Inhibits Α-synuclein Aggregation Research Articles

Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation

Parkinson’s disease (PD) is one of the most prevalent forms of synucleinopathies, and it is characterized neuropathologically by the presence of intracellular inclusions composed primarily of the protein α-synuclein (α-syn) in neurons. The previous immunotherapy targeting the α-syn in PD models with monoclonal antibodies has established α-syn protein as an effective target for neuronal cell death. However, due to the essential weaknesses of antibody and the unique features of aptamers, the aptamers could represent a promising alternative to the currently used antibodies in immunotherapy for PD. In this study, the purified human α-syn was used as the target for in vitro selection of aptamers using systematic evolution by exponential enrichment. This resulted in the identification of two 58-base DNA aptamers with a high binding affinity and good specificity to the α-syn, with KD values in the nanomolar range. Both aptamers could effectively reduce α-syn aggregation in vitro and in cells and target the α-syn to intracellular degradation through the lysosomal pathway. These effects consequently rescued the mitochondrial dysfunction and cellular defects caused by α-syn overexpression. To our knowledge, this is the first study to employ aptamers to block the aberrant cellular effects of the overexpressed α-syn in cells.

LncRNA SNHG1 promotes α-synuclein aggregation and toxicity by targeting miR-15b-5p to activate SIAH1 in human neuroblastoma SH-SY5Y cells

Numerous long non-coding RNAs (lncRNAs) have been identified as aberrantly expressed in Parkinson’s disease (PD). However, limited knowledge is available concerning the roles of dysregulated lncRNAs and the underlying molecular regulatory mechanism in the pathological process of PD. In this study, we found that lncRNA small nucleolar RNA host gene 1 (SNHG1) and seven in absentia homolog 1 (SIAH1) were upregulated, but microRNA-15b-5p (miR-15b-5p) was downregulated in SH-SY5Y cells pretreated with MPP+, as well as in MPTP-induced mouse model of PD. Overexpression of SIAH1 enhanced cellular toxicity of α-synuclein in SH-SY5Y cells, as indicated by the reduction of cell viability and elevation of LDH release. The percentage of α-synuclein aggregate-positive cells and the number of α-synuclein aggregates per cell were increased in SH-SY5Y cells transfected with pcDNA-SIAH1, while decreased after transfection with short interfering RNA specific for SIAH1 (si-SIAH1). Bioinformatics and luciferase reporter assay revealed that SIAH1 was a direct target of miR-15b-5p. We also found that SNHG1 could directly bind to miR-15-5p and repress miR-15-5p expression. Upregulation of miR-15b-5p alleviated α-synuclein aggregation and apoptosis by targeting SIAH1 in SH-SY5Y cells overexpressing α-synuclein. Overexpression of SNHG1 enhanced, whereas SNHG1 knockdown inhibited α-synuclein aggregation and α-synuclein-induced apoptosis. Moreover, the neuroprotective effect of si-SNHG1 was abrogated by downregulation of miR-15b-5p. In summary, our data suggest that SNHG1, as a pathogenic factor, promotes α-synuclein aggregation and toxicity by targeting the miR-15b-5p/SIAH1 axis, contributing to a better understanding of the mechanisms of Lewy body formation and loss of dopaminergic neurons in PD.

Delivery of Native Proteins into C. elegans Using a Transduction Protocol Based on Lipid Vesicles

The nematode worm Caenorhabditis elegans (C. elegans) is a versatile and widely used animal model for in vivo studies of a broad range of human diseases, in particular for understanding their genetic origins and for screening drug candidates. Nevertheless, the challenges associated with the administration of native proteins to C. elegans have limited the range of applications of this animal model in protein-based drug discovery programs. Here, we describe a readily usable protocol for the transduction of native proteins in C. elegans, which is based on the encapsulation of the proteins of interest within cationic lipid vesicles, prior to their administration to worms. This procedure limits the degradation of the proteins in the guts of the animals, and promotes their adsorption into body tissues. To illustrate the efficacy of this approach we apply it to deliver an antibody designed to inhibit α-synuclein aggregation, and show that it can lead to the rescue of the disease phenotype in a C. elegans model of Parkinson’s disease. As this transduction protocol is fast and inexpensive, we anticipate that it will be readily applicable to protein-based drug discovery studies that utilize C. elegans as a model organism.

Curcumin affords neuroprotection and inhibits α-synuclein aggregation in lipopolysaccharide-induced Parkinson's disease model.

Parkinson's disease (PD) pathology is characterized by the abnormal accumulation and aggregation of the pre-synaptic protein α-synuclein in the dopaminergic neurons as Lewy bodies (LBs). Curcumin, which plays a neuroprotective role in various animal models of PD, was found to directly modulate the aggregation of α-synuclein in in vitro as well as in in vivo studies. While curcumin has been shown to exhibit strong anti-oxidant and anti-inflammatory properties, there are a number of other possible mechanisms by which curcumin may alter α-synuclein aggregation which still remains obscure. Therefore, the present study was designed to understand such concealed mechanisms behind neuroprotective effects of curcumin. An animal model of PD was established by injecting lipopolysaccharide (LPS, 5µg/5µl PBS) into the substantia nigra (SN) of rats which was followed by curcumin administration (40mg/kg b.wt (i.p.)) daily for a period of 21 days. Modulatory functions of curcumin were evident from the inhibition of astrocytic activation (GFAP) by immunofluorescence and NADPH oxidase complex activation by RT-PCR. Curcumin supplementation prevented the LPS-induced upregulation in the protein activity of transcription factor NFκB, proinflammatory cytokines (TNF-α, IL-1β, and IL-1α), inducible nitric oxide synthase (iNOS) as well as the regulating molecules of the intrinsic apoptotic pathway (Bax, Bcl-2, Caspase 3 and Caspase 9) by ELISA. Curcumin also resulted in significant improvement in the glutathione system (GSH, GSSG and redox ratio) and prevented iron deposition in the dopaminergic neurons as depicted from atomic absorption spectroscopy (AAS) and Prussian blue staining, respectively. Curcumin also prevented α-synuclein aggregates in the dopaminergic neurons as observed from gene as well as protein activity of α-synuclein using RT-PCR and IHC. Collectively, our results suggest that curcumin can be further pursued as a candidate drug in the molecules targeted therapy for PD and other related synucleopathies.

DJ-1 Inhibits α-Synuclein Aggregation by Regulating Chaperone-Mediated Autophagy.

α-Synuclein misfolding and aggregation play an important role in the pathogenesis of Parkinson’s disease (PD). Loss of function and mutation of the PARK7/DJ-1 gene cause early-onset familial PD. DJ-1 can inhibit α-synuclein aggregation, and may function at an early step in the aggregation process. Soluble wild-type (WT) α-synuclein is mainly degraded by chaperone-mediated autophagy (CMA), and impairment of CMA is closely related to the pathogenesis of PD. Here, we investigated whether DJ-1 could reduce α-synuclein accumulation and aggregation by CMA. DJ-1 knockout mice and DJ-1 siRNA knockdown SH-SY5Y cells were used to investigate the potential mechanisms underlying the relationship between DJ-1 deficiency and α-synuclein aggregation. First, we confirmed that DJ-1 deficiency increased the accumulation and aggregation of α-synuclein in both SH-SY5Y cells and PD animal models, and overexpression of DJ-1 in vitro effectively decreased α-synuclein levels. α-Synuclein overexpression activated CMA by elevating the levels of lysosome-associated membrane protein type-2A (LAMP2A), but DJ-1 deficiency suppressed upregulation of LAMP2A. DJ-1 deficiency downregulated the level of lysosomal 70 kDa heat-shock cognate protein (HSC70) but not the levels of that in homogenates. Further studies showed that DJ-1 deficiency accelerated the degradation of LAMP2A in lysosomes, leading to the aggregation of α-synuclein. Our study suggests that DJ-1 deficiency aggravates α-synuclein aggregation by inhibiting the activation of CMA and provides further evidence of the molecular interaction between PD-related proteins via the CMA pathway.

Diarylheptanoids from Rhizomes of Alpinia officinarum Inhibit Aggregation of α-Synuclein.

Two new diarylheptanoids, alpinin A (1) and alpinin B (2), together with 18 known diarylheptanoids (3-20), were isolated from the rhizomes of Alpinia officinarum. Their structures were elucidated by comprehensive spectroscopic analysis, including high-resolution mass spectrometry, infrared spectroscopy, and one- and two-dimensional nuclear magnetic resonance spectroscopy. Structurally, alpinin A is a new member of the small family of oxa-bridged diarylheptanoids and contains the characteristic 2,6-cis-configured tetrahydropyran motif (C1-C5 oxa bridge). The absolute configuration of alpinin A was confirmed by asymmetric total synthesis of the enantiomer (ent-1), corroborating the assignment of the molecular structure. The absolute configuration of alpinin B was determined on the basis of the analysis of the circular dichroism exciton chirality spectrum. We evaluated the inhibitory activity of all isolated diarylheptanoids against α-synuclein aggregation at 10 μM. Alpinins A and B significantly inhibited α-synuclein aggregation by 66 and 67%, respectively.

Enzymatic O-GlcNAcylation of α-synuclein reduces aggregation and increases SDS-resistant soluble oligomers.

Neurodegenerative diseases including dementia with Lewy bodies, Lewy body variant of Alzheimer's disease, and Parkinson's disease are associated with the aberrant aggregation of α-synuclein, which is influenced by several post-translational modifications (PTMs). O-GlcNAcylation is one PTM that has an important role in many fundamental processes. The O-GlcNAcylation of endogenous α-synuclein at residues 53, 64, 72 and 87 has been reported in an unbiased mass spectrum analysis. The consequences of O-GlcNAcylation at residues 72 or 87 have been studied by using a synthetic α-synuclein bearing O-GlcNAcylation at threonine residue 72 or serine 87, respectively. O-GlcNAcylation at Thr72 or Ser87 suppresses the aggregation of α-synuclein. However, the effect of enzymatic O-GlcNAcylation of α-synuclein at multiple residues is not clear. Here, we successfully generated O-GlcNAcylated α-synuclein by co-expressing a shorter form of OGT (sOGT) with α-synuclein. The O-GlcNAcylation inhibited α-synuclein aggregation and promoted the formation of soluble SDS-resistant and Thioflavine T negative oligomers. Our data warrant further studies on the role of O-GlcNAcylation in the progression/treatment of Parkinson's disease in animal models.

Inhibitory Role of β-Casein on the α-Synuclein Aggregation Associated with Parkinson’s Disease In Vitro

Large soluble oligomeric species are observed as probable intermediates during fibril formation in aggregations of Parkinson’s disease (PD). Fibrillar deposits occur in PD. Amyloid forms α-Synuclein is one of the main compounds aggregations. β-Casein, a member of the Casein family, has been demonstrated to inhibit α-Synuclein aggregation by chaperone-like activity. In this study, we investigated the effect of chaperone activity of β-Casein in preventing the aggregation of α-Synuclein protein. We have examined the effect of β-Casein in preventing α-Synuclein aggregation by using from Thioflavin T-binding assay, transmission electron microscopy, ANS-binding assay, circular dichroism spectroscopy and FTIR spectroscopy. Results from the ThT binding assay demonstrated an increase in the ThT fluorescence intensity of α-Synuclein incubated in absence of β-Casein but in its presence fluorescence intensity is decreased. Electron microscopy data also indicated that β-Casein decreased the aggregation content of α-Synuclein. ANS results also showed that β-Casein significantly decreased the the hydrophobic area in α-Synuclein incubated. Circular dichroism spectroscopy (CD) results also showed that β-sheet structures of α-Synuclein incubated change to structural α-helical and β-turn in presence of β-Casein. FTIR spectroscopy indicates the presence of β-sheet structures in α-Synuclein incubated in absence of β-Casein and β-sheet structures decreased in its presence. Thus, our results suggest that in vitro, β-Casein interacts with α-Synuclein fibrils, changes the α-Synuclein structure and prevents amyloid fibril formation. This means that β-Casein could be essential for therapies inhibiting aggregation and to be an important therapeutic drug against PD.

O-GlcNAc modification inhibits the calpain-mediated cleavage of α-synuclein

The major protein associated with Parkinson’s disease (PD) is α-synuclein, as it can form toxic amyloid-aggregates that are a hallmark of many neurodegenerative diseases. α-Synuclein is a substrate for several different posttranslational modifications (PTMs) that have the potential to affect its biological functions and/or aggregation. However, the biophysical effects of many of these modifications remain to be established. One such modification is the addition of the monosaccharide N-acetyl-glucosamine, O-GlcNAc, which has been found on several α-synuclein serine and threonine residues in vivo. We have previously used synthetic protein chemistry to generate α-synuclein bearing two of these physiologically relevant O-GlcNAcylation events at threonine 72 and serine 87 and demonstrated that both of these modifications inhibit α-synuclein aggregation. Here, we use the same synthetic protein methodology to demonstrate that these same O-GlcNAc modifications also inhibit the cleavage of α-synuclein by the protease calpain. This further supports a role for O-GlcNAcylation in the modulation of α-synuclein biology, as proteolysis has been shown to potentially affect both protein aggregation and degradation.

O-GlcNAcylation of α-Synuclein at Serine 87 Reduces Aggregation without Affecting Membrane Binding.

The aggregation of neurodegenerative-disease associated proteins can be affected by many factors, including a variety of post-translational modifications. One such modification, O-GlcNAcylation, has been found on some of these aggregation prone proteins, including α-synuclein, the major protein that plays a causative role in synucleinopathies like Parkinson's disease. We previously used synthetic protein chemistry to prepare α-synuclein bearing a homogeneous O-GlcNAc modification at threonine 72 and showed that this modification inhibits protein aggregation. However, the effects of the other eight O-GlcNAcylation sites that have been identified were unknown. Here, we use a similar synthetic strategy to investigate the consequences of this modification at one of these sites, serine 87. We show that O-GlcNAcylation at this site also inhibits α-synuclein aggregation but to a lesser extent than that for the same modification at threonine 72. However, we also find that this modification does not affect the membrane-binding properties of α-synuclein, which differentiates it from phosphorylation at the same site. These results further support the development of therapies that can elevate O-GlcNAcylation of α-synuclein to slow the progression of Parkinson's disease.

Tetrahydroxystilbene glucoside inhibits α-synuclein aggregation and apoptosis in A53T α-synuclein-transfected cells exposed to MPP.

Increasing evidence has solidified the involvement of α-synuclein (α-Syn) and neurotoxins in the pathogenesis of Parkinson's disease (PD), suggesting a combination of genetic and environmental influences. 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside (TSG) is one of the main active components extracted from Polygonum multiflorum. The purpose of the present study was to investigate the effects of TSG on α-Syn aggregation, mitochondrial dysfunction, oxidative stress, and apoptosis in vitro. A53T mutant α-synuclein-transfected cells (A53T AS cells) plus MPP+ exposure were used as a complex cell model of PD. The expression of proteins was determined by Western blot assay. Flow cytometry was utilized to measure mitochondrial membrane potential and apoptosis. The results showed that MPP+ exposure for 24 h induced more severe damage in A53T AS cells than in vector control cells. Pretreatment of TSG for 24 h significantly increased the cell viability; decreased lactate dehydrogenase leakage; inhibited α-Syn over-expression and aggregation; elevated mitochondrial membrane potential; decreased reactive oxygen species, Bax/Bcl-2 ratio, and caspase-3 activity; and inhibited apoptosis in A53T AS cells exposed to MPP+. These results suggest that TSG may be an attractive candidate for PD therapy.

β-Synuclein suppresses both the initiation and amplification steps of α-synuclein aggregation via competitive binding to surfaces.

α-Synuclein is an intrinsically disordered protein that is associated with the pathogenesis of Parkinson’s disease through the processes involved in the formation of amyloid fibrils. α and β-synuclein are homologous proteins found at comparable levels in presynaptic terminals but β-synuclein has a greatly reduced propensity to aggregate and indeed has been found to inhibit α-synuclein aggregation. In this paper, we describe how sequence differences between α- and β-synuclein affect individual microscopic processes in amyloid formation. In particular, we show that β-synuclein strongly suppresses both lipid-induced aggregation and secondary nucleation of α-synuclein by competing for binding sites at the surfaces of lipid vesicles and fibrils, respectively. These results suggest that β-synuclein can act as a natural inhibitor of α-synuclein aggregation by reducing both the initiation of its self-assembly and the proliferation of its aggregates.

Baicalein attenuates α-synuclein aggregation, inflammasome activation and autophagy in the MPP+-treated nigrostriatal dopaminergic system in vivo

Ethnopharmacological relevanceNeuroinflammation, oxidative stress, and protein aggregation form a vicious cycle in the pathophysiology of Parkinson's disease (PD); activated microglia is the main location of neuroinflammation. A Chinese medicine book, “Shanghan Lun”, known as the “Treatises on Cold damage Diseases” has suggested that Scutellaria baicalensis Georgi is effective in treating CNS diseases. The anti-inflammatory mechanisms of baicalein, a phenolic flavonoid in the dried root of Scutellaria baicalensis Georgi, remain to be explored. Aim of the studyThe neuroprotective mechanisms of baicalein involving α-synuclein aggregation, inflammasome activation, and programmed cell death were investigated in the nigrostriatal dopaminergic system of rat brain in vivo. Materials and methodsIntranigral infusion of 1-methyl-4-phenylpyridinium (MPP+, a Parkinsonian neurotoxin) was performed on anesthetized Sprague-Dawley rats. Baicalein was daily administered via intraperitoneal injection. Striatal dopamine levels were measured using high performance liquid chromatography coupled with electrochemical detection. Cellular signalings were measured by Western blot assay, immunofluorescent staining assay and enzyme-linked immunosorbent assay. ResultsSystemic administration of baicalein attenuated MPP+-induced reductions in striatal dopamine content and tyrosine hydroxylase (a biomarker of dopaminergic neurons) in the infused substantia nigra (SN). Furthermore, MPP+-induced elevations in α-synuclein aggregates (a pathological hallmark of PD), ED-1 (a biomarker of activated microglia), activated caspase-1 (a proinflammatory caspase), IL-1β and cathepsin B (a cysteine lysosomal protease) in the infused SN were attenuated in the baicalein-treated rats. Moreover, intense immunoreactivities of caspase 1 and cathepsin B were co-localized with that of ED-1 in the MPP+-infused SN. At the same time, baicalein inhibited MPP+-induced increases in active caspases 9 and 12 (biomarkers of apoptosis) as well as LC3-II levels (a biomarker of autophagy) in the rat nigrostriatal dopaminergic system. ConclusionOur in vivo study showed that baicalein possesses anti-inflammatory activities by inhibiting α-synuclein aggregation, inflammasome activation and cathepsin B production in the MPP+-infused SN. Moreover, baicalein is of therapeutic significance because it inhibits MPP+-induced apoptosis and autophagy in the nigrostriatal dopaminergic system of rat brain.

Erratum to: The PROMESA-protocol: progression rate of multiple system atrophy under EGCG supplementation as anti-aggregation-approach.

Formation of toxic α-synuclein oligomers appears to be a key underlying pathological mechanism of synucleinopathies such as Parkinson's disease or multiple system atrophy (MSA). Given that Epigallocatechin-gallate has been shown to inhibit α-synuclein aggregation, it might represent a causal treatment option. Therefore, we set out to evaluate the safety, tolerability and a potential disease-modifying effect of Epigallocatechin-gallate in patients with MSA after 48 weeks of treatment. Power calculation was performed on existing natural history data on the progression of the Unified MSA Rating Scale as primary readout parameter. To assess the efficacy of Epigallocatechin-gallate versus placebo regarding the reduction of disease progression measured during the study period (80 % power, 5 % p level, 50 % effect size) 36 patients per group are needed. Considering a drop-out rate of 20 % a total of 86 patients will be recruited in this multicentre study. These data provide a solid rationale to investigate whether supplementation of Epigallocatechin-gallate can delay the progression of the MSA-related disability.

Intracellular formation of α-synuclein oligomers and the effect of heat shock protein 70 characterized by confocal single particle spectroscopy

Synucleinopathies such as dementia with Lewy bodies or Parkinson’s disease are characterized by intracellular deposition of pathologically aggregated α-synuclein. The details of the molecular pathogenesis of PD and especially the conditions that lead to intracellular aggregation of α-synuclein and the role of these aggregates in cell death remain unknown. In cell free in vitro systems considerable knowledge about the aggregation processes has been gathered. In comparison, the knowledge about these aggregation processes in cells is far behind. In cells α-synuclein aggregates can be toxic. However, the crucial particle species responsible for decisive steps in pathogenesis such as seeding a continuing aggregation process and triggering cell death remain to be identified. In order to understand the complex nature of intracellular α-synuclein aggregate formation, we analyzed fluorescent particles formed by venus and α-synuclein-venus fusion proteins and α-synuclein-hemi-venus fusion proteins derived from gently lyzed cells. With these techniques we were able to identify and characterize α-synuclein oligomers formed in cells. Especially the use of α-synuclein-hemi-venus fusion proteins enabled us to identify very small α-synuclein oligomers with high sensitivity. Furthermore, we were able to study the molecular effect of heat shock protein 70, which is known to inhibit α-synuclein aggregation in cells. Heat shock protein 70 does not only influence the size of α-synuclein oligomers, but also their quantity. In summary, this approach based on fluorescence single particle spectroscopy, that is suited for high throughput measurements, can be used to detect and characterize intracellularly formed α-synuclein aggregates and characterize the effect of molecules that interfere with α-synuclein aggregate formation.

Interactions of GFAP with ceftriaxone and phenytoin: SRCD and molecular docking and dynamic simulation

BackgroundGFAP is the major intermediate filament protein in mature astrocytes. Its increased expression and aggregation was firstly associated to Alexander's disease, and successively in different neurological diseases including scrapie, Alzheimer's and Creutzfeld–Jacob diseases. Recently, ceftriaxone a multi-potent β-lactam antibiotic able to overcome the blood–brain barrier, successfully eliminated the cellular toxic effects of misfolded mutated GFAP, similarly to phenytoin sodium, in a cellular model of Alexander's disease and inhibited α-synuclein aggregation protecting PC12 cells from the exposure to 6-hydroxydopamine. MethodsIn this study, synchrotron radiation circular dichroism spectroscopy has been used to obtain structural information about the GFAP-ceftriaxone (phenytoin) interactions, while computational methods allowed the identification of the relevant putative binding site of either ceftriaxone or phenytoin on the dimer structure of GFAP, permitting to rationalize the spectroscopic experimental results. ResultsWe found that GFAP exhibited enhanced stability upon the addition of two equivalents of each ligands with ceftriaxone imparting a more spontaneous interactions and a more ordered complex system than phenytoin. ConclusionsSRCD data and MD models indicate a stronger protective effect of ceftriaxone in neurological disorders characterized by an increased production and polymerization of GFAP. General significanceThis result, in addition to our previous works in which we documented that ceftriaxone interacts with α-synuclein inhibiting its pathological aggregation and that a cyclical treatment with this molecule in a patient with adult-onset Alexander's disease halted, and partly reversed, the progression of neurodegeneration, suggests the possibility of a chaperone-like effect of ceftriaxone on protein involved in specific neurodegenerative diseases.

The role of NUB1 in α-synuclein degradation in Lewy body disease model mice

Abnormal α-synuclein is deposited in neuronal cytoplasmic inclusions and presynapses in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Previously we have shown that NUB1 is accumulated in these specific regions together with abnormal α-synuclein and that NUB1 is able to inhibit α-synuclein aggregation in cultured cells. We therefore created transgenic (Tg) mice expressing both NUB1 and abnormal α-synuclein to investigate the role of NUB1 on degradation of abnormal α-synuclein in vivo. Immunohistochemical and biochemical studies confirmed that NUB1 was over-expressed in neurons of mice expressing NUB1 (NUB1 Tg), and both NUB1 and abnormal α-synuclein (double Tg). NUB1 levels were increased by 4.7-fold in NUB1 Tg mice compared with wild type mice. Unexpectedly, normal and abnormal α-synuclein levels were unchanged between abnormal α-synuclein Tg mice (Lewy body disease model mice) and double Tg mice, and pathological observations were almost similar between them. Finally, we found that the levels of insoluble α-synuclein were lower and those of some chaperone molecules were higher in double Tg mice compared with abnormal α-synuclein Tg mice. These results suggest that increased levels of NUB1 play a potential role in degradation of detergent-insoluble α-synuclein in vivo, although it is insufficient to degrade abnormal α-synuclein in Lewy body disease model mice.

Isoliquiritigenin and liquiritin from Glycyrrhiza uralensis inhibit α-synuclein amyloid formation

Bioactive isoliquiritigenin and liquiritin inhibit α-synuclein aggregation and extend the life span of theC. elegansmodel of Parkinson's disease.

Tetrahydroxystilbene glucoside ameliorates memory and movement functions, protects synapses and inhibits α-synuclein aggregation in hippocampus and striatum in aged mice.

To investigate the effects of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) on the memory and movement functions and its mechanisms related to synapses and α-synuclein in aged mice. The memory ability of mice was detected by step-through passive avoidance task. The movement function was measured by the pole test and rotarod test. Transmission electron microscopy was used to observe the synaptic ultrastructure. Western blotting was applied to measure the expression of synapse-related proteins and α-synuclein. Intragastrical administration of TSG for 3 months significantly improved the memory and movement functions in aged mice. TSG treatment obviously protected the synaptic ultrastructure and increased the number of synaptic connections in the hippocampal CA1 region and striatum; enhanced the expression of synaptophysin, phosphorylated synapsin I and postsynaptic density protein 95 (PSD95), elevated phosphorylated calcium/calmodulin-dependent protein kinase II (p-CaMKII) expression, and inhibited the overexpression and aggregation of α-synuclein in the hippocampus, striatum and cerebral cortex of aged mice. TSG improved the memory and movement functions in aged mice through protecting synapses and inhibiting α-synuclein overexpression and aggregation in multiple brain regions. The results suggest that TSG may be beneficial to the treatment of ageing-related neurodegenerative diseases.

Definition of a molecular pathway mediating α-synuclein neurotoxicity.

α-Synuclein physiologically chaperones SNARE-complex assembly at the synapse but pathologically misfolds into neurotoxic aggregates that are characteristic for neurodegenerative disorders, such as Parkinson's disease, and that may spread from one neuron to the next throughout the brain during Parkinson's disease pathogenesis. In normal nerve terminals, α-synuclein is present in an equilibrium between a cytosolic form that is natively unfolded and monomeric and a membrane-bound form that is composed of an α-helical multimeric species that chaperones SNARE-complex assembly. Although the neurotoxicity of α-synuclein is well established, the relationship between the native conformations of α-synuclein and its pathological aggregation remain incompletely understood; most importantly, it is unclear whether α-synuclein aggregation originates from its monomeric cytosolic or oligomeric membrane-bound form. Here, we address this question by introducing into α-synuclein point mutations that block membrane binding and by then assessing the effect of blocking membrane binding on α-synuclein aggregation and neurotoxicity. We show that membrane binding inhibits α-synuclein aggregation; conversely, blocking membrane binding enhances α-synuclein aggregation. Stereotactic viral expression of wild-type and mutant α-synuclein in the substantia nigra of mice demonstrated that blocking α-synuclein membrane binding significantly enhanced its neurotoxicity in vivo. Our data delineate a folding pathway for α-synuclein that ranges from a physiological multimeric, α-helical, and membrane-bound species that acts as a SNARE-complex chaperone over a monomeric, natively unfolded form to an amyloid-like aggregate that is neurotoxic in vivo.