Spike Protein S1 Subunit in Pulmonary Endothelial Cells

SARS-CoV-2 primarily infects epithelial airway cells that express the host entry receptor angiotensin-converting enzyme 2 (ACE2), which binds to the S1 spike protein on the surface of the virus. To delineate the impact of S1 spike protein interaction with the ACE2 receptor, we incubated the S1 spike protein with human pulmonary arterial endothelial cells (HPAEC). HPAEC treatment with the S1 spike protein caused disruption of endothelial barrier function, increased levels of numerous inflammatory molecules (VCAM-1, ICAM-1, IL-1β, CCL5, CXCL10), elevated mitochondrial reactive oxygen species (ROS), and a mild rise in glycolytic reserve capacity. Because low oxygen tension (hypoxia) is associated with severe cases of COVID-19, we also evaluated treatment with hemoglobin (HbA) as a potential countermeasure in hypoxic and normal oxygen environments in analyses with the S1 spike protein. We found hypoxia downregulated the expression of the ACE2 receptor and increased the critical oxygen homeostatic signaling protein, hypoxia-inducible factor (HIF-1α); however, treatment of the cells with HbA yielded no apparent change in the levels of ACE2 or HIF-1α. Use of quantitative proteomics revealed that S1 spike protein-treated cells have few differentially regulated proteins in hypoxic conditions, consistent with the finding that ACE2 serves as the host viral receptor and is reduced in hypoxia. However, in normoxic conditions, we found perturbed abundance of proteins in signaling pathways related to lysosomes, extracellular matrix receptor interaction, focal adhesion, and pyrimidine metabolism. We conclude that the spike protein alone without the rest of the viral components is sufficient to elicit cell signaling in HPAEC, and that treatment with HbA failed to reverse the vast majority of these spike protein-induced changes.     

Bacterial Outer Membrane Protein OmpX Regulates β1 Integrin and Epidermal Growth Factor Receptor (EGFR) Involved in Invasion of M-HeLa Cells by Serratia proteamaculans

Opportunistic pathogen Serratia proteamaculans are able to penetrate the eukaryotic cells. The penetration rate can be regulated by bacterial surface protein OmpX. OmpX family proteins are able to bind to host cell surface to the epidermal growth factor receptor (EGFR) and the extracellular matrix protein fibronectin, whose receptors are in return the α5 β1 integrins. Here we elucidated the involvement of these host cell proteins in S. proteamaculans invasion. We have shown that, despite the absence of fibronectin contribution to S. proteamaculans invasion, β1 integrin was directly involved in invasion of M-HeLa cells. Herewith β1 integrin was not the only receptor that determines sensitivity of host cells to bacterial invasion. Signal transfer from EGFR was also involved in the penetration of these bacteria into M-HeLa cells. However, M-HeLa cells have not been characterized by large number of these receptors. It turned out that S. proteamaculans attachment to the host cell surface resulted in an increment of EGFR and β1 integrin genes expression. Such gene expression increment also caused Escherichia coli attachment, transformed with a plasmid encoding OmpX from S. proteamaculans. Thus, an OmpX binding to the host cell surface caused an increase in the EGFR and β1 integrin expression involved in S. proteamaculans invasion.     

The Bacterial Amyloids Phenol Soluble Modulins from Staphylococcus aureus Catalyze Alpha-Synuclein Aggregation

Aggregated α-synuclein (α-syn) is the main constituent of Lewy bodies, which are a pathological hallmark of Parkinson’s disease (PD). Environmental factors are thought to be potential triggers capable of initiating the aggregation of the otherwise monomeric α-syn. Braak’s seminal work redirected attention to the intestine and recent reports of dysbiosis have highlighted the potential causative role of the microbiome in the initiation of pathology of PD. Staphylococcus aureus is a bacterium carried by 30–70% of the general population. It has been shown to produce functional amyloids, called phenol soluble modulins (PSMαs). Here, we studied the kinetics of α-syn aggregation under quiescent conditions in the presence or absence of four different PSMα peptides and observed a remarkable shortening of the lag phase in their presence. Whereas pure α-syn monomer did not aggregate up to 450 h after initiation of the experiment in neither neutral nor mildly acidic buffer, the addition of different PSMα peptides resulted in an almost immediate increase in the Thioflavin T (ThT) fluorescence. Despite similar peptide sequences, the different PSMα peptides displayed distinct effects on the kinetics of α-syn aggregation. Kinetic analyses of the data suggest that all four peptides catalyze α-syn aggregation through heterogeneous primary nucleation. The immunogold electron microscopic analyses showed that the aggregates were fibrillar and composed of α-syn. In addition of the co-aggregated materials to a cell model expressing the A53T α-syn variant fused to GFP was found to catalyze α-syn aggregation and phosphorylation in the cells. Our results provide evidence of a potential trigger of synucleinopathies and could have implications for the prevention of the diseases.     

Chronic Exposure to Paraquat Induces Alpha-Synuclein Pathogenic Modifications in Drosophila

Parkinson’s disease (PD) is characterized by the progressive accumulation of neuronal intracellular aggregates largely composed of alpha-Synuclein (αSyn) protein. The process of αSyn aggregation is induced during aging and enhanced by environmental stresses, such as the exposure to pesticides. Paraquat (PQ) is an herbicide which has been widely used in agriculture and associated with PD. PQ is known to cause an increased oxidative stress in exposed individuals but the consequences of such stress on αSyn conformation remains poorly understood. To study αSyn pathogenic modifications in response to PQ, we exposed Drosophila expressing human αSyn to a chronic PQ protocol. We first showed that PQ exposure and αSyn expression synergistically induced fly mortality. The exposure to PQ was also associated with increased levels of total and phosphorylated forms of αSyn in the Drosophila brain. Interestingly, PQ increased the detection of soluble αSyn in highly denaturating buffer but did not increase αSyn resistance to proteinase K digestion. These results suggest that PQ induces the accumulation of toxic soluble and misfolded forms of αSyn but that these toxic forms do not form fibrils or aggregates that are detected by the proteinase K assay. Collectively, our results demonstrate that Drosophila can be used to study the effect of PQ or other environmental neurotoxins on αSyn driven pathology.