Α-synuclein Protein Expression Research Articles

MiR-101a-3p/ROCK2 axis regulates neuronal injury in Parkinson's disease models.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic neurons in substantia nigra pars compacta (SNpc). This study focuses on deciphering the role of microRNA (miR)-101a-3p in the neuronal injury of PD and its regulatory mechanism. We constructed a mouse model of PD by intraperitoneal injection of 1-methyl 4-phenyl 1, 2, 3, 6-tetrahydropyridine hydrochloride (MPTP), and used 1-methyl-4-phenylpyridinium (MPP+) to treat Neuro-2a cells to construct an in-vitro PD model. Neurological dysfunction in mice was evaluated by swimming test and traction test. qRT-PCR was utilized to examine miR-101a-3p expression and ROCK2 expression in mouse brain tissues and Neuro-2a cells. Western blot was conducted to detect the expression of α-synuclein protein and ROCK2 in mouse brain tissues and Neuro-2a cells. The targeting relationship between miR-101a-3p and ROCK2 was determined by dual-luciferase reporter gene assay. The apoptosis of neuro-2a cells was assessed by flow cytometry. Low miR-101a-3p expression and high ROCK2 expression were found in the brain tissues of PD mice and MPP+-treated Neuro-2a cells; PD mice showed decreased neurological disorders, and apoptosis of Neuro-2a cells was increased after MPP+ treatment, both of which were accompanied by increased accumulation of α-synuclein protein. After miR-101a-3p was overexpressed, the neurological function of PD mice was improved, and the apoptosis of Neuro-2a cells induced by MPP+ was alleviated, and the accumulation of α-synuclein protein was reduced; ROCK2 overexpression counteracted the protective effect of miR-101a-3p. Additionally, ROCK2 was identified as the direct target of miR-101a-3p. MiR-101a-3p can reduce neuronal apoptosis and neurological deficit in PD mice by inhibiting ROCK2 expression, suggesting that miR-101a-3p is a promising therapeutic target for PD.

CRISPR-Cas9-Edited SNCA Knockout Human Induced Pluripotent Stem Cell-Derived Dopaminergic Neurons and Their Vulnerability to Neurotoxicity.

Parkinson's disease (PD) is an age-related disorder with selective dopaminergic (DA) neuronal degeneration in the substantia nigra pars compacta. The presence of mainly α-synuclein-composed Lewy bodies in DA neurons is among the disease hallmarks in the brain of patients with PD. Human induced pluripotent stem cells (hiPSCs) are powerful tools to investigate PD pathophysiology and understand its molecular and cellular mechanisms better. In this study, we generated an α-synuclein-null hiPSC line introducing a nonsense mutation in the α-synuclein-encoding SNCA alleles using clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9)-mediated gene editing. Our Western blotting analysis revealed the lack of α-synuclein protein expression in SNCA knockout hiPSC-derived cells. In addition, SNCA knockout hiPSCs retained healthy cell morphology, undifferentiated marker gene (e.g., NANOG, POU5F1, and SOX2) expression, and differentiation ability (based on the marker gene expression levels of the three germ layers). Finally, SNCA knockout hiPSC-derived DA neurons exhibited reduced vulnerability to the DA neurotoxin, 1-methyl-4-phenylpyridinium. In conclusion, the SNCA knockout hiPSC line we generated would provide a useful experimental tool for studying the physiological and pathological role of α-synuclein in PD.

Hydroxychloroquine attenuated motor impairment and oxidative stress in a rat 6-hydroxydopamine model of Parkinson’s disease

Purpose Parkinson’s disease (PD) is associated with the destruction of dopaminergic neurons in the substantia nigra (SN). Hydroxychloroquine (HCQ) has the capability to cross the blood–brain barrier and promote a neuroprotective potential. This study evaluated the effects of HCQ on the 6-hydroxydopamine (6-OHDA)-induced PD model in rats. Methods Wistar rats were randomly divided into sham, PD, PD + levodopa and PD + HCQ groups. The PD model was induced by a stereotactic administration of 6-OHDA into the left SN pars compacta (SNpc) and confirmed by rotation and the Murprogo’s tests. HCQ (100 mg/kg, p.o.) and levodopa (12 mg/kg, p.o.) were administered once a day for 21 days. Three weeks after surgery, the behavioral tests were performed. Brain lipid peroxidation index (MDA), glutathione peroxidase activity (GPx), total antioxidant capacity (TAC) levels and α-synuclein protein expression in the SN were also measured. Results The behavioral tests demonstrated that induction of PD increased the muscle rigidity and the number of rotations, which were reversed by HCQ treatment. Also, induction of PD was associated with an increase in α-synuclein protein levels and MDA and decreased TAC levels and GPx activity. However, HCQ decreased α-synuclein and MDA levels while increased TAC levels and GPx activity. In addition, histopathological data showed that HCQ protects dopaminergic neurons against 6-OHDA-induced toxicity. Conclusion According to the results, HCQ has a beneficial effect in improving PD-related pathophysiology, in part, by mitigating oxidative stress and protecting the dopaminergic neurons in the SN.

Administration of quercetin improves mitochondria quality control and protects the neurons in 6-OHDA-lesioned Parkinson's disease models.

Mounting evidence suggests that mitochondrial dysfunction and impaired mitophagy lead to Parkinson’s disease (PD). Quercetin, one of the most abundant polyphenolic flavonoids, displays many health-promoting biological effects in many diseases. We explored the neuroprotective effect of quercetin in vivo in the 6-hydroxydopamine (6-OHDA)-lesioned rat model of PD and in vitro in 6-OHDA-treated PC12 cells. In vitro, we found that quercetin (20 μM) treatment improved mitochondrial quality control, reduced oxidative stress, increased the levels of the mitophagy markers PINK1 and Parkin and decreased α-synuclein protein expression in 6-OHDA-treated PC12 cells. Moreover, our in vivo findings demonstrated that administration of quercetin also relieved 6-OHDA-induced progressive PD-like motor behaviors, mitigated neuronal death and reduced mitochondrial damage and α-synuclein accumulation in PD rats. Furthermore, the neuroprotective effect of quercetin was suppressed by knockdown of either Pink1 or Parkin.

Velvet Antler Methanol Extracts Ameliorate Parkinson's Disease by Inhibiting Oxidative Stress and Neuroinflammation: From C. elegans to Mice.

Velvet antler is the traditional tonic food or medicine used in East Asia for treating aging-related diseases. Herein, we try to dissect the pharmacology of methanol extracts (MEs) of velvet antler on Parkinson's disease (PD). Caenorhabditis elegans studies showed that MEs decreased the aggregation of α-synuclein and protected oxidative stress-induced DAergic neuron degeneration. In vitro cellular data indicated that MEs suppressed the LPS-induced MAPKs and NF-κB activation, therefore inhibiting overproduction of reactive oxygen species, nitric oxide, tumor necrosis factor-α, and interleukin-6; blocking microglia activation; and protecting DAergic neurons from the microglia-mediated neurotoxicity. In vivo MPTP-induced PD mouse investigations found that MEs prevented MPTP-induced neuron loss in the substantia nigra and improved the behavioral rotating rod performance in MPTP-treated mice by increasing the expression level of tyrosine hydroxylase (TH) and downregulating α-synuclein protein expression. In all, these results demonstrate that MEs ameliorate PD by inhibiting oxidative stress and neuroinflammation.

Isolongifolene mitigates rotenone-induced dopamine depletion and motor deficits through anti-oxidative and anti-apoptotic effects in a rat model of Parkinson's disease

Isolongifolene (ILF), a novel tricyclic sesquiterpene compound isolated from the Indian herb Murraya koenigii (M. koenigii), has been previously demonstrated to have a neuroprotective effect against rotenone-induced oxidative stress, mitochondrial dysfunction, and apoptosis in in vitro model. However, these neuroprotective and anti-apoptotic effects of ILF are not well understood and must be further investigated to elucidate the underlying molecular mechanism of ILF in animal experiments. The objective of this study was to evaluate the neuroprotective effect of ILF on motor impediments, neurochemical variables, anti-oxidative indices, and apoptotic protein expression in a rotenone-induced rat model of Parkinson's disease (PD). PD was induced in male albino Wistar rats via injection of 2.5 mg/kg rotenone for 4 weeks. Rotenone produces PD-like effects by promoting mitochondrial complex I inhibition and microglial activation properties. The protective effect of three different doses of ILF 5, 10 and 20 mg/kg were evaluated for spontaneous locomotion, rotarod performance, and striatal dopamine (DA) content. The results showed that ILF dose-dependently ameliorated the rotenone-induced striatal DA loss and motor impairment from 10 mg/kg. Therefore, we selected 10 mg/kg as the ILF dose for further investigation. Chronic administration of rotenone caused PD-related pathological processes like oxidative stress, and produced a significant decrease in tyrosine hydroxylase (TH), DA transporter (DAT), Vesicular monoamine transporter 2 (VMAT2), and a significant upregulated in α-synuclein and apoptotic protein expression of Bax, Cyt-C and caspases -3, -8 and −9 as well as by decreasing Bcl2 expression. Treatment with ILF 10 mg/kg mitigated oxidative stress in rotenone-treated rats. Furthermore, ILF dramatically alleviated rotenone-induced toxicity and cell death by increasing TH, DAT and VMAT2 expression and reducing the upregulation of α-synuclein, Bax, Cyt-C, caspases -3, -8 and -9. Together, our results confirm that ILF’s protective effect against rotenone-induced PD is mediated through anti-oxidant and anti-apoptotic properties. However, further in-depth investigations on ILF’s anti-inflammatory and mitochondrial protective abilities are needed to establish ILF as a potential drug candidate for the treatment of Parkinson’s disease.

운동 형태에 따른 알츠하이머질환과 봉입체근염의 아밀로이드 베타와 알파시뉴클린 단백질 수준에 미치는 영향

운동 형태에 따른 알츠하이머질환과 봉입체근염의 아밀로이드 베타와 알파시뉴클린 단백질 수준에 미치는 영향

Translation of the intrinsically disordered protein α-synuclein is inhibited by a small molecule targeting its structured mRNA

Many proteins are refractory to targeting because they lack small-molecule binding pockets. An alternative to drugging these proteins directly is to target the messenger (m)RNA that encodes them, thereby reducing protein levels. We describe such an approach for the difficult-to-target protein α-synuclein encoded by the SNCA gene. Multiplication of the SNCA gene locus causes dominantly inherited Parkinson's disease (PD), and α-synuclein protein aggregates in Lewy bodies and Lewy neurites in sporadic PD. Thus, reducing the expression of α-synuclein protein is expected to have therapeutic value. Fortuitously, the SNCA mRNA has a structured iron-responsive element (IRE) in its 5' untranslated region (5' UTR) that controls its translation. Using sequence-based design, we discovered small molecules that target the IRE structure and inhibit SNCA translation in cells, the most potent of which is named Synucleozid. Both in vitro and cellular profiling studies showed Synucleozid directly targets the α-synuclein mRNA 5' UTR at the designed site. Mechanistic studies revealed that Synucleozid reduces α-synuclein protein levels by decreasing the amount of SNCA mRNA loaded into polysomes, mechanistically providing a cytoprotective effect in cells. Proteome- and transcriptome-wide studies showed that the compound's selectivity makes Synucleozid suitable for further development. Importantly, transcriptome-wide analysis of mRNAs that encode intrinsically disordered proteins revealed that each has structured regions that could be targeted with small molecules. These findings demonstrate the potential for targeting undruggable proteins at the level of their coding mRNAs. This approach, as applied to SNCA, is a promising disease-modifying therapeutic strategy for PD and other α-synucleinopathies.

Loss of MINAR2 impairs motor function and causes Parkinson's disease-like symptoms in mice.

Parkinson’s disease is the second most common human neurodegenerative disease. Motor control impairment represents a key clinical hallmark and primary clinical symptom of the disease, which is further characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of α-synuclein aggregations. We have identified major intrinsically disordered NOTCH2-associated receptor 2 encoded by KIAA1024L, a previously uncharacterized protein that is highly conserved in humans and other species. In this study, we demonstrate that major intrinsically disordered NOTCH2-associated receptor 2 expression is significantly down-regulated in the frontal lobe brain of patients with Lewy body dementia. Major intrinsically disordered NOTCH2-associated receptor 2 is predominantly expressed in brain tissue and is particularly prominent in the midbrain. Major intrinsically disordered NOTCH2-associated receptor 2 interacts with neurogenic locus notch homologue protein 2 and is localized at the endoplasmic reticulum compartments. We generated major intrinsically disordered NOTCH2-associated receptor 2 knockout mouse and demonstrated that the loss of major intrinsically disordered NOTCH2-associated receptor 2 in mouse results in severe motor deficits such as rigidity and bradykinesia, gait abnormalities, reduced spontaneous locomotor and exploratory behaviour, symptoms that are highly similar to those observed in human Parkinson’s spectrum disorders. Analysis of the major intrinsically disordered NOTCH2-associated receptor 2 knockout mice brain revealed significant anomalies in neuronal function and appearance including the loss of tyrosine hydroxylase-positive neurons in the pars compacta, which was accompanied by an up-regulation in α-synuclein protein expression. Taken together, these data demonstrate a previously unknown function for major intrinsically disordered NOTCH2-associated receptor 2 in the pathogenesis of Parkinson’s spectrum disorders.

MicroRNA Expression Profiling Screen miR-3557/324-Targeted CaMK/mTOR in the Rat Striatum of Parkinson's Disease in Regular Aerobic Exercise.

This study aimed to screen the target miRNAs and to investigate the differential miR-3557/324-targeted signal mechanisms in the rats' model of Parkinson's disease (PD) with regular aerobic exercise. Rats were divided into sedentary control PD group (SED-PD, n = 18) and aerobic exercise PD group (EX-PD, n = 22). After 8 weeks of regular aerobic exercise, a 6-hydroxydopamine- (6-OHDA-) induced PD lesion model was constructed. Preregular aerobic exercises enhanced the injury resistance of rats with 6-OHDA-induced PD. The rotational behavior after injection of apomorphine hydrochloride was alleviated. Under the scanning electron microscopy, we found the neurons, axons, and villi of the striatum were clearly and tightly arranged, and neurons and axons significantly becoming larger. Tyrosine hydroxylase (TH) was increased significantly and α-synuclein protein expression was reduced in the EX-PD group compared to the SED-PD group. Screening from miRNA microarray chip, we further found upregulation of miR-3557 and downregulation of miR-324 were closely related to the calcium-modulating signaling pathway, remitting the progress of Parkinson's disease on aerobic exercise. Compared to the SED-PD group, Ca2+/calmodulin dependent protein kinase II (CaMK2α) was upregulated, but CaMKV and voltage-dependent anion-selective channel protein 1 (Vdac1) were significantly downregulated in the EX-PD group. Additionally, phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) expression were activated, and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) expression was upregulated in the EX-PD group. Conclusions: the adaptive mechanism of regular aerobic exercise delaying neurodegenerative diseases and lesions was that miR-3557/324 was activated to regulate one of its targets CaMKs signaling pathways. CaMKs, coordinated with mTOR pathway-related gene expression, improved UCH-L1 level to favor for delaying neurodegeneration or improving the pathogenesis of PD lesions.

Regulator of G protein Signaling 6 (RGS6) Suppresses Late‐age‐onset Nigral Dopaminergic Neurodegeneration, α‐Synuclein Accumulation and Motor Dysfunction

Parkinson's disease (PD) is devastating, primarily non‐familial, age‐related neurodegenerative disorder caused by the progressive loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Why these neurons die with aging in sporadic PD is unknown. However, accumulation of α‐synuclein aggregates in Lewy bodies is believed to play a crucial role in PD pathogenesis. In support of this hypothesis, cAMP‐elevating β‐agonists dramatically reduce both α‐synuclein expression and the incidence of human PD as well as repress MPTP‐induced SNc DA neuron loss in mice (Mittal et al., Science 2017). Neuronal cAMP levels are controlled by neurotransmitter GPCRs coupled to Gs or Gi/o which stimulate or inhibit adenylyl cyclases (AC), respectively. RGS proteins powerfully modulate signaling by GPCRs including DA receptors. Remarkably, we have recently discovered that RGS6 is restrictively expressed in human SNc DA neurons that are lost in PD and knockout of RGS6 in mice recapitulates key hallmarks of sporadic PD, including: late‐age‐onset SNc DA neuron degeneration, reduced nigrostriatal DA, and motor deficits. We also demonstrated that RGS6 functions as a critical negative regulator of SNc D2 autoreceptor signaling allowing for proper modulation of DA‐dependent motor behaviors. Given that β‐agonists repress α‐synuclein expression through their activation of β2‐adrenorecetor‐Gαs, a GPCR that increases cAMP, and RGS6's role in inhibiting D2 autoreceptor signaling, a Gi/o GPCR that reduces cAMP, we hypothesize that RGS6, like β‐agonists, may function to prevent the pathological accumulation of α‐synuclein through modulation of cAMP‐PKA signaling. Here we show that RGS6 is a key negative modulator of Gi/o signaling in SNc DA neurons, with its loss provoking enhance D2 autoreceptor activity and a subsequent reduction in cAMP‐PKA signaling. These data are important as cAMP‐PKA signaling has not only been linked to SNc DA neuron survival in PD but also pathological α‐synuclein accumulation. In agreement with these previous reports, we observed elevated SNc α‐synuclein protein expression in 12 and 18mo RGS6−/− mice relative to RGS6+/+ controls. The aberrant elevation in α‐synuclein expression in the SNc of RGS6−/− mice was accompanied by a transition in α‐synuclein expression patterns from largely intracellular, as seen in RGS6+/+ mice, to largely extracellular. Furthermore, western blot analysis revealed increased expression of oligomeric α‐synuclein isoforms in the SNc of RGS6−/− mice, another hallmark of PD pathophysiology. Together, these data identify RGS6 as a key G protein regulator that protects against PD‐related neurodegeneration and α‐synuclein pathology through inhibition of D2R‐Gi/o signaling in SNc DA neurons.Support or Funding InformationNIH AA025919 and Interdisciplinary Research Fellowship T32HL007121This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects and mechanism of epigallocatechin-3-gallate on apoptosis and mTOR/AKT/GSK-3β pathway in substantia nigra neurons in Parkinson rats.

The aim of this study is to investigate the protective effect of epigallocatechin-3-gallate (EGCG) on apoptosis and mTOR/AKT/GSK-3β pathway in substantia nigra neurons in 6-dopamine-induced Parkinson rats. A total of 30 healthy male SD rats were randomly divided into control group, the Parkinson model group, and Parkinson model+EGCG treatment group. The model and EGCG groups were injected into the right striatum with 6-OHDA to establish the Parkinson model, and the control group was injected with saline only. The EGCG group was intragastrically administered with EGCG 50 mg/kg daily for 4 weeks. The rats' turns, speed, and left forelimb usage; neuron apoptosis by TUNEL; and the α-synuclein protein expression in substantia nigra by immunohistochemical staining were studied. Western blotting was used to detect the relative protein (mTOR, AKT and GSK-3β) expressions. Compared with the model group, the EGCG group significantly reduced the rotation speed; increased the left forelimb usage (P<0.01); reduced the neuron apoptosis (P<0.01); decreased α-synuclein expression (P<0.01); and decreased the mTOR, AKT, and GSK-3β protein expressions (P<0.01). EGCG can reduce neuron cell apoptosis in substantia nigra neurons in 6-OHDA-induced Parkinson rats. The mechanism might be related to mTOR/AKT/GSK-3β activation.

Sir-2.1 mediated attenuation of \u03b1-synuclein expression by Alaskan bog blueberry polyphenols in a transgenic model of Caenorhabditis elegans

Misfolding and accumulation of cellular protein aggregates are pathological hallmarks of aging and neurodegeneration. One such protein is α-synuclein, which when misfolded, forms aggregates and disrupts normal cellular functions of the neurons causing Parkinson’s disease. Nutritional interventions abundant in pharmacologically potent polyphenols have demonstrated a therapeutic role for combating protein aggregation associated with neurodegeneration. The current study hypothesized that Alaskan bog blueberry (Vaccinum uliginosum), which is high in polyphenolic content, will reduce α-synuclein expression in a model of Caenorhabditis elegans (C. elegans). We observed that blueberry extracts attenuated α-synuclein protein expression, improved healthspan in the form of motility and restored lipid content in the transgenic strain of C. elegans expressing human α-synuclein. We also found reduced gene expression levels of sir-2.1 (ortholog of mammalian Sirtuin 1) in blueberry treated transgenic animals indicating that the beneficial effects of blueberries could be mediated through partial reduction of sirtuin activity. This therapeutic effect of the blueberries was attributed to its xenohormetic properties. The current results highlight the role of Alaskan blueberries in mediating inhibition of sir-2.1 as a novel therapeutic approach to improving pathologies of protein misfolding diseases. Finally, our study warrants further investigation of the structure, and specificity of such small molecules from indigenous natural compounds and its role as sirtuin regulators.

Down-regulation of natural resistance-associated macrophage protein-1 (Nramp1) is associated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/1-methyl-4-phenylpyridinium (MPP+ )-induced α-synuclein accumulation and neurotoxicity.

The accumulation of α-synuclein is a hallmark in the pathogenesis of Parkinson's disease (PD). Natural resistance-associated macrophage protein-1 (Nramp1) was previously shown to contribute to the degradation of extracellular α-synuclein in microglia under conditions of iron overload. This study was aimed at investigating the role of Nramp1 in α-synuclein pathology in the neurone under 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/1-methyl-4-phenylpyridinium (MPP+ ) treatment. The expression of Nramp1 and pathological features (including iron and α-synuclein accumulation) were examined in the dopaminergic neurones of humans (with and without PD) and of mice [with and without receiving chronic MPTP intoxication]. The effects of Nramp1 expression on low-dose MPP+ -induced α-synuclein expression and neurotoxicity were determined in human dopaminergic neuroblastoma SH-SY5Y cells. Similar to the findings in the substantia nigra of human PD, lower expression of Nramp1 but higher levels of iron and α-synuclein were identified in the dopaminergic neurones of mice receiving chronic MPTP intoxication, compared to controls. In parallel to the loss of dopaminergic neurones, the numbers of glial fibrillary acidic protein- and ionized calcium-binding adapter molecule-1-positive cells were significantly increased in the substantia nigra of MPTP-treated mice. Likewise, in human neuroblastoma SH-SY5Y cells exposed to low-dose MPP+ , Nramp1 expression and cathepsin D activity were decreased, along with an increase in α-synuclein protein expression and aggregation. Overexpression of functional Nramp1 restored cathepsin D activity and attenuated α-synuclein up-regulation and neuronal cell death caused by MPP+ treatment. These data suggest that the neuronal expression of Nramp1 is important for protecting against the development of MPTP/MPP+ -induced α-synuclein pathology and neurotoxicity.

MicroRNA-30e regulates neuroinflammation in MPTP model of Parkinson\u2019s disease by targeting Nlrp3

Accumulating evidences suggest that neuroinflammation is a pathological hallmark of Parkinson’s disease (PD), a neurodegenerative disorder characterized by loss of dopaminergic neurons in substantia nigra pars compacta (SNpc). MicroRNAs have been recently recognized as crucial regulators of inflammatory responses. Here, we found significant downregulation of microRNA-30e (miR-30e) in SNpc of MPTP-induced PD mice. Next, we employed miR-30e agomir to upregulate miR-30e expression in MPTP-treated mice. Our results showed that delivery of miR-30e agomir remarkably improved motor behavioral deficits and neuronal activity, and inhibited the loss of dopamine neurons. Moreover, the increased α-synuclein protein expression in SNpc of MPTP-PD mice was alleviated by the upregulation of miR-30e. Further, miR-30e agomir administration also attenuated the marked increase of inflammatory cytokines, such as TNF-α, COX-2, iNOS, and restored the decreased secretion of BDNF in SNpc. In addition, we demonstrated for the first time that miR-30e directly targeted to Nlrp3, thus suppressing Nlrp3 mRNA and protein expression. Finally, miR-30e upregulation significantly inhibited the activation of Nlrp3 inflammasome as evident from the decreased Nlrp3, Caspase-1 and ASC expressions and IL-18 and IL-1β secretions. Taken together, our study demonstrates that miR-30e ameliorates neuroinflammation in the MPTP model of PD by decreasing Nlrp3 inflammasome activity. These findings suggesting that miR30e may be a key inflammation-mediated molecule that could be a potential target for PD therapeutics.

Tau and α‐synuclein protein expression, purification, and their amyloid inhibitor discovery

Alzheimer's and Parkinson's diseases (AD/PD) are two major neurodegenerative diseases. AD is characterized by the accumulation in brain of two types of abnormal structures, extracellular amyloid plaques and intraneuronal neurofibrillary tangles. Prevalent evidence now indicates that the respective building blocks of plaques and tangles – oligomers of Aβ peptide and the neuron‐specific, axon‐enriched microtubule‐associated protein, tau, represent the most toxic molecular species contributing to AD pathogenesis. Similarly, alpha‐synuclein protein amyloid is a major constitute of Lewy body, protein aggregates that are the pathological hallmark of Parkinson's disease.Currently there are no effective drugs to prevent or cure AD and PD. Because of protein aggregates, including oligomers, mediate neuronal toxicity in AD/PD, small molecule aggregation inhibitors are attractive drug candidates. Many drug trials aimed at preventing or clearing Aβ pathology have failed in recent years. Investigations on tau yielded innovative targets backed by recent findings about the central role of tau in AD pathogenesis (1,2). Preliminary data from a phase 2 trial with the small molecule, methylene blue chloride, have shown some promise and thereby validate the potential of tau aggregation inhibitors (TAIs), but superior TAIs are needed.In this poster we will present our work on expression and purification of human tau and α‐synuclein proteins and their amyloid formation and inhibitor discovery. Tau and α‐synuclein are expressed as recombinant proteins in E. coli and were purified to homogeneity using multiple chromatographic steps. They are further validated by western blot analysis and mass spectrometry. We have developed multiple biochemical, biophysical, and cell‐based assays to monitor oligomer/amyloid formation and amyloid‐induced cytotoxicity assays in characterizing amyloidogenic proteins (3). We use ThT fluorescence‐based assay for semi‐high throughput library screens. Upon identification of “hit” compounds, we validate them with multiple secondary assays including transmission electron microscopy (TEM), photo‐induced crosslinking of unmodified proteins (PICUP), IC50 measurements, and cell‐based cytotoxicity assays. We will further perform mechanistic studies for these validated inhibitors.Support or Funding InformationThis work is in part supported by Virginia Tech new faculty start‐up funds, Commonwealth Health Research Board (CHRB), Alzheimer's and Related Diseases Research Award Fund (ARDRAF) from Virginia Center on Aging, and Diabetes Action Research and Education Foundation (DAREF) to B.X.

Beta-asarone protects against MPTP-induced Parkinson's disease via regulating long non-coding RNA MALAT1 and inhibiting α-synuclein protein expression.

Numerous long non-coding RNAs (lncRNA) have been identified in neurodegenerative disorders including Parkinson's disease (PD). Emerging evidence demonstrates that β-asarone functions as neuroprotective effects in both in vitro and in vivo models. However, the role of β-asarone and its potential mechanism in PD remain not completely clear. MPTP-induced PD mouse model and SH-SY5Y cells subjected to MPP+ as its in vitro model were used to evaluate the effects of β-asarone on PD. LncRNA MALAT1 and α-synuclein expression were determined by real-time PCR and western blot methods. β-Asarone significantly increased the TH+ cells number and decreased the expression levels of MALAT1 and α-synuclein in midbrain tissue of PD mice. RNA pull-down and immunoprecipitation assays confirmed that MALAT1 associated with α-synuclein, leading to the increased stability of α-synuclein and its expression in SH-SY5Y cells. β-asarone elevated the viability of cells exposed to MPP+. Either overexpressed MALAT1 or α-synuclein could canceled the protective effect of β-asarone on cell viability. In PD mice, pcDNA-MALAT1 also decreased the TH+ cells number and increased the α-synuclein expression in PD mice with treatment of β-asarone. β-Asarone functions as a neuroprotective effect in both in vivo and in vitro models of PD via regulating MALAT1 and α-synuclein expression.

Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells

Increased expression and aggregation of α-synuclein (α-syn) protein plays a critical role in mediating the toxic effects of a number of neurodegenerative substances including metals. Thus, knockdown expression of α-syn is proposed as a possible modality for treatment of Parkinson disease (PD). Aluminum (Al) is a neurotoxic metal that contributes to pathogenesis of PD. The aim of this study was to investigate the role of α-syn protein in mediating Al-induced toxicity in PC12 cells. Specific α-syn small interference RNA (siRNA) was applied to knockdown the expression of α-syn protein in PC12 cells. The effects of different concentrations of Al-maltolate (Almal) were then evaluated on cell viability and oxidative stress in the α-syn downregulated cells. The results showed that Almal dose dependently induced apoptosis and increased malondialdehyde (MDA) and catalase activity in PC12 cells. Downregulation of α-syn protein significantly increased cell viability and decreased oxidative markers in Almal-treated cells. These findings suggest that α-syn protein may mediate Al-induced apoptosis and oxidative stress in PC12 cells.

DNA methylation levels of α-synuclein intron 1 in the aging brain

DNA methylation patterns change with age, and aging itself is a major confounding risk factor for Parkinson's disease (PD). Duplication and triplication, that is, increased expression of the α-synuclein (SNCA) gene, cause familial PD, and demethylation of SNCA intron 1 has been shown to result in increased expression of SNCA. We thus hypothesized that age-related alterations of SNCA methylation might underly the increased susceptibility toward PD in later life. The present study sought to determine (1) whether alterations of SNCA intron 1 methylation occurred during aging, (2) whether the methylation pattern differed between men and women, and (3) whether purified neurons compared with non-neuronal cells exhibited different methylation patterns. The analysis of DNA from brain tissue and fluorescence activated cell sorting-sorted purified neurons of 41 individuals revealed only a minor increase of SNCA intron 1 DNA methylation levels in presumably healthy individuals during aging but no significant difference between men and women. Interestingly enough, methylation of SNCA intron 1 was higher in neurons compared with non-neuronal cells, although non-neuronal cells express lower levels of SNCA. Therefore, the normal pattern of SNCA methylation during aging should not result in increased expression of α-synuclein protein. It is thus likely that additional, yet not identified, mechanisms contribute to the tissue specificity of SNCA expression and the presumed dysregulation in PD.

Elevated neuronal α-synuclein promotes microglia activation after spinal cord ischemic/reperfused injury.

The present study aimed to investigate the mechanism of injured neurons caused by ischemia/reperfusion in the induction of microglia activation. Spinal neurons were prepared and exposed to ischemic/reperfused conditions. The α-synuclein protein levels in these cells were analyzed by western blot, immunofluorescence, or enzyme-linked immunosorbent assay. Ischemia/reperfusion exposure led to elevated α-synuclein protein expression and release. Furthermore, when cocultured with injured neurons or supernatants from injured neurons, nitric oxide generation, H2O2 production, and tumor necrosis factor-α expression were promoted in microglia. Nevertheless, this effect was impeded by pretreatment of the α-synuclein antibody in the supernatants from injured neurons. Moreover, toll-like receptor 2 (TLR2) rather than TLR3 or TLR4 mediated microglia activation by α-synuclein. This process involved p38 MAPK and NF-κB activation, the inhibition of which resulted in reduced NADPH oxidase 2 (Nox2) in microglia. In conclusion, ischemia/reperfusion-injured neurons could express and release increased levels of α-synuclein and cause microglia activation through TLR2 both in vitro and in vivo.