Cooperative inhibition of SNARE-mediated vesicle fusion by α-synuclein monomers and oligomers.

The primary hallmark of Parkinson's disease (PD) is the generation of Lewy bodies of which major component is α-synuclein (α-Syn). Because of increasing evidence of the fundamental roles of α-Syn oligomers in disease progression, α-Syn oligomers have become potential targets for therapeutic interventions for PD. One of the potential toxicities of α-Syn oligomers is their inhibition of SNARE-mediated vesicle fusion by specifically interacting with vesicle-SNARE protein synaptobrevin-2 (Syb2), which hampers dopamine release. Here, we show that α-Syn monomers and oligomers cooperatively inhibit neuronal SNARE-mediated vesicle fusion. α-Syn monomers at submicromolar concentrations increase the fusion inhibition by α-Syn oligomers. This cooperative pathological effect stems from the synergically enhanced vesicle clustering. Based on this cooperative inhibition mechanism, we reverse the fusion inhibitory effect of α-Syn oligomers using small peptide fragments. The small peptide fragments, derivatives of α-Syn, block the binding of α-Syn oligomers to Syb2 and dramatically reverse the toxicity of α-Syn oligomers in vesicle fusion. Our findings demonstrate a new strategy for therapeutic intervention in PD and related diseases based on this specific interaction of α-Syn.

Fibrillar α-synuclein toxicity depends on functional lysosomes.

Neurodegeneration in Parkinson's disease (PD) can be recapitulated in animals by administration of α-synuclein preformed fibrils (PFFs) into the brain. However, the mechanism by which these PFFs induce toxicity is unknown. Iron is implicated in PD pathophysiology, so we investigated whether α-synuclein PFFs induce ferroptosis, an iron-dependent cell death pathway. A range of ferroptosis inhibitors were added to a striatal neuron-derived cell line (STHdhQ7/7 cells), a dopaminergic neuron-derived cell line (SN4741 cells), and WT primary cortical neurons, all of which had been intoxicated with α-synuclein PFFs. Viability was not recovered by these inhibitors except for liproxstatin-1, a best-in-class ferroptosis inhibitor, when used at high doses. High-dose liproxstatin-1 visibly enlarged the area of a cell that contained acidic vesicles and elevated the expression of several proteins associated with the autophagy-lysosomal pathway similarly to the known lysosomal inhibitors, chloroquine and bafilomycin A1. Consistent with high-dose liproxstatin-1 protecting via a lysosomal mechanism, we further de-monstrated that loss of viability induced by α-synuclein PFFs was attenuated by chloroquine and bafilomycin A1 as well as the lysosomal cysteine protease inhibitors, leupeptin, E-64D, and Ca-074-Me, but not other autophagy or lysosomal enzyme inhibitors. We confirmed using immunofluorescence microscopy that heparin prevented uptake of α-synuclein PFFs into cells but that chloroquine did not stop α-synuclein uptake into lysosomes despite impairing lysosomal function and inhibiting α-synuclein toxicity. Together, these data suggested that α-synuclein PFFs are toxic in functional lysosomes in vitro. Therapeutic strategies that prevent α-synuclein fibril uptake into lysosomes may be of benefit in PD.

A study on the modulation of alpha-synuclein fibrillation by Scutellaria pinnatifida extracts and its neuroprotective properties.

Aggregation of alpha-synuclein (α-SN) is a key pathogenic event in Parkinson's disease (PD) leading to dopaminergic degeneration. The identification of natural compounds inhibiting α-SN aggregation may have a major role in treating PD. Different Scutellaria species are known as valuable medicinal plants, primarily due to their high flavonoid levels. Scutellaria pinnatifida (S. pinnatifida) is endemic to Iran; however, the knowledge of its pharmaceutical properties is limited. Here we report that S. pinnatifida extracts have an anti-fibrillation effect on α-SN aggregation and neuroprotective properties on PC12 and primary dopaminergic neurons. Treatment during α-SN fibril formation with S. pinnatifida extracts showed that the extractions performed with dichloromethane (DCMEx) and n-butanol (BuOHEx) strongly inhibited α-SN fibrillation. TLC-based analysis revealed that S. pinnatifida contains a great amount of flavonoids with high antioxidant properties as shown using a radical scavenging assay. Further analysis using HPLC and Mass spectroscopy on the DCMEx revealed the presence of baicalein in this extract. We then selected the more efficient extracts based on cell viability and ROS scavenging on PC12 cells and tested their neuroprotective properties on primary dopaminergic neurons. Our results showed the extracts strongly protected against α-SN oligomers. Surprisingly, they also neutralized the severe toxicity of paraquat. Therefore, S. pinnatifida may be a potential valuable medicinal herb for further studies related to the treatment of PD.

Attenuation of neuromotor deficits by natural antioxidants of Decalepis hamiltonii in transgenic Drosophila model of Parkinson's disease.

Oxidative stress is believed to be a major factor for the onset of Parkinson's disease (PD). In this study, we have investigated oxidative status in transgenic Drosophila model of PD. Our results revealed elevated levels of reactive oxygen species (ROS) and lipid peroxidation (LPO) in A30P and A53T α-synuclein PD model flies compared to control. We have demonstrated for the first time the ameliorating potential of natural antioxidants characterized from the roots of Dh in A30P and A53T α-synuclein PD model flies. Feeding of transgenic flies with aqueous Dh root extract for 21 days significantly improved their climbing ability and circadian rhythm of locomotor activity which was associated with reduction in levels of ROS and LPO and enhancement in the activities of catalase (CAT) and superoxide dismutase (SOD). Dh protected against paraquat (PQ) sensitivity in α-synuclein transgenic flies and delayed the onset of PD-like symptoms which appears to be mediated by suppression of oxidative stress.

Ginsenoside Rb1 inhibits fibrillation and toxicity of alpha-synuclein and disaggregates preformed fibrils.

Compelling evidence indicates that α-synuclein (α-syn) aggregation plays a central role in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies. Identification of compounds that inhibit or reverse the aggregation process may thus represent a viable therapeutic strategy against PD and related disorders. Ginseng is a well-known medicinal plant that has been used in East Asia for more than two thousand years to treat several conditions. It is now understood that the pharmacological properties of ginseng can be attributed to its biologically active components, the ginsenosides, which in turn have been shown to have neuroprotective properties. We therefore sought to determine for the first time, the potential of the most frequently used and studied ginsenosides, namely Rg1, Rg3 and Rb1, as anti-amyloidogenic agents. The effect of Rg1, Rg3 and Rb1 on α-syn aggregation and toxicity was determined by an array of biophysical, biochemical and cell-culture-based techniques. Among the screened ginsenosides, only Rb1 was shown to be a potent inhibitor of α-syn fibrillation and toxicity. Additionally, Rb1 exhibited a strong ability to disaggregate preformed fibrils and to inhibit the seeded polymerization of α-syn. Interestingly, Rb1 was found to stabilize soluble non-toxic oligomers with no ß-sheet content, that were susceptible to proteinase K digestion, and the binding of Rb1 to those oligomers may represent a potential mechanism of action. Thus, Rb1 could represent the starting point for designing new molecules that could be utilized as drugs for the treatment of PD and related disorders.

iTRAQ-based quantitative proteomics study on the neuroprotective effects of extract of Acanthopanax senticosus harm on SH-SY5Y cells overexpressing A53T mutant α-synuclein.

Extract of Acanthopanax senticosus harms (EAS) has been shown to have neuroprotective effects on Parkinson's disease (PD) cell model against α-synuclein overexpression and toxicity. However, studies of its anti-PD mechanism are challenging, owing to the complex pathophysiology of PD, and complexity of EAS with multiple constituents acting on different proteomic pathways. Here, we have investigated the proteomic profiles and potential biomarkers in a cell model of A53T mutant α-synuclein (A53T-α-Syn) overexpression after treatment of EAS. Using an iTRAQ (isobaric tags for relative and absolute quantitation)-based proteomics research approach, we identified 3425 modulated proteins, out of which 84 were found to be altered by A53T-α-Syn and considered as potential biomarkers. After treatment with EAS, the group showed the tendency to correct the abnormal expressions of 16 proteins out of 84 potential biomarkers, which were associated with the formation of Lewy body, mitochondrial energy metabolism, protein synthesis and apoptosis, etc. This study indicated that EAS might be a promising candidate for prevention or treatment of PD by regulating the related proteomic pathways in A53T-α-Syn transgenic SH-SY5Y cells.

N-terminal deletion does not affect α-synuclein membrane binding, self-association and toxicity in human neuroblastoma cells, unlike yeast.

α-Synuclein causes Parkinson's disease if mutated or aberrantly produced in neurons. α-Synuclein-lipid interactions are important for the normal function of the protein, but can also contribute to pathogenesis. We previously reported that deletion of the first 10 N-terminal amino acids dramatically reduced lipid binding in vitro, as well as membrane binding and toxicity in yeast. Here we extend this study to human neuroblastoma SHSY-5Y cells, and find that in these cells the first 10 N-terminal residues do not affect α-synuclein membrane binding, self-association and cell viability, contrary to yeast. Differences in lipid composition, membrane fluidity and cytosolic factors between yeast and neuronal cells may account for the distinct binding behavior of the truncated variant in these two systems. Retinoic acid promotes differentiation and α-synuclein oligomer formation in neuroblastoma cells, while addition of a proteasomal inhibitor induces neurite outgrowth and toxicity to certain wild-type and truncated α-synuclein clones. Yeast recapitulate several features of α-synuclein (patho)biology, but its simplicity sets limitations; verification of yeast results in more relevant model systems is, therefore, essential.

Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson's disease models.

alpha-Synuclein (alpha-syn) is a small lipid-binding protein involved in vesicle trafficking whose function is poorly characterized. It is of great interest to human biology and medicine because alpha-syn dysfunction is associated with several neurodegenerative disorders, including Parkinson's disease (PD). We previously created a yeast model of alpha-syn pathobiology, which established vesicle trafficking as a process that is particularly sensitive to alpha-syn expression. We also uncovered a core group of proteins with diverse activities related to alpha-syn toxicity that is conserved from yeast to mammalian neurons. Here, we report that a yeast strain expressing a somewhat higher level of alpha-syn also exhibits strong defects in mitochondrial function. Unlike our previous strain, genetic suppression of endoplasmic reticulum (ER)-to-Golgi trafficking alone does not suppress alpha-syn toxicity in this strain. In an effort to identify individual compounds that could simultaneously rescue these apparently disparate pathological effects of alpha-syn, we screened a library of 115,000 compounds. We identified a class of small molecules that reduced alpha-syn toxicity at micromolar concentrations in this higher toxicity strain. These compounds reduced the formation of alpha-syn foci, re-established ER-to-Golgi trafficking and ameliorated alpha-syn-mediated damage to mitochondria. They also corrected the toxicity of alpha-syn in nematode neurons and in primary rat neuronal midbrain cultures. Remarkably, the compounds also protected neurons against rotenone-induced toxicity, which has been used to model the mitochondrial defects associated with PD in humans. That single compounds are capable of rescuing the diverse toxicities of alpha-syn in yeast and neurons suggests that they are acting on deeply rooted biological processes that connect these toxicities and have been conserved for a billion years of eukaryotic evolution. Thus, it seems possible to develop novel therapeutic strategies to simultaneously target the multiple pathological features of PD.

Detection of compounds that rescue Rab1-synuclein toxicity.

Recent studies implicate a disruption in Rab-mediated protein trafficking as a possible contributing factor to neurodegeneration in Parkinson's disease (PD). Misfolding of the neuronal protein alpha-synuclein (asyn) is implicated in PD. Overexpression of asyn results in cell death in a wide variety of model systems, and in several organisms, including yeast, worms, flies, and rodent primary neurons, this toxicity is suppressed by the overproduction of Rab proteins. These and other findings suggest that asyn interferes with Rab function and provide new avenues for PD drug discovery. This chapter describes two assay formats that have been used successfully to identify small molecules that rescue asyn toxicity in yeast. The 96-well format monitors rescue by optical density and is suitable for screening thousands of compounds. A second format measures viable cells by reduction of the dye alamarBlue, a readout that is compatible with 96-, 384-, and 1536-well plates allowing the screening of large libraries (>100,000 compounds). A secondary assay to eliminate mechanistically undesirable hits is also described.