Retinoic Acid Prevents α-Synuclein Preformed Fibrils-Induced Toxicity via Inhibiting STAT1-PARP1 Signaling.

Parkinson's disease (PD), the second-most prevalent neurodegenerative disorder, is characterized by the aberrant deposition of α-synuclein (α-Syn) aggregation in neurons. Recent reports have shown that retinoic acid (RA) ameliorates motor deficits. However, the underlying molecular mechanisms remain unclear. In this article, we investigated the effects of RA on cellular and animal models of PD. We found that RA is beneficial for neuronal survival in PD-associated models. In α-Syn preformed fibrils-treated mice, RA administration relieved the formation of intracellular inclusions, dopaminergic neuronal loss, and behavioral deficits. α-Syn preformed fibrils-treated SH-SY5Y cells manifested decreased cell viability, apoptosis, α-Syn aggregation, and autophagy defects. All these negative phenomena were alleviated by RA. More importantly, RA could inhibit the neurotoxicity via inhibiting α-Syn preformed fibrils-induced STAT1-PARP1 signaling, which could also be antagonized by IFN-γ. In conclusion, RA could hinder α-Syn preformed fibrils-induced toxicity by inhibiting STAT1-PARP1 signaling. Thus, we present new insight into RA in PD management.

Neuroinflammation in Parkinson's Disease and its Treatment Opportunities.

Parkinson's disease (PD) is a complex, chronic, and progressive neurodegenerative disease that is characterized by irreversible dopaminergic neuronal loss in the substantia nigra. Alpha-synuclein is normally a synaptic protein that plays a key role in PD due to pathological accumulation as oligomers or fibrils. Clustered alpha-synuclein binds to the Toll-like receptors and activates the microglia, which initiates a process that continues with pro-inflammatory cytokine production and secretion. Pro-inflammatory cytokine overproduction and secretion induce cell death and accelerate PD progression. Microglia are found in a resting state in physiological conditions. Microglia became activated by stimulating Toll-like receptors on it under pathological conditions, such as alpha-synuclein aggregation, environmental toxins, or oxidative stress. The interaction between Toll-like receptors and its downstream pathway triggers an activation series, leads to nuclear factor-kappa B activation, initiates the inflammasome formation, and increases cytokine levels. This consecutive inflammatory process leads to dopaminergic cell damage and cell death. Microglia become overactive in response to chronic inflammation, which is observed in PD and causes excessive cytotoxic factor production, such as reactive oxidase, nitric oxide, and tumor necrosis factor-alpha. This inflammatory process contributes to the exacerbation of pathology by triggering neuronal damage or death. Current treatments, such as dopaminergic agonists, anticholinergics, or monoamine oxidase inhibitors alleviate PD symptoms, but they can not stop the disease progression. Finding a radical treatment option or stopping the progression is essential when considering that PD is the second most reported neurodegenerative disorder. Many cytokines are released during inflammation, and they can start the phagocytic process, which caused the degradation of infected cells along with healthy ones. Therefore, targeting the pathological mechanisms, such as microglial activation, mitochondrial dysfunction, and oxidative stress, that should be involved in the treatment program is important. Neuroinflammation is one of the key factors involved in PD pathogenesis as well as alpha-synuclein accumulation, synaptic dysfunction, or dopaminergic neuronal loss, especially in the substantia nigra. Therefore, evaluating the therapeutic efficiency of the mechanisms is important, such as microglial activation and nuclear factor-kappa B pathway or inflammasome formation inhibition, and cytokine release interruption against neuroinflammation may create new treatment possibilities for PD. This study examined the pathological relation between PD and neuroinflammation, and targeting neuroinflammation as an opportunity for PD treatments, such as Toll-like receptor antagonists, NOD-like receptor family pyrin domain containing-3 inflammasome inhibitors, cytokine inhibitors, peroxisome proliferator-activated receptor-γ agonists, reactive oxygen species inhibitors, and nonsteroidal anti-inflammatory drugs.

Interleukin-6 triggers toxic neuronal iron sequestration in response to pathological α-synuclein.

α-synuclein (α-syn) aggregation and accumulation drive neurodegeneration in Parkinson's disease (PD). The substantia nigra of patients with PD contains excess iron, yet the underlying mechanism accounting for this iron accumulation is unclear. Here, we show that misfolded α-syn activates microglia, which release interleukin 6 (IL-6). IL-6, via its trans-signaling pathway, induces changes in the neuronal iron transcriptome that promote ferrous iron uptake and decrease cellular iron export via a pathway we term the cellular iron sequestration response, or CISR. The brains of patients with PD exhibit molecular signatures of the IL-6-mediated CISR. Genetic deletion of IL-6, or treatment with the iron chelator deferiprone, reduces pathological α-syn toxicity in a mouse model of sporadic PD. These data suggest that IL-6-induced CISR leads to toxic neuronal iron accumulation, contributing to synuclein-induced neurodegeneration.

Kaempferol Has Potent Protective and Antifibrillogenic Effects for α-Synuclein Neurotoxicity In Vitro.

Aggregation of α-synuclein (α-Syn) is implicated in the pathogenesis of Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Therefore, the removal of α-Syn aggregation could lead to the development of many new therapeutic agents for neurodegenerative diseases. In the present study, we succeeded in generating a new α-Syn stably expressing cell line using a piggyBac transposon system to investigate the neuroprotective effect of the flavonoid kaempferol on α-Syn toxicity. We found that kaempferol provided significant protection against α-Syn-related neurotoxicity. Furthermore, kaempferol induced autophagy through an increase in the biogenesis of lysosomes by inducing the expression of transcription factor EB and reducing the accumulation of α-Syn; thus, kaempferol prevented neuronal cell death. Moreover, kaempferol directly blocked the amyloid fibril formation of α-Syn. These results support the therapeutic potential of kaempferol in diseases such as synucleinopathies that are characterized by α-Syn aggregates.

Cortical alpha-synuclein preformed fibrils do not affect interval timing in mice.

One hallmark feature of Parkinson's disease (PD) is Lewy body pathology associated with misfolded alpha-synuclein. Previous studies have shown that striatal injection of alpha-synuclein preformed fibrils (PFF) can induce misfolding and aggregation of native alpha-synuclein in a prion-like manner, leading to cell death and motor dysfunction in mouse models. Here, we tested whether alpha-synuclein PFFs injected into the medial prefrontal cortex results in deficits in interval timing, a cognitive task which is disrupted in human PD patients and in rodent models of PD. We injected PFF or monomers of human alpha-synuclein into the medial prefrontal cortex of mice pre-injected with adeno-associated virus (AAV) coding for overexpression of human alpha-synuclein or control protein. Despite notable medial prefrontal cortical synucleinopathy, we did not observe consistent deficits in fixed-interval timing. These results suggest that cortical alpha-synuclein does not reliably disrupt fixed-interval timing.

Brain iron enrichment attenuates α-synuclein spreading after injection of preformed fibrils.

Regional iron accumulation and α-synuclein (α-syn) spreading pathology within the central nervous system are common pathological findings in Parkinson's disease (PD). Whereas iron is known to bind to α-syn, facilitating its aggregation and regulating α-syn expression, it remains unclear if and how iron also modulates α-syn spreading. To elucidate the influence of iron on the propagation of α-syn pathology, we investigated α-syn spreading after stereotactic injection of α-syn preformed fibrils (PFFs) into the striatum of mouse brains after neonatal brain iron enrichment. C57Bl/6J mouse pups received oral gavage with 60, 120, or 240 mg/kg carbonyl iron or vehicle between postnatal days 10 and 17. At 12 weeks of age, intrastriatal injections of 5-µg PFFs were performed to induce seeding of α-syn aggregates. At 90 days post-injection, PFFs-injected mice displayed long-term memory deficits, without affection of motor behavior. Interestingly, quantification of α-syn phosphorylated at S129 showed reduced α-syn pathology and attenuated spreading to connectome-specific brain regions after brain iron enrichment. Furthermore, PFFs injection caused intrastriatal microglia accumulation, which was alleviated by iron in a dose-dependent way. In primary cortical neurons in a microfluidic chamber model in vitro, iron application did not alter trans-synaptic α-syn propagation, possibly indicating an involvement of non-neuronal cells in this process. Our study suggests that α-syn PFFs may induce cognitive deficits in mice independent of iron. However, a redistribution of α-syn aggregate pathology and reduction of striatal microglia accumulation in the mouse brain may be mediated via iron-induced alterations of the brain connectome.

Mechanistic basis for receptor-mediated pathological α-synuclein fibril cell-to-cell transmission in Parkinson's disease.

The spread of pathological α-synuclein (α-syn) is a crucial event in the progression of Parkinson's disease (PD). Cell surface receptors such as lymphocyte activation gene 3 (LAG3) and amyloid precursor-like protein 1 (APLP1) can preferentially bind α-syn in the amyloid over monomeric state to initiate cell-to-cell transmission. However, the molecular mechanism underlying this selective binding is unknown. Here, we perform an array of biophysical experiments and reveal that LAG3 D1 and APLP1 E1 domains commonly use an alkaline surface to bind the acidic C terminus, especially residues 118 to 140, of α-syn. The formation of amyloid fibrils not only can disrupt the intramolecular interactions between the C terminus and the amyloid-forming core of α-syn but can also condense the C terminus on fibril surface, which remarkably increase the binding affinity of α-syn to the receptors. Based on this mechanism, we find that phosphorylation at serine 129 (pS129), a hallmark modification of pathological α-syn, can further enhance the interaction between α-syn fibrils and the receptors. This finding is further confirmed by the higher efficiency of pS129 fibrils in cellular internalization, seeding, and inducing PD-like α-syn pathology in transgenic mice. Our work illuminates the mechanistic understanding on the spread of pathological α-syn and provides structural information for therapeutic targeting on the interaction of α-syn fibrils and receptors as a potential treatment for PD.

Cannabinoid receptor-1 has an effect on CD200 under rotenone and alpha-synuclein induced stress.

Decades after identifying cannabinoids and their beneficial effects on Parkinson's disease (PD), many gaps are still missing. Although, CB2-dependent actions have been shown as underlying positive effects of cannabinoid treatment, in recent years, another receptor of cannabinoids, CB1, emerged as a valuable player in cannabinoid-induced neuroprotection. Remarkably, the effects of CB1 are mainly related to immune cells in the CNS, microglia, and astrocytes. However, oxidative stress, α-syn accumulation, and immune disbalance are essential aspects of both neurons and glial cells. Therefore, in this study, we investigated the effects of the CB1 on both α-syn and rotenone-treated SH-SY5Y and C8-D1A cells. ACEA and AM-251 were used as CB1 agonists and antagonists. Cell viability, IL-1ß, IL-6, TNF-α levels, and CD200 expressions were determined in culture mediums. Our results demonstrated that preformed fibril form (pFF) of α-syn did not cause any significant change in SH-SY5Y cells compared to C8-D1A cells. Rotenone significantly increased the expression of IL-1ß, IL-6, and TNF-α levels in both cells. pFF α-syn and rotenone treatment caused a decrease in CD200 expression. Surprisingly both ACEA and AM-251 alleviated rotenone-induced increase in cytokine levels in both cell lines. Although ACEA prevented pFF α-syn induced increase in cytokine levels and decrease in CD200 expression in C8-D1A cells, AM-251 failed to affect CD200 expression levels. Additionally, ACEA + AM-251 abolished the protective effects of both ACEA and AM-251 against rotenone and α-syn insults in both cell lines. The current study suggests that cannabinoid receptor agonism alleviates rotenone and α-syn-dependent inflammation in neurons and astrocytes.

Neuroprotection of Exendin-4 by Enhanced Autophagy in a Parkinsonian Rat Model of α-Synucleinopathy.

Glucagon-like peptide-1 (GLP-1) receptor stimulation ameliorates parkinsonian motor and non-motor deficits in both experimental animals and patients; however, the disease-modifying mechanisms of GLP-1 receptor activation have remained unknown. The present study investigated whether exendin-4 (a GLP-1 analogue) can rescue motor deficits and exert disease-modifying effects in a parkinsonian rat model of α-synucleinopathy. This model was established by unilaterally injecting AAV-9-A53T-α-synuclein into the right substantia nigra pars compacta, followed by 4 or 8 weeks of twice-daily intraperitoneal injections of exendin-4 (5 µg/kg/day) starting at 2 weeks after AAV-9-A53T-α-synuclein injections. Positron emission tomography/computed tomography (PET/CT) scanning and immunostaining established that treatment with exendin-4 attenuated tyrosine-hydroxylase-positive neuronal loss and terminal denervation and mitigated the decrease in expression of vesicular monoamine transporter 2 within the nigrostriatal dopaminergic systems of rats injected with AAV-9-A53T-α-synuclein. It also mitigated the parkinsonian motor deficits assessed in behavioral tests. Furthermore, through both in vivo and in vitro models of Parkinson's disease, we showed that exendin-4 promoted autophagy and mediated degradation of pathological α-synuclein, the effects of which were counteracted by 3-methyladenine or chloroquine, the autophagic inhibitors. Additionally, exendin-4 attenuated dysregulation of the PI3K/Akt/mTOR pathway in rats injected with AAV-9-A53T-α-synuclein. Taken together, our results demonstrate that exendin-4 treatment relieved behavioral deficits, dopaminergic degeneration, and pathological α-synuclein aggregation in a parkinsonian rat model of α-synucleinopathy and that these effects were mediated by enhanced autophagy via inhibiting the PI3K/Akt/mTOR pathway. In light of the safety and tolerance of exendin-4 administration, our results suggest that exendin-4 may represent a promising disease-modifying treatment for Parkinson's disease.

Pharmacological Inhibition of Brain EGFR Activation By a BBB-penetrating Inhibitor, AZD3759, Attenuates α-synuclein Pathology in a Mouse Model of α-Synuclein Propagation.

Aggregation and deposition of α-synuclein (α-syn) in Lewy bodies within dopamine neurons of substantia nigra (SN) is the pathological hallmark of Parkinson's disease (PD). These toxic α-syn aggregates are believed to propagate from neuron-to-neuron and spread the α-syn pathology throughout the brain beyond dopamine neurons in a prion-like manner. Targeting propagation of such α-syn aggregates is of high interest but requires identifying pathways involving in this process. Evidence from previous Alzheimer's disease reports suggests that EGFR may be involved in the prion-like propagation and seeding of amyloid-ß. We show here that EGFR regulates the uptake of exogenous α-syn-PFFs and the levels of endogenous α-syn in cell cultures and a mouse model of α-syn propagation, respectively. Thus, we tested the therapeutic potentials of AZD3759, a highly selective BBB-penetrating EGFR inhibitor, in a preclinical mouse model of α-syn propagation. AZD3759 decreases activated EGFR levels in the brain and reduces phosphorylated α-synuclein (pSyn) pathology in brain sections, including striatum and SN. As AZD3759 is already in the clinic, this paper's results suggest a possible repositioning of AZD3759 as a disease-modifying approach for PD.

Quinacrine and Niclosamide Promote Neurite Growth in Midbrain Dopaminergic Neurons Through the Canonical BMP-Smad Pathway and Protect Against Neurotoxin and α-Synuclein-Induced Neurodegeneration.

Parkinson's disease is a neurodegenerative disorder characterised by nigrostriatal dopaminergic degeneration, and intracellular α-synuclein aggregation. Current pharmacological treatments are solely symptomatic so there is a need to identify agents that can slow or stop dopaminergic degeneration. One proposed class of therapeutics are neurotrophic factors which promote the survival of nigrostriatal dopaminergic neurons. However, neurotrophic factors need to be delivered directly to the brain. An alternative approach may be to identify pharmacological agents which can reach the brain to stimulate neurotrophic factor expression and/or their signalling pathways in dopaminergic neurons. BMP2 is a neurotrophic factor that is expressed in the human substantia nigra; exogenous BMP2 administration protects against dopaminergic degeneration in in vitro models of PD. In this study, we investigated the neurotrophic potential of two FDA-approved drugs, quinacrine and niclosamide, that are modulators of BMP2 signalling. We report that quinacrine and niclosamide, like BMP2, significantly increased neurite length, as a readout of neurotrophic action, in SH-SY5Y cells and dopaminergic neurons in primary cultures of rat ventral mesencephalon. We also show that these effects of quinacrine and niclosamide require the activation of BMP-Smad signalling. Finally, we demonstrate that quinacrine and niclosamide are neuroprotective against degeneration induced by the neurotoxins, MPP+ and 6-OHDA, and by viral-mediated overexpression of α-synuclein in vitro. Collectively, this study identifies two drugs, that are safe for use in patients' to 'are approved for human use, that exert neurotrophic effects on dopaminergic neurons through modulation of BMP-Smad signalling. This rationalises the further study of drugs that target the BMP-Smad pathway as potential neuroprotective pharmacotherapy for Parkinson's disease.

Prion-like C-Terminal Domain of TDP-43 and α-Synuclein Interact Synergistically to Generate Neurotoxic Hybrid Fibrils.

Aberrant aggregation and amyloid formation of tar DNA binding protein (TDP-43) and α-synuclein (αS) underlie frontotemporal dementia (FTD) and Parkinson's disease (PD), respectively. Amyloid inclusions of TDP-43 and αS are also commonly co-observed in amyotrophic lateral sclerosis (ALS), dementia with Lewy bodies (DLB) and Alzheimer disease (AD). Emerging evidence from cellular and animal models show colocalization of the TDP-43 and αS aggregates, raising the possibility of direct interactions and co-aggregation between the two proteins. In this report, we set out to answer this question by investigating the interactions between αS and prion-like pathogenic C-terminal domain of TDP-43 (TDP-43 PrLD). PrLD is an aggregation-prone fragment generated both by alternative splicing as well as aberrant proteolytic cleavage of full length TDP-43. Our results indicate that two proteins interact in a synergistic manner to augment each other's aggregation towards hybrid fibrils. While monomers, oligomers and sonicated fibrils of αS seed TDP-43 PrLD monomers, TDP-43 PrLD fibrils failed to seed αS monomers indicating selectivity in interactions. Furthermore, αS modulates liquid droplets formed by TDP-43 PrLD and RNA to promote insoluble amyloid aggregates. Importantly, the cross-seeded hybrid aggregates show greater cytotoxicity as compared to the individual homotypic aggregates suggesting that the interactions between the two proteins have a discernable impact on cellular functions. Together, these results bring forth insights into TDP-43 PrLD - αS interactions that could help explain clinical and pathological presentations in patients with co-morbidities involving the two proteins.

Brazilin Removes Toxic Alpha-Synuclein and Seeding Competent Assemblies from Parkinson Brain by Altering Conformational Equilibrium.

Alpha-synuclein (α-syn) fibrils, a major constituent of the neurotoxic Lewy Bodies in Parkinson's disease, form via nucleation dependent polymerization and can replicate by a seeding mechanism. Brazilin, a small molecule derived from red cedarwood trees in Brazil, has been shown to inhibit the fibrillogenesis of amyloid-beta (Aß) and α-syn as well as remodel mature fibrils and reduce cytotoxicity. Here we test the effects of Brazilin on both seeded and unseeded α-syn fibril formation and show that the natural polyphenol inhibits fibrillogenesis of α-syn by a unique mechanism that alters conformational equilibria in two separate points of the assembly mechanism: Brazilin preserves the natively unfolded state of α-syn by specifically binding to the compact conformation of the α-syn monomer. Brazilin also eliminates seeding competence of α-syn assemblies from Parkinson's disease patient brain tissue, and reduces toxicity of pre-formed assemblies in primary neurons by inducing the formation of large fibril clusters. Molecular docking of Brazilin shows the molecule to interact both with unfolded α-syn monomers and with the cross-ß sheet structure of α-syn fibrils. Our findings suggest that Brazilin has substantial potential as a neuroprotective and therapeutic agent for Parkinson's disease.

Crowded organelles, lipid accumulation, and abnormal membrane tubulation in cellular models of enhanced α-synuclein membrane interaction.

Previous work from our group showed that certain engineered missense mutations to the α-synuclein (αS) KTKEGV repeat motifs abrogate the protein's ability to form native multimers. The resultant excess monomers accumulate in lipid-membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial Parkinson's disease mutants such as E46K. We presented an initial characterization of the lipid-rich inclusions and found similarities to the αS- and vesicle-rich inclusions that form in baker's yeast when αS is expressed. We also discussed, with some caution, a possible role of membrane-rich inclusions as precursors to filamentous Lewy bodies, the widely accepted hallmark pathology of Parkinson's disease and other synucleinopathies. In the meantime, advances in the microscopic characterization of Lewy bodies have highlighted the presence of crowded organelles and lipid membranes in addition to αS accumulation. This prompted us to revisit the αS inclusions caused by our repeat motif variants in neuroblastoma cells. In addition to our previous characterization, we found that these inclusions can often be seen by brightfield microscopy, overlap with endogenous vesicle markers in immunofluorescence experiments, stain positive for lipid dyes, and can be found to be closely associated with mitochondria. We also observed abnormal tubulation of membranes, which was subtle in inducible lines and pronounced in cells that transiently expressed high amounts of the highly disruptive KTKEGV motif mutant "KLKEGV". Membrane tubulation had been reported before as an αS activity in reductionist systems. Our in-cellulo demonstration now suggests that this mechanism could possibly be a relevant aspect of aberrant αS behavior in cells.

The Cytotoxic Effect of α-Synuclein Aggregates.

Parkinson's disease is a neurodegenerative disorder involving a functional protein, α-synuclein, whose primary function is related to vesicle trafficking. However, α-synuclein is prone to form aggregates, and these inclusions, known as Lewy bodies, are the hallmark of Parkinson's disease. α-synuclein can alter its conformation and acquire aggregating capacity, forming aggregates containing ß-sheets. This protein's pathogenic importance is based on its ability to form oligomers that impair synaptic transmission and neuronal function by increasing membrane permeability and altering homeostasis, generating a deleterious effect over cells. First, we establish that oligomers interfere with the mechanical properties of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane, as demonstrated by nanoindentation curves. In contrast, nanoindentation revealed that the α-synuclein monomer's presence leads to a much more resistant lipid bilayer. Moreover, the oligomers' interaction with cell membranes can promote lactate dehydrogenase (LDH) release, suggesting the activation of cytotoxic events.

LncRNA OIP5-AS1 reduces α-synuclein aggregation and toxicity by targeting miR-126 to activate PLK2 in human neuroblastoma SH-SY5Y cells.

It has been reported that many long noncoding RNAs (lncRNA) are abnormally expressed in Parkinson's disease (PD). However, the knowledge about the role of dysregulated lncRNA in the pathological process of PD and the potential molecular regulation mechanism is still limited. Our immunofluorescence data show that miR-126 enhances the aggregation and toxicity of synuclein, while lncRNA OIP5-AS1 reduces the aggregation and toxicity of MPP + induced α-synuclein by targeting miR-126. Luciferase experiments have found that miR-126 regulates α-synuclein by targeting PLK2. Western blot and IP experimental analysis showed that this process is achieved by regulating PLK2/α-synuclein autophagy. In conclusion, our data indicate that OIP5-AS1 promotes the autophagy of PLK2-α-synuclein by targeting the miR-126 axis with pathogenic factors, thus reducing the aggregation toxicity of α-synuclein, which It will help better to understand the mechanism of dopaminergic neuron loss in PD and provide novel treatment options.

Two C-terminal sequence variations determine differential neurotoxicity between human and mouse α-synuclein.

BACKGROUND: α-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinson's disease (PD). Mouse aSyn contains a threonine residue at position 53 that mimics the human familial PD substitution A53T, yet in contrast to A53T patients, mice show no evidence of aSyn neuropathology even after aging. Here, we studied the neurotoxicity of human A53T, mouse aSyn, and various human-mouse chimeras in cellular and in vivo models, as well as their biochemical properties relevant to aSyn pathobiology. METHODS: Primary midbrain cultures transduced with aSyn-encoding adenoviruses were analyzed immunocytochemically to determine relative dopaminergic neuron viability. Brain sections prepared from rats injected intranigrally with aSyn-encoding adeno-associated viruses were analyzed immunohistochemically to determine nigral dopaminergic neuron viability and striatal dopaminergic terminal density. Recombinant aSyn variants were characterized in terms of fibrillization rates by measuring thioflavin T fluorescence, fibril morphologies via electron microscopy and atomic force microscopy, and protein-lipid interactions by monitoring membrane-induced aSyn aggregation and aSyn-mediated vesicle disruption. Statistical tests consisted of ANOVA followed by Tukey's multiple comparisons post hoc test and the Kruskal-Wallis test followed by a Dunn's multiple comparisons test or a two-tailed Mann-Whitney test. RESULTS: Mouse aSyn was less neurotoxic than human aSyn A53T in cell culture and in rat midbrain, and data obtained for the chimeric variants indicated that the human-to-mouse substitutions D121G and N122S were at least partially responsible for this decrease in neurotoxicity. Human aSyn A53T and a chimeric variant with the human residues D and N at positions 121 and 122 (respectively) showed a greater propensity to undergo membrane-induced aggregation and to elicit vesicle disruption. Differences in neurotoxicity among the human, mouse, and chimeric aSyn variants correlated weakly with differences in fibrillization rate or fibril morphology. CONCLUSIONS: Mouse aSyn is less neurotoxic than the human A53T variant as a result of inhibitory effects of two C-terminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization. Our findings highlight the importance of membrane-induced self-assembly in aSyn neurotoxicity and suggest that inhibiting this process by targeting the C-terminal domain could slow neurodegeneration in PD and other synucleinopathy disorders.

Trodusquemine displaces protein misfolded oligomers from cell membranes and abrogates their cytotoxicity through a generic mechanism.

The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-ß (Aß) in Alzheimer's disease and α-synuclein (αS) in Parkinson's disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aß and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases.

Maysin plays a protective role against α-Synuclein oligomers cytotoxicity by triggering autophagy activation.

Parkinson's disease (PD) is a widespread neurodegenerative disorder characterized by the progressive loss of neurons. The accumulation of aggregated forms of the α-Synuclein (Syn) protein is the main cause of neurotoxicity in PD by disrupting cellular homeostasis until neuronal death. Scientific research is constantly looking for natural products as preventive agents against the progression of several neurodisorders due their safety and non-toxic nature. Neuroprotective phytochemicals include Maysin (Mys), the most abundant C-glycosilflavone in corn silk. In this work, the Mys protective role against damage by Syn amyloid aggregates - oligomers and fibrils - was investigated in SH-SY5Y human neuroblastoma cells obtaining novel and interesting information concerning the Mys molecular mechanism of action. Mys showed effectiveness in preventing the typical toxic events induced by Syn amyloid aggregates, i.e. oxidative stress and imbalance of intracellular calcium homeostasis. Mys exhibited a cytoprotective role, especially against Syn oligomers injury, activating an autophagic degradative process, thus playing a key role on several features of amyloid neurotoxicity. Therefore, Mys could be proposed for the first time to the scientific community as an interesting novel natural compound that might allow to develop alternative strategies to prevent the damage of Syn oligomers involved in Parkinson's disease.

Alleviating toxic α-Synuclein accumulation by membrane depolarization: evidence from an in vitro model of Parkinson's disease.

Parkinson's disease (PD) is characterized by the formation of toxic, fibrillar form alpha-synuclein (α-Syn) protein aggregates in dopaminergic neurons. Accumulating evidence has shown a multifactorial interplay between the intracellular calcium elevation and α-Syn dynamics. However, whether membrane depolarization regulates toxic α-Syn aggregates remains unclear. To understand this better, we used an in vitro α-Syn preformed fibrils (PFF) model of PD in human neural cells. We demonstrated functional membrane depolarization in differentiated SH-SY5Y cells induced by two independent treatments: high extracellular K+ and the GABAA receptor blocker picrotoxin. We then observed that these treatments significantly alleviated toxic α-Syn aggregation in PFF-treated SH-SY5Y cells. Moreover, clinically relevant direct current stimulation (DCS) also remarkably decreased toxic α-Syn aggregation in PFF-treated SH-SY5Y cells. Taken together, our findings suggest that membrane depolarization plays an important role in alleviating PFF-induced toxic α-Syn aggregates, and that it may represent a novel therapeutic mechanism for PD.