α-Synuclein misfolding and Parkinson's disease

Substantial evidence links α-synuclein, a small highly conserved presynaptic protein with unknown function, to both familial and sporadic Parkinson's disease (PD). α-Synuclein has been identified as the major component of Lewy bodies and Lewy neurites, the characteristic proteinaceous deposits that are the hallmarks of PD. α-Synuclein is a typical intrinsically disordered protein, but can adopt a number of different conformational states depending on conditions and cofactors. These include the helical membrane-bound form, a partially-folded state that is a key intermediate in aggregation and fibrillation, various oligomeric species, and fibrillar and amorphous aggregates. The molecular basis of PD appears to be tightly coupled to the aggregation of α-synuclein and the factors that affect its conformation. This review examines the different aggregation states of α-synuclein, the molecular mechanism of its aggregation, and the influence of environmental and genetic factors on this process.     

Convergence of miRNA expression profiling, α-synuclein interacton and GWAS in Parkinson's disease

miRNAs were recently implicated in the pathogenesis of numerous diseases, including neurological disorders such as Parkinson''s disease (PD). miRNAs are abundant in the nervous system, essential for efficient brain function and play important roles in neuronal patterning and cell specification. To further investigate their involvement in the etiology of PD, we conducted miRNA expression profiling in peripheral blood mononuclear cells (PBMCs) of 19 patients and 13 controls using microarrays. We found 18 miRNAs differentially expressed, and pathway analysis of 662 predicted target genes of 11 of these miRNAs revealed an over-representation in pathways previously linked to PD as well as novel pathways. To narrow down the genes for further investigations, we undertook a parallel approach using chromatin immunoprecipitation-sequencing (ChIP-seq) analysis to uncover genome-wide interactions of α-synuclein, a molecule with a central role in both monogenic and idiopathic PD. Convergence of ChIP-seq and miRNomics data highlighted the glycosphingolipid biosynthesis and the ubiquitin proteasome system as key players in PD. We then tested the association of target genes belonging to these pathways with PD risk, and identified nine SNPs in USP37 consistently associated with PD susceptibility in three genome-wide association studies (GWAS) datasets (0.46≤OR≤0.63) and highly significant in the meta-dataset (3.36×10⁻⁴−3). A SNP in ST8SIA4 was also highly associated with PD (p = 6.15×10⁻³) in the meta-dataset. These findings suggest that several miRNAs may act as regulators of both known and novel biological processes leading to idiopathic PD.     

α-Synuclein misfolding and aggregation: Implications in Parkinson’s disease pathogenesis

α-Synuclein (α-Syn) has been extensively studied for its structural and biophysical properties owing to its pathophysiological role in Parkinson’s disease (PD). Lewy bodies and Lewy neurites are the pathological hallmarks of PD and contain α-Syn aggregates as their major component. It was therefore hypothesized that α-Syn aggregation is actively associated with PD pathogenesis. The central role of α-Syn aggregation in PD is further supported by the identification of point mutations in α-Syn protein associated with rare familial forms of PD. However, the correlation between aggregation propensities of α-Syn mutants and their association with PD phenotype is not straightforward. Recent evidence suggested that oligomers, formed during the initial stages of aggregation, are the potent neurotoxic species causing cell death in PD. However, the heterogeneous and unstable nature of these oligomers limit their detailed characterization. α-Syn fibrils, on the contrary, are shown to be the infectious agents and propagate in a prion-like manner. Although α-Syn is an intrinsically disordered protein, it exhibits remarkable conformational plasticity by adopting a range of structural conformations under different environmental conditions. In this review, we focus on the structural and functional aspects of α-Syn and role of potential factors that may contribute to the underlying mechanism of synucleinopathies. This information will help to identify novel targets and develop specific therapeutic strategies to combat Parkinson’s and other protein aggregation related neurodegenerative diseases.     

α-Synuclein genetic variants predict faster motor symptom progression in idiopathic Parkinson disease

Currently, there are no reported genetic predictors of motor symptom progression in Parkinson's disease (PD). In familial PD, disease severity is associated with higher α-synuclein (SNCA) expression levels, and in postmortem studies expression varies with SNCA genetic variants. Furthermore, SNCA is a well-known risk factor for PD occurrence. We recruited Parkinson's patients from the communities of three central California counties to investigate the influence of SNCA genetic variants on motor symptom progression in idiopathic PD. We repeatedly assessed this cohort of patients over an average of 5.1 years for motor symptom changes employing the Unified Parkinson's Disease Rating Scale (UPDRS). Of 363 population-based incident PD cases diagnosed less than 3 years from baseline assessment, 242 cases were successfully re-contacted and 233 were re-examined at least once. Of subjects lost to follow-up, 69% were due to death. Adjusting for covariates, risk of faster decline of motor function as measured by annual increase in motor UPDRS exam score was increased 4-fold in carriers of the REP1 263bp promoter variant (OR 4.03, 95%CI:1.57-10.4). Our data also suggest a contribution to increased risk by the G-allele for rs356165 (OR 1.66; 95%CI:0.96-2.88), and we observed a strong trend across categories when both genetic variants were considered (p for trend = 0.002). Our population-based study has demonstrated that SNCA variants are strong predictors of faster motor decline in idiopathic PD. SNCA may be a promising target for therapies and may help identify patients who will benefit most from early interventions. This is the first study to link SNCA to motor symptom decline in a longitudinal progression study.     

Redistribution of DAT/α-synuclein complexes visualized by "in situ" proximity ligation assay in transgenic mice modelling early Parkinson's disease

Alpha-synuclein, the major component of Lewy bodies, is thought to play a central role in the onset of synaptic dysfunctions in Parkinson's disease (PD). In particular, α-synuclein may affect dopaminergic neuron function as it interacts with a key protein modulating dopamine (DA) content at the synapse: the DA transporter (DAT). Indeed, recent evidence from our "in vitro" studies showed that α-synuclein aggregation decreases the expression and membrane trafficking of the DAT as the DAT is retained into α-synuclein-immunopositive inclusions. This notwithstanding, "in vivo" studies on PD animal models investigating whether DAT distribution is altered by the pathological overexpression and aggregation of α-synuclein are missing. By using the proximity ligation assay, a technique which allows the "in situ" visualization of protein-protein interactions, we studied the occurrence of alterations in the distribution of DAT/α-synuclein complexes in the SYN120 transgenic mouse model, showing insoluble α-synuclein aggregates into dopaminergic neurons of the nigrostriatal system, reduced striatal DA levels and an altered distribution of synaptic proteins in the striatum. We found that DAT/α-synuclein complexes were markedly redistributed in the striatum and substantia nigra of SYN120 mice. These alterations were accompanied by a significant increase of DAT striatal levels in transgenic animals when compared to wild type littermates. Our data indicate that, in the early pathogenesis of PD, α-synuclein acts as a fine modulator of the dopaminergic synapse by regulating the subcellular distribution of key proteins such as the DAT.     

IL-1α/IL-1R1 expression in chronic obstructive pulmonary disease and mechanistic relevance to smoke-induced neutrophilia in mice

Background

Cigarette smoking is the main risk factor for the development of chronic obstructive pulmonary disease (COPD), a major cause of morbidity and mortality worldwide. Despite this, the cellular and molecular mechanisms that contribute to COPD pathogenesis are still poorly understood.

Methodology and Principal Findings

The objective of this study was to assess IL-1 α and β expression in COPD patients and to investigate their respective roles in perpetuating cigarette smoke-induced inflammation. Functional studies were pursued in smoke-exposed mice using gene-deficient animals, as well as blocking antibodies for IL-1α and β. Here, we demonstrate an underappreciated role for IL-1α expression in COPD. While a strong correlation existed between IL-1α and β levels in patients during stable disease and periods of exacerbation, neutrophilic inflammation was shown to be IL-1α-dependent, and IL-1β- and caspase-1-independent in a murine model of cigarette smoke exposure. As IL-1α was predominantly expressed by hematopoietic cells in COPD patients and in mice exposed to cigarette smoke, studies pursued in bone marrow chimeric mice demonstrated that the crosstalk between IL-1α+ hematopoietic cells and the IL-1R1+ epithelial cells regulates smoke-induced inflammation. IL-1α/IL-1R1-dependent activation of the airway epithelium also led to exacerbated inflammatory responses in H1N1 influenza virus infected smoke-exposed mice, a previously reported model of COPD exacerbation.

Conclusions and Significance

This study provides compelling evidence that IL-1α is central to the initiation of smoke-induced neutrophilic inflammation and suggests that IL-1α/IL-1R1 targeted therapies may be relevant for limiting inflammation and exacerbations in COPD.     

LncRNA DQ786243 affects Treg related CREB and Foxp3 expression in Crohn’s disease

Background

Long non-coding RNAs (lncRNAs) have different functions in cells. They work as signals, decoys, guides, and scaffolds. Altered lncRNA levels can affect the expression of gene products. There are seldom studies on the role of lncRNAs in inflammatory bowel disease (IBD).

Results

Quantitative RT-PCR showed that {"type":"entrez-nucleotide","attrs":{"text":"DQ786243","term_id":"110631570","term_text":"DQ786243"}}DQ786243 was significantly overexpressed in clinical active CD patients compared with clinical inactive CD patients (P = 0.0118) or healthy controls (P = 0.002). CREB was also more highly expressed in active CD than in inactive CD (P = 0.0034) or controls (P = 0.0241). Foxp3 was interestingly lower in inactive CD than in active CD (P = 0.0317) or controls (P = 0.0103), but there were no apparent differences between active CD and controls. CRP was well correlated with {"type":"entrez-nucleotide","attrs":{"text":"DQ786243","term_id":"110631570","term_text":"DQ786243"}}DQ786243 (r = 0.489, P = 0.034), CREB (r = 0.500, P = 0.029) and Foxp3 (r = 0.546, P = 0.016). At 48 hours after {"type":"entrez-nucleotide","attrs":{"text":"DQ786243","term_id":"110631570","term_text":"DQ786243"}}DQ786243 transfection, qRT-PCR showed both CREB (P = 0.017) and Foxp3 (P = 0.046) had an increased mRNA expression in Jurkat cells. Western blot showed the same pattern. After {"type":"entrez-nucleotide","attrs":{"text":"DQ786243","term_id":"110631570","term_text":"DQ786243"}}DQ786243 transfection, CREB phosphorylation ratio (p-CREB/t-CREB) was increased (P = 0.0043).

Conclusion

{"type":"entrez-nucleotide","attrs":{"text":"DQ786243","term_id":"110631570","term_text":"DQ786243"}}DQ786243 can be related with severity of CD. It can affect the expression of CREB and Foxp3 through which regulates the function of Treg. CREB itself seems not the mediator of {"type":"entrez-nucleotide","attrs":{"text":"DQ786243","term_id":"110631570","term_text":"DQ786243"}}DQ786243 to up-regulate Foxp3. The phosphorylation of CREB might play a more important role in the process.     

α-synuclein reactive antibodies as diagnostic biomarkers in blood sera of Parkinson's disease patients

Background

Auto-antibodies with specificity to self-antigens have been implicated in a wide variety of neurological diseases, including Parkinson''s (PD) and Alzheimer''s diseases, being sensitive indicators of neurodegeneration and focus for disease prevention. Of particular interest are the studies focused on the auto-immune responses to amyloidogenic proteins associated with diseases and their applications in therapeutic treatments such as vaccination with amyloid antigens and antibodies in PD, Alzheimer''s disease and potentially other neurodegeneration ailments.

Methodology/Principal Findings

Generated auto-antibodies towards the major amyloidogenic protein involved in PD Lewy bodies – α-synuclein and its amyloid oligomers and fibrils were measured in the blood sera of early and late PD patients and controls by using ELISA, Western blot and Biacore surface plasmon resonance. We found significantly higher antibody levels towards monomeric α-synuclein in the blood sera of PD patients compared to controls, though the responses decreased with PD progression (P<0.0001). This indicates potential protective role of autoimmunity in maintaining the body homeostasis and clearing protein species whose disbalance may lead to amyloid assembly. There were no noticeable immune responses towards amyloid oligomers, but substantially increased levels of IgGs towards α-synuclein amyloid fibrils both in PD patients and controls, which subsided with the disease progression (P<0.0001). Pooled IgGs from PD patients and controls interacted also with the amyloid fibrils of Aβ (1–40) and hen lysozyme, however the latter were recognized with lower affinity. This suggests that IgGs bind to the generic amyloid conformational epitope, displaying higher specificity towards human amyloid species associated with neurodegeneration.

Conclusions/Significance

Our findings may suggest the protective role of autoimmunity in PD and therefore immune reactions towards PD major amyloid protein – α-synuclein can be of value in the development of treatment and diagnostic strategies, especially during the early disease stages.     

A blastocyst biopsy approach for preimplantation genetic diagnosis technique that affects the expression of SNAP-α in mice

Preimplantation genetic diagnosis (PGD) is a technique that is commonly used during assisted reproduction in the clinics to eliminate genetically abnormal embryos before implantation. The blastomere biopsy technique has risks related to the embryo, but blastocyst biopsy has not been systematically evaluated in relation to effects after birth, and the resulting offspring have not been followed up on. We designed a series of experiments to evaluate the risk of blastocyst biopsy on the resulting progeny. Mice were divided into a PGD group and a control group. The former was the progeny of mice that underwent blastocyst biopsy and the latter was delivered through a normal pregnancy without blastocyst biopsy. Each group consisted of 15 animals. We found no effects of blastocyst biopsy on reproductive capacities and weight gain. As for neurobehavioral evaluation between both groups, there were no significant differences in tail suspension test, sucrose preference test, the open field test and the elevated plus maze. Western blotting, immunohistochemistry and quantitative RT-PCR results showed that the expression levels of MBP, PRDX5 and UCHL1 in the PGD group were not significantly different compared to the control group, but SNAP-α expression in the PGD group was lower than that in control group. In summary, we concluded that blastocyst biopsy had no adverse effect on the general growth and behavior in mice. However, blastocyst biopsy effected the expression of SNAP-α. Therefore, the safety of blastocyst biopsy requires further evaluation.     

α-Synuclein modifies huntingtin aggregation in living cells

Several neurodegenerative disorders are characterized by the accumulation of proteinaceous inclusions in the central nervous system. These inclusions are frequently composed of a mixture of aggregation-prone proteins. Here, we used a bimolecular fluorescence complementation assay to study the initial steps of the co-aggregation of huntingtin (Htt) and α-synuclein (α-syn), two aggregation-prone proteins involved in Huntington's disease (HD) and Parkinson's disease (PD), respectively. We found that Htt (exon 1) oligomerized with α-syn and sequestered it in the cytosol. In turn, α-syn increased the number of cells displaying aggregates, decreased the number of aggregates per cell and increased the average size of the aggregates. Our results support the idea that co-aggregation of aggregation-prone proteins can contribute to the histopathology of neurodegenerative disorders.     

Gene expression of proteins of the vesicle cycle in dopaminergic neurons in modeling of Parkinson’s disease

It is assumed that one of the causes of the degeneration of dopaminergic neurons is the dysregulation of the vesicle cycle, which is ensured by a number of proteins including syntaxin I, synaptotagmin I, complexins I and II, and Rab5. It was shown that there is a compensatory increase in gene expression of proteins responsible for exocytosis at the preclinical stage of Parkinson’s disease (PD) in the in substantia nigra (SN) in mice. Conversely, in the model of the clinical stage of PD, the decreases of gene expression of proteins responsible for exocytosis, endocytosis, and neuronal survival, which may be among the triggers of motor dysfunctions.     

Ubiquitination of α-synuclein and autophagy in Parkinson's disease

α-synuclein is mutated in Parkinson's disease (PD) and is found in cytosolic inclusions, called Lewy bodies, in sporadic forms of the disease. A fraction of α-synuclein purified from Lewy bodies is monoubiquitinated, but the role of this monoubiquitination has been obscure. We now review recent data indicating a role of α-synuclein monoubiquitination in Lewy body formation and implicating the autophagic pathway in regulating these processes. The E3 ubiquitin-ligase SIAH is present in Lewy bodies and monoubiquitinates α-synuclein at the same lysines that are monoubiquitinated in Lewy bodies. Monoubiquitination by SIAH promotes the aggregation of α-synuclein into amorphous aggregates and increases the formation of inclusions within dopaminergic cells. Such effect is observed even at low monoubiquitination levels, suggesting that monoubiquitinated α-synuclein may work as a seed for aggregation. Accumulation of monoubiquitinated α-synuclein and formation of cytosolic inclusions is promoted by autophagy inhibition and to a lesser extent by proteasomal and lysosomal inhibition. Monoubiquitinated α-synuclein inclusions are toxic to cells and recruit PD-related proteins, such as synphilin-1 and UCH-L1. Altogether, the new data indicate that monoubiquitination might play an important role in Lewy body formation. Decreasing α-synuclein monoubiquitination, by preventing SIAH function or by stimulating autophagy, constitutes a new therapeutic strategy for Parkinson's disease.

Addendum to: Rott R, Szargel R, Haskin J, Shani V, Shainskaya A, Manov I, Liani E, Avraham E, Engelender S. Monoubiquitination of α-synuclein by SIAH promotes its aggregation in dopaminergic cells. J Biol Chem 2007; Epub ahead of print.     

A Spirulina-Enhanced Diet Provides Neuroprotection in an α-Synuclein Model of Parkinson's Disease

Inflammation in the brain plays a major role in neurodegenerative diseases. In particular, microglial cell activation is believed to be associated with the pathogenesis of neurodegenerative diseases, including Parkinson’s disease (PD). An increase in microglia activation has been shown in the substantia nigra pars compacta (SNpc) of PD models when there has been a decrease in tyrosine hydroxylase (TH) positive cells. This may be a sign of neurotoxicity due to prolonged activation of microglia in both early and late stages of disease progression. Natural products, such as spirulina, derived from blue green algae, are believed to help reverse this effect due to its anti-inflammatory/anti-oxidant properties. An adeno-associated virus vector (AAV9) for α-synuclein was injected in the substantia nigra of rats to model Parkinson''s disease and to study the effects of spirulina on the inflammatory response. One month prior to surgeries, rats were fed either a diet enhanced with spirulina or a control diet. Immunohistochemistry was analyzed with unbiased stereological methods to quantify lesion size and microglial activation. As hypothesized, spirulina was neuroprotective in this α-synuclein model of PD as more TH+ and NeuN+ cells were observed; spirulina concomitantly decreased the numbers of activated microglial cells as determined by MHCII expression. This decrease in microglia activation may have been due, in part, to the effect of spirulina to increase expression of the fractalkine receptor (CX3CR1) on microglia. With this study we hypothesize that α-synuclein neurotoxicity is mediated, at least in part, via an interaction with microglia. We observed a decrease in activated microglia in the rats that received a spirulina- enhanced diet concomitant to neuroprotection. The increase in CX3CR1 in the groups that received spirulina, suggests a potential mechanism of action.     

Timed high-fat diet in the evening affects the hepatic circadian clock and PPARα-mediated lipogenic gene expressions in mice

A long-term high-fat diet may result in a fatty liver. However, whether or not high-fat diets affect the hepatic circadian clock is controversial. The objective of this study is to investigate the effects of timed high-fat diet on the hepatic circadian clock and clock-controlled peroxisome proliferator-activated receptor (PPAR) α-mediated lipogenic gene expressions. Mice were orally administered high-fat milk in the evening for 4 weeks. The results showed that some hepatic clock genes, such as Clock, brain-muscle-Arnt-like 1 (Bmal1), Period 2 (Per2), and Cryptochrome 2 (Cry2) exhibited obvious changes in rhythms and/or amplitudes. Alterations in the expression of clock genes, in turn, further altered the circadian rhythm of PPARα expression. Among the PPARα target genes, cholesterol 7α-hydroxylase (CYP7A1), 3-hydroxy-3-methylglutaryl-coenzyme A reductase, low-density lipoprotein receptor, lipoprotein lipase, and diacylglycerol acyltransferase (DGAT) showed marked changes in rhythms and/or amplitudes. In particular, significant changes in the expressions of DGAT and CYP7A1 were observed. The effects of a high-fat diet on the expression of lipogenic genes in the liver were accompanied by increased hepatic cholesterol and triglyceride levels. These results suggest that timed high-fat diets at night could change the hepatic circadian expressions of clock genes Clock, Bmal1, Per2, and Cry2 and subsequently alter the circadian expression of PPARα-mediated lipogenic genes, resulting in hepatic lipid accumulation.     

Can cellular models revolutionize drug discovery in Parkinson's disease?

The study of mechanisms that underlie Parkinson's disease (PD), as well as translational drug development, has been hindered by the lack of appropriate models. Both cell culture systems and animal models have limitations, and to date none faithfully recapitulate all of the clinical and pathological phenotypes of the disease. In this review we examine the various cell culture model systems of PD, with a focus on different stem cell models that can be used for investigating disease mechanisms as well as drug discovery for PD. We conclude with a discussion of recent discoveries in the field of stem cell biology that have led to the ability to reprogram somatic cells to a pluripotent state via the use of a combination of genetic factors; these reprogrammed cells are termed “induced pluripotent stem cells” (iPSCs). This groundbreaking technique allows for the derivation of patient-specific cell lines from individuals with sporadic forms of PD and also those with known disease-causing mutations. Such cell lines have the potential to serve as a human cellular model of neurodegeneration and PD when differentiated into dopaminergic neurons. The hope is that these iPSC-derived dopaminergic neurons can be used to replicate the key molecular aspects of neural degeneration associated with PD. If so, this approach could lead to transformative new tools for the study of disease mechanisms. In addition, such cell lines can be potentially used for high-throughput drug screening. While not the focus of this review, ultimately it is envisioned that techniques for reprogramming of somatic cells may be optimized to a point sufficient to provide potential new avenues for stem cell-based restorative therapies.