Exploring Intrinsic Disorder in Human Synucleins and Associated Proteins.

In this work, we explored the intrinsic disorder status of the three members of the synuclein family of proteins-α-, ß-, and γ-synucleins-and showed that although all three human synucleins are highly disordered, the highest levels of disorder are observed in γ-synuclein. Our analysis of the peculiarities of the amino acid sequences and modeled 3D structures of the human synuclein family members revealed that the pathological mutations A30P, E46K, H50Q, A53T, and A53E associated with the early onset of Parkinson's disease caused some increase in the local disorder propensity of human α-synuclein. A comparative sequence-based analysis of the synuclein proteins from various evolutionary distant species and evaluation of their levels of intrinsic disorder using a set of commonly used bioinformatics tools revealed that, irrespective of their origin, all members of the synuclein family analyzed in this study were predicted to be highly disordered proteins, indicating that their intrinsically disordered nature represents an evolutionary conserved and therefore functionally important feature. A detailed functional disorder analysis of the proteins in the interactomes of the human synuclein family members utilizing a set of commonly used disorder analysis tools showed that the human α-synuclein interactome has relatively higher levels of intrinsic disorder as compared with the interactomes of human ß- and γ- synucleins and revealed that, relative to the ß- and γ-synuclein interactomes, α-synuclein interactors are involved in a much broader spectrum of highly diversified functional pathways. Although proteins interacting with three human synucleins were characterized by highly diversified functionalities, this analysis also revealed that the interactors of three human synucleins were involved in three common functional pathways, such as the synaptic vesicle cycle, serotonergic synapse, and retrograde endocannabinoid signaling. Taken together, these observations highlight the critical importance of the intrinsic disorder of human synucleins and their interactors in various neuronal processes.

Brain Region-Specific Expression Levels of Synuclein Genes in an Acid Sphingomyelinase Knockout Mouse Model: Correlation with Depression-/Anxiety-Like Behavior and Locomotor Activity in the Absence of Genotypic Variation.

Accumulating evidence suggests an involvement of sphingolipids, vital components of cell membranes and regulators of cellular processes, in the pathophysiology of both Parkinson's disease and major depressive disorder, indicating a potential common pathway in these neuropsychiatric conditions. Based on this interaction of sphingolipids and synuclein proteins, we explored the gene expression patterns of α-, ß-, and γ-synuclein in a knockout mouse model deficient for acid sphingomyelinase (ASM), an enzyme catalyzing the hydrolysis of sphingomyelin to ceramide, and studied associations with behavioral parameters. Normalized Snca, Sncb, and Sncg gene expression was determined by quantitative PCR in twelve brain regions of sex-mixed homozygous (ASM-/-, n = 7) and heterozygous (ASM+/-, n = 7) ASM-deficient mice, along with wild-type controls (ASM+/+, n = 5). The expression of all three synuclein genes was brain region-specific but independent of ASM genotype, with ß-synuclein showing overall higher levels and the least variation. Moreover, we discovered correlations of gene expression levels between brain regions and depression- and anxiety-like behavior and locomotor activity, such as a positive association between Snca mRNA levels and locomotion. Our results suggest that the analysis of synuclein genes could be valuable in identifying biomarkers and comprehending the common pathological mechanisms underlying various neuropsychiatric disorders.

Synuclein in neurodegeneration.

Neurodegenerative diseases are a heterogeneous group of disorders characterized by gradual progressive neuronal loss in the central nervous system. Unfortunately, the pathogenesis of many of these diseases remains unknown. Synucleins are a family of small, highly charged proteins expressed predominantly in neurons. Following their discovery, much has been learned about their structure, function, interaction with other proteins and role in neurodegenerative disease over the last two decades. One of these proteins, α-Synuclein (α-Syn), appears to be involved in many neurodegenerative disorders. These include Parkinson's disease (PD), dementia with Lewy bodies (DLB), Rapid Eye Movement Sleep Behavior Disorder (RBD) and Pure Autonomic Failure (PAF), i.e., collectively termed α-synucleinopathies. This review focuses on α-Syn dysfunction in neurodegeneration and assesses its role in synucleinopathies from a biochemical, genetic and neuroimaging perspective.

Triple-Knockout, Synuclein-Free Mice Display Compromised Lipid Pattern.

Recent studies have implicated synucleins in several reactions during the biosynthesis of lipids and fatty acids in addition to their recognised role in membrane lipid binding and synaptic functions. These are among aspects of decreased synuclein functions that are still poorly acknowledged especially in regard to pathogenesis in Parkinson's disease. Here, we aimed to add to existing knowledge of synuclein deficiency (i.e., the lack of all three family members), with respect to changes in fatty acids and lipids in plasma, liver, and two brain regions in triple synuclein-knockout (TKO) mice. We describe changes of long-chain polyunsaturated fatty acids (LCPUFA) and palmitic acid in liver and plasma, reduced triacylglycerol (TAG) accumulation in liver and non-esterified fatty acids in plasma of synuclein free mice. In midbrain, we observed counterbalanced changes in the relative concentrations of phosphatidylcholine (PC) and cerebrosides (CER). We also recorded a notable reduction in ethanolamine plasmalogens in the midbrain of synuclein free mice, which is an important finding since the abnormal ether lipid metabolism usually associated with neurological disorders. In summary, our data demonstrates that synuclein deficiency results in alterations of the PUFA synthesis, storage lipid accumulation in the liver, and the reduction of plasmalogens and CER, those polar lipids which are principal compounds of lipid rafts in many tissues. An ablation of all three synuclein family members causes more profound changes in lipid metabolism than changes previously shown to be associated with γ-synuclein deficiency alone. Possible mechanisms by which synuclein deficiency may govern the reported modifications of lipid metabolism in TKO mice are proposed and discussed.

NF­κB is negatively associated with Nurr1 to reduce the inflammatory response in Parkinson's disease.

Parkinson's disease (PD) is one of the most disabling diseases of the central nervous system, seriously affecting health and quality of life for the elderly. The present study aimed to explore the effects of nuclear receptor subfamily 4 group A member 2 (Nurr1) and nuclear factor­κB (NF­κB) on the progression of Parkinson's disease (PD). Pheochromocytoma (PC12) cells were pretreated with the NF­κB inhibitor quinazoline (QNZ) or transfected with small interfering (si)RNA­NF­κB, followed by the addition of lipopolysaccharide (LPS). After culturing for 24 h, Cell Counting Kit­8 (CCK­8) was utilized to measure cell viability. Next, the expression levels of interleukin (IL)­1ß, IL­6 and tumor necrosis factor (TNF)­α were determined using the relevant Enzyme­linked immunosorbent assay kits. Expression levels of p65, tyrosine hydroxylase (TH), α­Synuclein (A­SYN) and Nurr1 were examined by immunofluorescence and western blotting. CCK­8 results showed that the cell viability was significantly reduced in the LPS group than in the control group (P<0.05), whereas QNZ and si­NF­κB demonstrated significantly enhanced viability induced by LPS (P<0.05). After LPS induction, the levels of IL­1ß, IL­6 and TNF­α were significantly elevated when compared with those in the control group (P<0.05), whereas QNZ and NF­κB interference partially restored their levels. Additionally, after LPS induction, the expression of p65 and A­SYN was higher, while the expression of TH and Nurr1 was lower. However, QNZ and NF­κB treatment significantly reversed the expression levels induced by LPS (P<0.05). Finally, it was observed that NF­κB may be negatively associated with Nurr1. In conclusion, inhibition of NF­κB may reduce the production of inflammatory factors by upregulating Nurr1 and TH and downregulating A­SYN, thus relieving the inflammatory response in PD.

The Membrane Interactions of Synuclein: Physiology and Pathology.

Specific proteins accumulate in neurodegenerative disease, and human genetics has indicated a causative role for many. In most cases, however, the mechanisms remain poorly understood. Degeneration is thought to involve a gain of abnormal function, although we do not know the normal function of many proteins implicated. The protein α-synuclein accumulates in the Lewy pathology of Parkinson's disease and related disorders, and mutations in α-synuclein cause degeneration, but we have not known its normal function or how it triggers disease. α-Synuclein localizes to presynaptic boutons and interacts with membranes in vitro. Overexpression slows synaptic vesicle exocytosis, and recent data suggest a normal role for the endogenous synucleins in dilation of the exocytic fusion pore. Disrupted membranes also appear surprisingly prominent in Lewy pathology. Synuclein thus interacts with membranes under both physiological and pathological conditions, suggesting that the normal function of synuclein may illuminate its role in degeneration.

Genetics of synucleins in neurodegenerative diseases.

The SNCA locus currently has an indisputable role in Parkinson's disease and other synucleinopathies. The role of genetic variability in the other members of the synuclein family (SNCB and SNCG) in disease is far less clear. In this review, we critically assess the pathogenicity, main characteristics, and roles of genetic variants in these genes reported to be causative of synucleinopathies. We also summarize the different association signals identified in the SNCA locus that have been associated with risk for disease. We take a bird's eye view of the variability currently reported in the general population for the three genes and use these data to infer on the potential relationship between each of the genes and human disease.

Synuclein Deficiency Decreases the Efficiency of Dopamine Uptake by Synaptic Vesicles.

In the present study, we analyzed the uptake of radiolabeled dopamine by intact synaptosomes and purified synaptic vesicles isolated from the dorsal striatum of mice with constitutive inactivation of all three synuclein-coding genes and wild-type mice. Synuclein deficiency substantially compromised the uptake of this neurotransmitter by synaptic vesicles but had no effect on synaptosomal dopamine uptake.

Role of tangeritin against cognitive impairments in transgenic Drosophila model of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease. As there is no permanent cure for the disease, the use of herbal compounds with antioxidant potential will be an effective approach for controlling the progression of disease. In this context the effect of tangeritin (a polymethoxy flavone concentrated in the peels of citrus fruits) was studied at final doses of 5, 10 and 20 µM on PD model flies. The doses were established in diet and the PD flies were allowed to feed on it for 24 days. The effect was studied on cognitive impairments. Immunostaining of brain sections for tyrosine hydroxylase was also performed. The docking studies were also carried out to give a plausible binding site of tangeritin on alpha synuclein molecule. The results of the study showed that tangeritin is effective in improving the cognitive impairments.

Therapeutic efficacy of regulable GDNF expression for Huntington's and Parkinson's disease by a high-induction, background-free "GeneSwitch" vector.

Gene therapy is currently an irreversible approach, without possibilities to fine-tune or halt the expression of a therapeutic gene product. Especially when expressing neurotrophic factors to treat neurodegenerative disorders, options to regulate transgene expression levels might be beneficial. We thus developed an advanced single-genome inducible AAV vector for expression of GDNF, under control of the approved small molecule drug mifepristone. In the rat brain, GDNF expression can be induced over a wide range up to three hundred-fold over endogenous background, and completely returns to baseline within 3-4 weeks. When applied with appropriate serotype and titre, the vector is absolutely free of any non-induced background expression. In the BACHD model of Huntington's disease we demonstrate that the vector can be kept in a continuous ON-state for extended periods of time. In a model of Parkinson's disease we demonstrate that repeated short-term expression of GDNF restores motor capabilities in 6-OHDA-lesioned rats. We also report on sex-dependent pharmacodynamics of mifepristone in the rodent brain. Taken together, we show that wide-range and high-level induction, background-free, fully reversible and therapeutically active GDNF expression can be achieved under tight pharmacological control by this novel AAV - "Gene Switch" vector.

GBA-Associated Parkinson's Disease and Other Synucleinopathies.

PURPOSE OF REVIEW: GBA mutations are the most common known genetic cause of Parkinson's disease (PD). Its biological pathway may be important in idiopathic PD, since activity of the enzyme encoded by GBA, glucocerebrosidase, is reduced even among PD patients without GBA mutations. This article describes the structure and function of GBA, reviews recent literature on the clinical phenotype of GBA PD, and suggests future directions for research, counseling, and treatment. RECENT FINDINGS: Several longitudinal studies have shown that GBA PD has faster motor and cognitive progression than idiopathic PD and that this effect is dose dependent. New evidence suggests that GBA mutations may be important in multiple system atrophy. Further, new interventional studies focusing on GBA PD are described. These studies may increase the interest of PD patients and caregivers in genetic counseling. GBA mutation status may help clinicians estimate PD progression, though mechanisms underlying GBA and synucleinopathy require further understanding.

The GBA p.Trp378Gly mutation is a probable French-Canadian founder mutation causing Gaucher disease and synucleinopathies.

Biallelic GBA mutations cause Gaucher disease (GD), and heterozygous carriers are at risk for synucleinopathies. No founder GBA mutations in French-Canadians are known. GBA was fully sequenced using targeted next generation and Sanger sequencing in French-Canadian Parkinson disease (PD) patients (n = 436), rapid eye movement (REM)-sleep behavior disorder (RBD) patients (n = 189) and controls (n = 891). Haplotype, identity-by-descent (IBD) and principal component analyses (PCA) were performed using single nucleotide polymorphism-chip data. Data on GD patients from Toronto and Montreal were collected from patients' files. A GBA p.Trp378Gly mutation was identified in two RBD and four PD patients (1% of all patients combined), and not in controls. The two RBD patients had converted to DLB within 3 years of their diagnosis. Haplotype, IBD and PCA analysis demonstrated that this mutation is from a single founder. Out of 167 GD patients screened, 15 (9.0%) carried the p.Trp378Gly mutation, all in trans with p.Asn370Ser. Three (20%) of the GD patients with the p.Trp378Gly mutation had developed Parkinsonism, and 11 patients had family history of PD. The p.Trp378Gly mutation is the first French-Canadian founder GBA mutation to be described, which leads to synucleinopathies and to GD type 1 when in compound heterozygosity with p.Asn370Ser.

Synuclein impairs trafficking and signaling of BDNF in a mouse model of Parkinson's disease.

Recent studies have demonstrated that hyperphosphorylation of tau protein plays a role in neuronal toxicities of α-synuclein (ASYN) in neurodegenerative disease such as familial Alzheimer's disease (AD), dementia with Lewy bodies (DLB) and Parkinson's disease. Using a transgenic mouse model of Parkinson's disease (PD) that expresses GFP-ASYN driven by the PDGF-ß promoter, we investigated how accumulation of ASYN impacted axonal function. We found that retrograde axonal trafficking of brain-derived neurotrophic factor (BDNF) in DIV7 cultures of E18 cortical neurons was markedly impaired at the embryonic stage, even though hyperphosphorylation of tau was not detectable in these neurons at this stage. Interestingly, we found that overexpressed ASYN interacted with dynein and induced a significant increase in the activated levels of small Rab GTPases such as Rab5 and Rab7, both key regulators of endocytic processes. Furthermore, expression of ASYN resulted in neuronal atrophy in DIV7 cortical cultures of either from E18 transgenic mouse model or from rat E18 embryos that were transiently transfected with ASYN-GFP for 72 hrs. Our studies suggest that excessive ASYN likely alters endocytic pathways leading to axonal dysfunction in embryonic cortical neurons in PD mouse models.

Neurotoxicity of the Parkinson Disease-Associated Pesticide Ziram Is Synuclein-Dependent in Zebrafish Embryos.

BACKGROUND: Exposure to the commonly used dithiocarbamate (DTC) pesticides is associated with an increased risk of developing Parkinson disease (PD), although the mechanisms by which they exert their toxicity are not completely understood. OBJECTIVE: We studied the mechanisms of ziram's (a DTC fungicide) neurotoxicity in vivo. METHODS: Zebrafish (ZF) embryos were utilized to determine ziram's effects on behavior, neuronal toxicity, and the role of synuclein in its toxicity. RESULTS: Nanomolar-range concentrations of ziram caused selective loss of dopaminergic (DA) neurons and impaired swimming behavior. Because ziram increases α-synuclein (α-syn) concentrations in rat primary neuronal cultures, we investigated the effect of ziram on ZF γ-synuclein 1 (γ1). ZF express 3 synuclein isoforms, and ZF γ1 appears to be the closest functional homologue to α-syn. We found that recombinant ZF γ1 formed fibrils in vitro, and overexpression of ZF γ1 in ZF embryos led to the formation of neuronal aggregates and neurotoxicity in a manner similar to that of α-syn. Importantly, knockdown of ZF γ1 with morpholinos and disruption of oligomers with the molecular tweezer CLR01 prevented ziram's DA toxicity. CONCLUSIONS: These data show that ziram is selectively toxic to DA neurons in vivo, and this toxicity is synuclein-dependent. These findings have important implications for understanding the mechanisms by which pesticides may cause PD. Citation: Lulla A, Barnhill L, Bitan G, Ivanova MI, Nguyen B, O'Donnell K, Stahl MC, Yamashiro C, Klärner FG, Schrader T, Sagasti A, Bronstein JM. 2016. Neurotoxicity of the Parkinson disease-associated pesticide ziram is synuclein-dependent in zebrafish embryos. Environ Health Perspect 124:1766-1775; http://dx.doi.org/10.1289/EHP141.

SNCA Gene Polymorphism may Contribute to an Increased Risk of Alzheimer's Disease.

BACKGROUND: The purpose of this study is to elucidate the association between α-synuclein (SNCA) polymorphisms and the risk of Alzheimer's disease (AD). METHODS: The PCR-RFLP was applied to detect SNCA gene rs6532190, rs3775430, and rs10516846 polymorphisms in 98 AD patients and 105 healthy elderly. RESULTS: The GG frequency of rs10516846 was evidently increased in AD group than control group (P < 0.05). There was a significant difference in SNCA level between the AD and control groups (P < 0.01). In the AD group, the SNCA level in cerebrospinal fluid of GG (rs10516846) carriers was increased as compared with AA carriers (P < 0.05). The GG (rs10516846) frequency of the early-onset AD group is significantly higher than that of the late-onset AD group (P < 0.05). The frequency of rs3775430 GG was lower in the early-onset group than that in the late-onset group (0% vs. 16.7%). The SNCA level in cerebrospinal fluid of GG (rs10516846) carriers in the early-onset AD group is higher than that of AA carriers (P < 0.05). CONCLUSION: SNCA gene polymorphism may be associated with an increased risk of AD and GG genotype of rs10516846 and elevated SNCA level in CSF may increase the risk of early-onset AD.

Progress and potential of non-inhibitory small molecule chaperones for the treatment of Gaucher disease and its implications for Parkinson disease.

Gaucher disease, caused by pathological mutations GBA1, encodes the lysosome-resident enzyme glucocerebrosidase, which cleaves glucosylceramide into glucose and ceramide. In Gaucher disease, glucocerebrosidase deficiency leads to lysosomal accumulation of substrate, primarily in cells of the reticulo-endothelial system. Gaucher disease has broad clinical heterogeneity, and mutations in GBA1 are a risk factor for the development of different synucleinopathies. Insights into the cell biology and biochemistry of glucocerebrosidase have led to new therapeutic approaches for Gaucher disease including small chemical chaperones. Such chaperones facilitate proper enzyme folding and translocation to lysosomes, thereby preventing premature breakdown of the enzyme in the proteasome. This review discusses recent progress in developing chemical chaperones as a therapy for Gaucher disease, with implications for the treatment of synucleinopathies. It focuses on the development of non-inhibitory glucocerebrosidase chaperones and their therapeutic advantages over inhibitory chaperones, as well as the challenges involved in identifying and validating chemical chaperones.

Repurposing the NRF2 Activator Dimethyl Fumarate as Therapy Against Synucleinopathy in Parkinson's Disease.

AIMS: This preclinical study was aimed at determining whether pharmacological targeting of transcription factor NRF2, a master controller of many homeostatic genes, might provide a disease-modifying therapy in the animal model of Parkinson's disease (PD) that best reproduces the main hallmark of this pathology, that is, α-synucleinopathy, and associated events, including nigral dopaminergic cell death, oxidative stress, and neuroinflammation. RESULTS: Pharmacological activation of NRF2 was achieved at the basal ganglia by repurposing dimethyl fumarate (DMF), a drug already in use for the treatment of multiple sclerosis. Daily oral gavage of DMF protected nigral dopaminergic neurons against α-SYN toxicity and decreased astrocytosis and microgliosis after 1, 3, and 8 weeks from stereotaxic delivery to the ventral midbrain of recombinant adeno-associated viral vector expressing human α-synuclein. This protective effect was not observed in Nrf2-knockout mice. In vitro studies indicated that this neuroprotective effect was correlated with altered regulation of autophagy markers SQTSM1/p62 and LC3 in MN9D, BV2, and IMA 2.1 and with a shift in microglial dynamics toward a less pro-inflammatory and a more wound-healing phenotype. In postmortem samples of PD patients, the cytoprotective proteins associated with NRF2 expression, NQO1 and p62, were partly sequestered in Lewy bodies, suggesting impaired neuroprotective capacity of the NRF2 signature. INNOVATION: These experiments provide a compelling rationale for targeting NRF2 with DMF as a therapeutic strategy to reinforce endogenous brain defense mechanisms against PD-associated synucleinopathy. CONCLUSION: DMF is ready for clinical validation in PD. Antioxid. Redox Signal. 25, 61-77.

Phage M13 for the treatment of Alzheimer and Parkinson disease.

The studies of microbes have been instrumental in combatting infectious diseases, but they have also led to great insights into basic biological mechanism like DNA replication, transcription, and translation of mRNA. In particular, the studies of bacterial viruses, also called bacteriophage, have been quite useful to study specific cellular processes because of the ease to isolate their DNA, mRNA, and proteins. Here, I review the recent discovery of how properties of the filamentous phage M13 emerge as a novel approach to combat neurodegenerative diseases.

Protection of mice against pandemic H1N1 influenza virus challenge after immunization with baculovirus-expressed stabilizing peptide fusion hemagglutinin protein.

Current influenza vaccines are produced in embryonated chicken eggs. However, egg-based vaccines have various problems. To address these problems, recombinant protein vaccines have been developed as new vaccine candidates. Unfortunately, recombinant proteins frequently encounter aggregation and low stability during their biogenesis. It has been previously demonstrated that recombinantly expressed proteins can be greatly stabilized with high solubility by fusing stabilizing peptide (SP) derived from the C-terminal acidic tail of human synuclein (ATS). To investigate whether SP fusion proteins can induce protective immunity in mice, we produced influenza HA and SP fusion protein using a baculovirus expression system. In in vitro tests, SP-fused recombinant HA1 (SP-rHA1) was shown to be more stable than recombinant HA1 (rHA1). Mice were immunized intramuscularly with baculovirus-expressed rHA1 protein or SP-rHA1 protein (2 µg/mouse) formulated with aluminum hydroxide. Antibody responses were determined by ELISA and hemagglutination inhibition assay. We observed that SP-rHA1 immunization elicited HA-specific antibody responses that were comparable to rHA1 immunization. These results indicate that fusion of SP to rHA1 does not negatively affect the immunogenicity of the vaccine candidate. Therefore, it is possible to apply SP fusion technology to develop stable recombinant protein vaccines with high solubility.

REM sleep behaviour disorder: how useful is it for the differential diagnosis of parkinsonism?

BACKGROUND: REM sleep behaviour disorder (RBD) is typically linked to synucleinopathies (SP). In this study we analyzed the utility and performance of RBD as a tool for the differential diagnosis of the most common forms of degenerative parkinsonism, including SPs and tauopathies. METHODS: Patients with a syndromic diagnosis of degenerative parkinsonism matched for gender, age, and disease stage were assessed using a structured protocol with demographic and clinical data, including the diagnosis of probable RBD (pRBD), ascertained clinically using established criteria. RESULTS: One hundred cases of Parkinson's disease (PD), 87 with progressive supranuclear palsy (PSP), 72 with the parkinsonian form of multiple system atrophy (MSA), 50 with dementia with Lewy bodies (DLB), and 18 with corticobasal degeneration (CBD) were included. pRBD was found in 58 (58%) of the PD patients, 59 (81.9%) of those with MSA, 37 (74%) with DLB, 32 (36.7%) with PSP, and one (5.5%) with CBD. Among the SPs, pRBD was significantly more common in MSA when compared with PD patients. Differences were also significant individually for all SPs when compared to PSP. The positive predictive value (PPV) of pRBD for a SP was 82.3%, but sensitivity was 69.4% and specificity 68.6%. CONCLUSIONS: In our sample, pRBD was more frequent in SPs than in PSP and CBD, however, its' frequency in PSP was significant. Although pRBD had a good PPV for a SP, all other measurements used for determine diagnostic performance were disappointing.