Alpha-synuclein Protein Levels Research Articles

Damage to the Locus Coeruleus Alters the Expression of Key Proteins in Limbic Neurodegeneration.

The present investigation was designed based on the evidence that, in neurodegenerative disorders, such as Alzheimer's dementia (AD) and Parkinson's disease (PD), damage to the locus coeruleus (LC) arising norepinephrine (NE) axons (LC-NE) is documented and hypothesized to foster the onset and progression of neurodegeneration within target regions. Specifically, the present experiments were designed to assess whether selective damage to LC-NE axons may alter key proteins involved in neurodegeneration within specific limbic regions, such as the hippocampus and piriform cortex, compared with the dorsal striatum. To achieve this, a loss of LC-NE axons was induced by the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) in C57 Black mice, as assessed by a loss of NE and dopamine-beta-hydroxylase within target regions. In these experimental conditions, the amount of alpha-synuclein (alpha-syn) protein levels were increased along with alpha-syn expressing neurons within the hippocampus and piriform cortex. Similar findings were obtained concerning phospho-Tau immunoblotting. In contrast, a decrease in inducible HSP70-expressing neurons and a loss of sequestosome (p62)-expressing cells, along with a loss of these proteins at immunoblotting, were reported. The present data provide further evidence to understand why a loss of LC-NE axons may foster limbic neurodegeneration in AD and limbic engagement during PD.

Sequential CRISPR screening reveals partial NatB inhibition as a strategy to mitigate alpha-synuclein levels in human neurons.

Alpha-synuclein (αSyn) protein levels correlate with the risk and severity of Parkinson's disease and related neurodegenerative diseases. Lowering αSyn is being actively investigated as a therapeutic modality. Here, we systematically map the regulatory network that controls endogenous αSyn using sequential CRISPR-knockout and -interference screens in an αSyn gene (SNCA)-tagged cell line and induced pluripotent stem cell-derived neurons (iNeurons). We uncover αSyn modifiers at multiple regulatory layers, with amino-terminal acetyltransferase B (NatB) enzymes being the most potent endogenous αSyn modifiers in both cell lines. Amino-terminal acetylation protects the cytosolic αSyn from rapid degradation by the proteasome in a Ube2w-dependent manner. Moreover, we show that pharmacological inhibition of methionyl-aminopeptidase 2, a regulator of NatB complex formation, attenuates endogenous αSyn in iNeurons carrying SNCA triplication. Together, our study reveals several gene networks that control endogenous αSyn, identifies mechanisms mediating the degradation of nonacetylated αSyn, and illustrates potential therapeutic pathways for decreasing αSyn levels in synucleinopathies.

6-Hydroxydopamine-Induced Neurotoxicity in Rat Model of Parkinson’s Disease: Is Reversed via Anti-Oxidative Activities of Curcumin and Aerobic Exercise Therapy

In the rat model, 6-hydroxydopamine (6-OHDA) known as a selective catecholaminergic neurotoxin used chiefly in modeling Parkinson's disease (PD). Continuous aerobic exercise and curcumin supplementations could play a vital role in neuroprotection. This study aimed to explore the neuroprotective roles of regular aerobic exercise and curcumin during PD. For this, rats were treated as follows for 8 consecutive weeks (5 d in a week): For this, animals were orally treated with curcumin (50 ml/kg) alone or in combination with aerobic exercise. Compared with a control group, induction of PD by 6-OHDA increased the amount of alpha-synuclein protein and malondialdehyde levels and decreased the number of substantia nigra neurons, total antioxidant capacity, and glutathione peroxidase activity in brain tissue. All these changes were abolished by the administration of curcumin with aerobic exercise treatments. Activity behavioral tests also confirmed the above-mentioned results by increasing the rod test time and the number of rotations due to apomorphine injection. Histopathology assays mimic the antioxidant activity and behavioral observations. Combined curcumin with aerobic exercise treatments is potentially an effective strategy for modifying the dopaminergic neuron dysfunction in 6-OHDA-induced rats modeling PD via dual inhibiting oxidative stress indices and regulating behavioral tasks.

Epigenome-Wide Analysis of DNA Methylation in Parkinson's Disease Cortex.

Background: Epigenetic factors including DNA methylation contribute to specific patterns of gene expression. Gene–environment interactions can change the methylation status in the brain, and accumulation of these epigenetic changes over a lifespan may be co-responsible for a neurodegenerative disease like Parkinson’s disease, which that is characterised by a late onset in life. Aims: To determine epigenetic modifications in the brains of Parkinson’s disease patients. Patients and Methods: DNA methylation patterns were compared in the cortex tissue of 14 male PD patients and 10 male healthy individuals using the Illumina Methylation 450 K chip. Subsequently, DNA methylation of candidate genes was evaluated using bisulphite pyrosequencing, and DNA methylation of cytochrome P450 2E1 (CYP2E1) was characterized in DNA from blood mononuclear cells (259 PD patients and 182 healthy controls) and skin fibroblasts (10 PD patients and 5 healthy controls). Protein levels of CYP2E1 were analysed using Western blot in human cortex and knock-out mice brain samples. Results: We found 35 hypomethylated and 22 hypermethylated genes with a methylation M-value difference >0.5. Decreased methylation of cytochrome P450 2E1 (CYP2E1) was associated with increased protein levels in PD brains, but in peripheral tissues, i.e., in blood cells and skin fibroblasts, DNA methylation of CYP2E1 was unchanged. In CYP2E1 knock-out mice brain alpha-synuclein (SNCA) protein levels were down-regulated compared to wild-type mice, whereas treatment with trichloroethylene (TCE) up-regulated CYP2E1 protein in a dose-dependent manner in cultured cells. We further identified an interconnected group of genes associated with oxidative stress, such as Methionine sulfoxide reductase A (MSRA) and tumour protein 73 (TP73) in the brain, which again were not paralleled in other tissues and appeared to indicate brain-specific changes. Conclusions: Our study revealed surprisingly few dysmethylated genes in a brain region less affected in PD. We confirmed hypomethylation of CYP2E1.

A preliminary study assessing the effect of isocyanate in neuroblastoma brain cells in vitro

Isocyanate is an intermediate compound used in the manufacturing of a number of pesticides. The aim of this study is to understand the mechanism of isocyanate in SHSY‑5Y neuroblastoma cells in vitro. Cells were treated with a chemical equivalent of isocyanate, i.e., N‑succinimidyl N‑methylcarbamate (NSNM). Cell cytotoxicity, as well as qualitative and quantitative alpha‑synuclein protein levels were analyzed using different molecular techniques. NSNM at a concentration of 0.005 μM significantly increased cell death, in a time‑dependent manner, as well as levels of alpha‑synuclein protein in SH‑SY5Y cells. These findings demonstrate the ability of low doses of isocyanate to increase neuronal vulnerability b y inducing cell cytotoxicity and protein dysfunction in vitro.

N-terminal acetylation mutants affect alpha-synuclein stability, protein levels and neuronal toxicity

Alpha-synuclein (aSyn) protein levels are sufficient to drive Parkinson's disease (PD) and other synucleinopathies. Despite the biomedical/therapeutic potential of aSyn protein regulation, little is known about mechanisms that limit/control aSyn levels. Here, we investigate the role of a post-translational modification, N-terminal acetylation, in aSyn neurotoxicity. N-terminal acetylation occurs in all aSyn molecules and has been proposed to determine its lipid binding and aggregation capacities; however, its effect in aSyn stability/neurotoxicity has not been evaluated. We generated N-terminal mutants that alter or block physiological aSyn N-terminal acetylation in wild-type or pathological mutant E46K aSyn versions and confirmed N-terminal acetylation status by mass spectrometry. By optical pulse-labeling in living primary neurons we documented a reduced half-life and accumulation of aSyn N-terminal mutants. To analyze the effect of N-terminal acetylation mutants in neuronal toxicity we took advantage of a neuronal model where aSyn toxicity was scored by longitudinal survival analysis. Salient features of aSyn neurotoxicity were previously investigated with this approach. aSyn-dependent neuronal death was recapitulated either by higher aSyn protein levels in the case of WT aSyn, or by the combined effect of protein levels and enhanced neurotoxicity conveyed by the E46K mutation. aSyn N-terminal mutations decreased E46K aSyn-dependent neuronal death both by reducing protein levels and, importantly, by reducing the intrinsic E46K aSyn toxicity, being the D2P mutant the least toxic. Together, our results illustrate that the N-terminus determines, most likely through its acetylation, aSyn protein levels and toxicity, identifying this modification as a potential therapeutic target.

Systemic Exosomal Delivery of shRNA Minicircles Prevents Parkinsonian Pathology.

The development of new therapies to slow down or halt the progression of Parkinson's disease is a health care priority. A key pathological feature is the presence of alpha-synuclein aggregates, and there is increasing evidence that alpha-synuclein propagation plays a central role in disease progression. Consequently, the downregulation of alpha-synuclein is a potential therapeutic target. As a chronic disease, the ideal treatment will be minimally invasive and effective in the long-term. Knockdown of gene expression has clear potential, and siRNAs specific to alpha-synuclein have been designed; however, the efficacy of siRNA treatment is limited by its short-term efficacy. To combat this, we designed shRNA minicircles (shRNA-MCs), with the potential for prolonged effectiveness, and used RVG-exosomes as the vehicle for specific delivery into the brain. We optimized this system using transgenic mice expressing GFP and demonstrated its ability to downregulate GFP protein expression in the brain for up to 6weeks. RVG-exosomes were used to deliver anti-alpha-synuclein shRNA-MC therapy to the alpha-synuclein preformed-fibril-induced model of parkinsonism. This therapy decreased alpha-synuclein aggregation, reduced the loss of dopaminergic neurons, and improved the clinical symptoms. Our results confirm the therapeutic potential of shRNA-MCs delivered by RVG-exosomes for long-term treatment of neurodegenerative diseases.

The effect of manganese exposure in Atp13a2-deficient mice

Loss of function mutations in the P5-ATPase ATP13A2 are associated with Kufor-Rakeb Syndrome and Neuronal Ceroid Lipofuscinosis. While the function of ATP13A2 is unclear, in vitro studies suggest it is a lysosomal protein that interacts with the metals manganese (Mn) and zinc and the presynaptic protein alpha-synuclein. Loss of ATP13A2 function in mice causes sensorimotor deficits, enhanced autofluorescent storage material, and accumulation of alpha-synuclein. The present study sought to determine the effect of Mn administration on these same outcomes in ATP13A2-deficient mice. Wildtype and ATP13A2-deficient mice received saline or Mn at 5–9 or 12–19 months for 45days. Sensorimotor function was assessed starting at day 30. Autofluorescence was quantified in multiple brain regions and alpha-synuclein protein levels were determined in the ventral midbrain. Brain Mn, iron, zinc, and copper concentrations were measured in 5–9 month old mice. The results show Mn enhanced sensorimotor function, increased autofluorescence in the substantia nigra, and increased insoluble alpha-synuclein in the ventral midbrain in older ATP13A2-deficient mice. In addition, the Mn regimen used increased Mn concentration in the brain and levels were higher in Mn-treated mutants than controls. These results indicate loss of ATP13A2 function leads to increased sensitivity to Mn in vivo.

259. Targeted Gene Silencing by U1 Adaptor Oligonucleotides in Preclinical Models of Parkinson's Disease and Huntington's Disease

Many candidate genes are implicated in neurodegenerative disease, but to study potential therapeutic effects of modifying their expression in the central nervous system of animal models has been difficult, often requiring slow, expensive transgenic methods. Transient gene silencing with synthetic oligonucleotides can be a fast, inexpensive alternative to making new transgenic animal models, and a complimentary technique to extend the utility of existing ones. For genes with products that have been validated as therapeutic targets, but are not amenable to small molecule drugs, gene silencing may also be the therapeutic modality of choice.U1 Adaptors are a third generation of oligonucleotide-mediated gene silencing technology, mechanistically distinct from antisense or siRNA. U1 Adaptors act by selectively interfering with a key step in mRNA maturation: the addition of a 3’ polyadenosine (polyA) tail. Nearly all protein-coding mRNAs require a polyA tail, and failure to add one results in rapid degradation of the nascent mRNA inside the nucleus, preventing expression of a protein product. U1 Adaptor oligonucleotides are well suited to in vivo applications because they can accept extensive chemical modifications to improve nuclease resistance and the attachment of bulky groups, such as tags for imaging or ligands for receptor-mediated uptake by target cells, without loss of silencing activity.To explore the feasibility of U1 Adaptor technology for CNS targets, we designed panels of candidate U1 Adaptor oligos for mouse Scna (alpha-synuclein) and human HTT (Huntingtin), and screened them in cell culture. We identified U1 Adaptors that robustly suppress mouse Scna mRNA and reduce alpha-synuclein protein levels in mouse cells. Similarly, we identified U1 Adaptors that suppress the predominant, full length human HTT mRNA and reduce HTT protein levels in human cells. We also identified U1 Adaptors that suppress the HTT exon-1 truncation isoform recently implicated in HD pathogenesis. For in-vivo PK/PD studies, U1 Adaptors were delivered into the CNS of mice, by intracerebroventricular (ICV) injection or by direct stereotaxic injections into the striatum. To examine distribution, cellular uptake and persistence over time, fluorescently tagged U1 Adaptors were administered, then visualized by confocal microscopy in brain sections. ICV injection achieved broad distribution of fluorescent U1 Adaptors throughout the brain, with uptake visible in most cells. Subcellular distribution 24 hours after injection was diffuse in both cytoplasmic and nuclear compartments, but became more punctate and perinuclear by 48 hours. U1 Adaptor oligonucleotides were detected on northern blots of small RNA recovered from brain tissue specimens. Their levels in tissue were estimated by comparison to a standard loading curve, and correlated well with ICV-injected dose. After direct stereotaxic injection to the striatum, U1 Adaptors diffused rapidly and widely, and were taken up by all striatial cells, though preferentially by neurons. Adaptors persisted in tissue for at least five days (the last time point assayed) and reached the nuclei of striatial cells. We then did a series of studies with U1 Adaptors specific for mouse Scna mRNA. RNAScope was used to visualize relative levels of mRNA in situ. Injection of U1 Adaptors directly into the striatum resulted in clearly reduced expression of Scna mRNA and also of mRNA for synaptophysin, known to be down-regulated when α-synuclein expression is reduced.

Rotenone down-regulates HSPA8/hsc70 chaperone protein in vitro: A new possible toxic mechanism contributing to Parkinson’s disease

HSPA8/hsc70 (70-kDa heat shock cognate) chaperone protein exerts multiple protective roles. Beside its ability to confer to the cells a generic resistance against several metabolic stresses, it is also involved in at least two critical processes whose activity is essential in preventing Parkinson’s disease (PD) pathology. Actually, hsc70 protein acts as the main carrier of chaperone-mediated autophagy (CMA), a selective catabolic pathway for alpha-synuclein, the main pathogenic protein that accumulates in degenerating dopaminergic neurons in PD. Furthermore, hsc70 efficiently fragments alpha-synuclein fibrils in vitro and promotes depolymerization into non-toxic alpha-synuclein monomers.Considering that the mitochondrial complex I inhibitor rotenone, used to generate PD animal models, induces alpha-synuclein aggregation, this study was designed in order to verify whether rotenone exposure leads to hsc70 alteration possibly contributing to alpha-synuclein aggregation. To this aim, human SH-SY5Y neuroblastoma cells were treated with rotenone and hsc70 mRNA and protein expression were assessed; the effect of rotenone on hsc70 was compared with that exerted by hydrogen peroxide, a generic oxidative stress donor with no inhibitory activity on mitochondrial complex I. Furthermore, the effect of rotenone on hsc70 was verified in primary mouse cortical neurons. The possible contribution of macroautophagy to rotenone-induced hsc70 modulation was explored and the influence of hsc70 gene silencing on neurotoxicity was assessed. We demonstrated that rotenone, but not hydrogen peroxide, induced a significant reduction of hsc70 mRNA and protein expression. We also observed that the toxic effect of rotenone on alpha-synuclein levels was amplified when macroautophagy was inhibited, although rotenone-induced hsc70 reduction was independent from macroautophagy. Finally, we demonstrated that hsc70 gene silencing up-regulated alpha-synuclein mRNA and protein levels without affecting cell viability and without altering rotenone- and hydrogen peroxide-induced cytotoxicity.These findings demonstrate the existence of a novel mechanism of rotenone toxicity mediated by hsc70 and indicate that dysfunction of both CMA and macroautophagy can synergistically exacerbate alpha-synuclein toxicity, suggesting that hsc70 up-regulation may represent a valuable therapeutic strategy for PD.

Rotenone Upregulates Alpha-Synuclein and Myocyte Enhancer Factor 2D Independently from Lysosomal Degradation Inhibition

Dysfunctions of chaperone-mediated autophagy (CMA), the main catabolic pathway for alpha-synuclein, have been linked to the pathogenesis of Parkinson's disease (PD). Since till now there is limited information on how PD-related toxins may affect CMA, in this study we explored the effect of mitochondrial complex I inhibitor rotenone on CMA substrates, alpha-synuclein and MEF2D, and effectors, lamp2A and hsc70, in a human dopaminergic neuroblastoma SH-SY5Y cell line. Rotenone induced an upregulation of alpha-synuclein and MEF2D protein levels through the stimulation of their de novo synthesis rather than through a reduction of their CMA-mediated degradation. Moreover, increased MEF2D transcription resulted in higher nuclear protein levels that exert a protective role against mitochondrial dysfunction and oxidative stress. These results were compared with those obtained after lysosome inhibition with ammonium chloride. As expected, this toxin induced the cytosolic accumulation of both alpha-synuclein and MEF2D proteins, as the result of the inhibition of their lysosome-mediated degradation, while, differently from rotenone, ammonium chloride decreased MEF2D nuclear levels through the downregulation of its transcription, thus reducing its protective function. These results highlight that rotenone affects alpha-synuclein and MEF2D protein levels through a mechanism independent from lysosomal degradation inhibition.

Alpha-synuclein and Protein Degradation Systems: a Reciprocal Relationship

An increasing wealth of data indicates a close relationship between the presynaptic protein alpha-synuclein and Parkinson's disease (PD) pathogenesis. Alpha-synuclein protein levels are considered as a major determinant of its neurotoxic potential, whereas secreted extracellular alpha-synuclein has emerged as an additional important factor in this regard. However, the manner of alpha-synuclein degradation in neurons remains contentious. Both the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP)-mainly macroautophagy and chaperone-mediated autophagy-have been suggested to contribute to alpha-synuclein turnover. Additionally, other proteases such as calpains, neurosin, and metalloproteinases have been also proposed to have a role in intracellular and extracellular alpha-synuclein processing. Both UPS and ALP activity decline with aging and such decline may play a pivotal role in many neurodegenerative conditions. Alterations in these major proteolytic pathways may result in alpha-synuclein accumulation due to impaired clearance. Conversely, increased alpha-synuclein protein burden promotes the generation of aberrant species that may impair further UPS or ALP function, generating thus a bidirectional positive feedback loop leading to neuronal death. In the current review, we summarize the recent findings related to alpha-synuclein degradation, as well as to alpha-synuclein-mediated aberrant effects on protein degradation systems. Identifying the factors that regulate alpha-synuclein association to cellular proteolytic pathways may represent potential targets for therapeutic interventions in PD and related synucleinopathies.

Role of Alpha-Synuclein Protein Levels in Mitochondrial Morphology and Cell Survival in Cell Lines

α-Synuclein is highly associated with some neurodegeneration and malignancies. Overexpressing wild-type or mutant α-synuclein promotes neuronal death by mitochondrial dysfunction, the underlying mechanisms of which remain poorly defined. It was recently reported that α-synuclein expression could directly lead to mitochondrial fragmentation in vitro and in vivo, which may be due to α-synuclein localization on mitochondria. Here, we applied a double staining method to demonstrate mitochondrial morphogenetic changes in cells overexpressed with α-synuclein. We show that mitochondrial localization of α-synuclein was increased following its overexpression in three distinct cell lines, including HeLa, SH-SY5Y, and PC12 cells, but no alteration in mitochondrial morphology was detected. However, α-synuclein knockdown prevents MPP+-induced mitochondrial fragmentation in SH-SY5Y and PC12 cells. These data suggest that α-synuclein protein levels hardly affect mitochondrial morphology in normal cell lines, but may have some influence on that under certain environmental conditions.

Dimebon (Latrepirdine) protects from cell death-induced by mitochondrial stressors and alpha-synuclein over-expression, and decreases alpha-synuclein protein levels in a Parkinson's disease cell model.

Dimebon (Latrepirdine) protects from cell death-induced by mitochondrial stressors and alpha-synuclein over-expression, and decreases alpha-synuclein protein levels in a Parkinson's disease cell model.

The Parkinson disease‐associated A30P mutation stabilizes α‐synuclein against proteasomal degradation triggered by heme oxygenase‐1 over‐expression in human neuroblastoma cells

Proteosomal degradation of proteins is one of the major mechanisms of intracellular protein turnover. Failure of the proteosome to degrade misfolded protein is implicated in the accumulation of alpha-synuclein in Parkinson's disease (PD). Heme oxygenase-1 (HO-1), an enzyme that converts heme to free iron, carbon monoxide (CO) and biliverdin (bilirubin precursor) is expressed in response to various stressors. HO-1 is up-regulated in PD- and Alzheimer's disease-affected neural tissues. In this study, we found that HO-1 over-expression engenders dose-dependent decreases in alpha-synuclein protein levels in human neuroblastoma M17 cells. When over-expression of HO-1 was silenced in HO-1 transfected cells, level of alpha-synuclein was restored. Likewise, treatment of HO-1 over-expressing cells with the HO-1 inhibitor, tin mesoporphyrin, the iron chelator deferoxamine or antagonist of CO-dependent cGMP activation, methylene blue, mitigated the HO-1-induced reduction in alpha-synuclein levels. Furthermore, when HO-1 over-expressing cells were treated with the proteosome inhibitors, lactacystin and MG132, level of alpha-synuclein was almost completely restored. In contrast to the effect on alpha-synuclein [wild-type (WT)] levels, HO-1 over-expression did not significantly impact PD-associated alpha-synuclein (A30P) levels in these cells. HO-1 also significantly reduced aggregation of alpha-synuclein (WT) but not that of A30P. Our results suggest that HO-1, which is expressed when neurons are exposed to toxic stimuli capable of inducing protein misfolding, triggers proteosomal degradation of proteins and prevents intracellular accumulation of protein aggregates and inclusions. Resistance to HO-1 induced proteosomal degradation may render the familial PD-associated A30P mutation prone to toxic intracellular aggregation.

FGF20 and Parkinson's disease: No evidence of association or pathogenicity via α‐synuclein expression

Genetic variation in fibroblast growth factor 20 (FGF20) has been associated with risk of Parkinson's disease (PD). Functional evidence suggested the T allele of one SNP, rs12720208 C/T, altered PD risk by increasing FGF20 and alpha-synuclein protein levels. Herein we report our association study of FGF20 and PD risk in four patient-control series (total: 1,262 patients and 1,881 controls), and measurements of FGF20 and alpha-synuclein protein levels in brain samples (nine patients). We found no evidence of association between FGF20 variability and PD risk, and no relationship between the rs12720208 genotype, FGF20 and alpha-synuclein protein levels.

Accelerated Proteasomal Activity Induced by Pb2+, Ga3+, or Cu2+ Exposure Does Not Induce Degradation of αt-Synuclein

The involvement of environmental heavy metals in Parkinson's disease (PD) has been suggested by epidemiologic studies; however, the mechanism of this effect is unknown. PD is characterized by the aggregation of alpha-synuclein in Lewy bodies. We previously showed that Pb2+ accelerates proteasomal activity. Therefore, we examined the effect of Pb2+, Ga3+, and Cu2+ on alpha-synuclein in human SH-SY5Y cells. The heavy metals induced an increase in heme-oxygenase-1 levels without significant cell death or ROS generation. The metals inhibited ALA-dehydratase, which is the inhibitory subunit of the proteasome, thereby accelerating proteasomal activity and decreasing protein levels of CDK-1 and PBGD. However, alpha-synuclein protein levels increased after exposure to metals, similar to the effect obtained with the proteasome inhibitor, hemin, suggesting that alpha-synuclein is inaccessible to proteasomal degradation. Indeed, electron microscopy revealed the formation of aggresomes in Pb2+- or hemin-treated cells. Thus, although heavy metals enhance proteasomal activity, alpha-synuclein is protected from degradation, and its protein levels and aggregation are increased.

Cerebellar αsynuclein levels are decreased in Parkinson's disease and do not correlate with SNCA polymorphisms associated with disease in a Swedish material

Alterations of brain and plasma alpha-synuclein levels and SNCA gene variability have been implicated in the pathogenesis of Parkinson's disease (PD). We therefore measured alpha-synuclein protein levels in postmortem PD and control cerebellum tissue using Western blot and investigated whether the levels correlated to SNCA genotype. We found markedly decreased alpha-synuclein levels in PD patients (n=16) compared to gender- and age-matched controls (n=14; P=0.004) normalized to alpha-tubulin. We also performed an association study of the noncoding polymorphisms rs2737029 (A/G) and rs356204 (A/G) (intron 4), and of rs356219 (T/C) (3'-region) of SNCA in a Swedish PD case-control material. Using a two-sided chi(2) test, we found significant association of rs2737029 (P=0.003; chi(2)=9.07) and rs356204 (P=0.048; chi(2)=3.91) with disease, strengthening the involvement of SNCA polymorphisms in sporadic PD. Stratification of the human postmortem brain material by genotype of the three investigated polymorphisms, did not indicate any influence of genotype on alpha-synuclein protein levels when comparing PD with controls. Taken together, our findings demonstrate that the investigated Parkinson patients have markedly reduced levels of alpha-synuclein in cerebellum, and that this reduction is general, rather then correlated to the investigated polymorphisms, although two of the polymorphisms also associated with disease in a Swedish material.

Alpha synuclein protein levels are increased in serum from recently abstinent cocaine abusers

Alpha synuclein is increased in dopamine neurons of cocaine abusers and in rats whose alcohol preference is inbred. Recent studies have shown increased alpha synuclein protein expression in serum of alcoholic patients that is linked with severity of alcohol craving. The aim of this study was to analyze the serum levels of alpha synuclein in healthy subjects and in recently abstinent cocaine abusers. Alpha synuclein protein expression was measured by enzyme-linked immunosorbent assay in serum specimens obtained from 38 recently abstinent cocaine dependent patients and 14 control subjects. The presence of cocaine dependence disorder was based on the Structured Clinical Interview (DSM-IV). Drug severity was assessed by the Addiction Severity Index ratings and composite measures. Scores of the intensity and frequency of cocaine craving episodes were obtained from the Minnesota Cocaine Craving Questionnaire. The serum concentrations of alpha synuclein in cocaine dependent patients were significantly higher as compared with age-matched drug-free controls ( p < 0.001). Alpha synuclein levels in blood were significantly correlated with the intensity ( r = 0.60, p < 0.001) and frequency ( r = 0.64, p < 0.001) of cocaine craving and with 30 days of cocaine use in the prior month before entry to treatment ( r = 0.56, p < 0.005). However, there was no correlation between the serum protein levels of alpha synuclein and age in either group. This report is the first demonstration of altered alpha synuclein levels in peripheral blood from cocaine abusers. These data agree with previous reports in postmortem brain of cocaine abusers and provide support for an association between alpha synuclein and cocaine dependence.

??-Synuclein Protein Levels Are Increased in Alcoholic Patients and Are Linked to Craving

Alpha synuclein has been found to be increased in dopamine neurones of cocaine abusers and in rats whose alcohol preference is inbred. Furthermore, increased alpha-synuclein messenger RNA expression has been linked to craving in patients with alcoholism. The aim of the current study was to investigate whether protein levels of alpha synuclein in alcoholics are changed and possibly influence alcohol craving. The alpha-synuclein protein expression level was measured by enzyme-linked immunosorbent assay in the serum of 49 male alcoholics and 50 nondrinking healthy controls. Alcohol craving was assessed by the Obsessive-Compulsive Drinking Scale total score, including subscales for obsessive and compulsive craving. Alpha-synuclein protein expression in patients with alcoholism (14.33 ng/ml; SD, 13.01 ng/ml) was significantly higher (t test, T = 3.66, p < 0.0001) when compared with that of healthy controls (5.92 ng/ml; SD, 9.72 ng/ml). Using a multivariate analysis, all craving scores (Obsessive-Compulsive Drinking Scale total score and obsessive and compulsive subscale scores) in alcoholics were significantly associated with their alpha-synuclein protein levels (multiple linear regression, p < 0.014). To our knowledge, this is the first study evaluating alpha-synuclein protein expression in alcoholics. The current study provides further evidence of altered alpha-synuclein levels in patients with alcoholism and their linkage to alcohol craving. Because alpha synuclein is involved in the modulation of dopaminergic neurotransmission, these results deliver further pathophysiological explanations of craving mechanisms.