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.     

Environmental toxins and α-synuclein in Parkinson’s disease

In recent years, environmental influences have been thought to play an important role in Parkinson’s disease (PD). Evidence from epidemiological investigations suggests that environmental factors might take part in the disease process. Intriguingly, most of environmental toxins share the common mechanism of causing mitochondria dysfunction by inhibiting complex I and promoting α-synuclein aggregation, a key factor in PD. Therefore, understanding the mechanism of interactions between α-synuclein and environmental factors could lead to new therapeutic approaches to PD.     

Synphilin suppresses α-synuclein neurotoxicity in a Parkinson's disease Drosophila model

Parkinson's disease (PD) is the second most common neurodegenerative disorder in humans. It affects 1% of the population over 65-years old. Its causes are environmental and genetic. As the world population ages, there is an urgent need for better and more detailed animal models for this kind of disease. In this work we show that the use of transgenic Drosophila is comparable to more complicated and costly animal models such as mice. The Drosophila model behaves very similar to the equivalent transgenic mice model. We show that both Synphilin-1 and α-synuclein are toxic by themselves, but when co-expressed, they suppress their toxicity reciprocally. Importantly, the symptoms induced in the fly can be treated and partially reverted using standard PD pharmacological treatments. This work showcases Drosophila as a detailed and multifaceted model for Parkinson's disease, providing a convenient platform in which to study and find new genetic modifiers of PD. genesis 49:392-402, 2011.     

Proteomic analysis of dopamine and α-synuclein interplay in a cellular model of Parkinson's disease pathogenesis

Altered dopamine homeostasis is an accepted mechanism in the pathogenesis of Parkinson's disease. α-Synuclein overexpression and impaired disposal contribute to this mechanism. However, biochemical alterations associated with the interplay of cytosolic dopamine and increased α-synuclein are still unclear. Catecholaminergic SH-SY5Y human neuroblastoma cells are a suitable model for investigating dopamine toxicity. In the present study, we report the proteomic pattern of SH-SY5Y cells overexpressing α-synuclein (1.6-fold induction) after dopamine exposure. Dopamine itself is able to upregulate α-synuclein expression. However, the effect is not observed in cells that already overexpress α-synuclein as a consequence of transfection. The proteomic analysis highlights significant changes in 23 proteins linked to specific cellular processes, such as cytoskeleton structure and regulation, mitochondrial function, energetic metabolism, protein synthesis, and neuronal plasticity. A bioinformatic network enrichment procedure generates a significant model encompassing all proteins and allows us to enrich functional categories associated with the combination of factors analyzed in the present study (i.e. dopamine together with α-synuclein). In particular, the model suggests a potential involvement of the nuclear factor kappa B pathway that is experimentally confirmed. Indeed, α-synuclein significantly reduces nuclear factor kappa B activation, which is completely quenched by dopamine treatment.     

Beyond α-synuclein transfer: pathology propagation in Parkinson's disease

α-Synuclein (α-syn) is the most abundant protein found in Lewy bodies, a hallmark of Parkinson's disease (PD), and can aggregate to form toxic oligomers and fibrillar structures. Recent studies have shown that α-syn can be transmitted between neurons and can seed the formation of toxic aggregates in recipient neurons in a prion-like manner. In addition, it is known that Lewy body pathology may spread gradually and systematically from the peripheral or enteric nervous system or olfactory bulb to specific brain regions during progression of idiopathic PD. It is therefore conceivable that α-syn species could act as seeds that drive PD progression. Here, we review recent advances from studies of α-syn cell-to-cell transfer, the current understanding of α-syn toxicity, and how these relate to progression of PD pathology.     

Influence of microRNA deregulation on chaperone-mediated autophagy and α-synuclein pathology in Parkinson's disease

The presence of α-synuclein aggregates in the characteristic Lewy body pathology seen in idiopathic Parkinson''s disease (PD), together with α-synuclein gene mutations in familial PD, places α-synuclein at the center of PD pathogenesis. Decreased levels of the chaperone-mediated autophagy (CMA) proteins LAMP-2A and hsc70 in PD brain samples suggests compromised α-synuclein degradation by CMA may underpin the Lewy body pathology. Decreased CMA protein levels were not secondary to the various pathological changes associated with PD, including mitochondrial respiratory chain dysfunction, increased oxidative stress and proteasomal inhibition. However, decreased hsc70 and LAMP-2A protein levels in PD brains were associated with decreases in their respective mRNA levels. MicroRNA (miRNA) deregulation has been reported in PD brains and we have identified eight miRNAs predicted to regulate LAMP-2A or hsc70 expression that were reported to be increased in PD. Using a luciferase reporter assay in SH-SY5Y cells, four and three of these miRNAs significantly decreased luciferase activity expressed upstream of the lamp-2a and hsc70 3′UTR sequences respectively. We confirmed that transfection of these miRNAs also decreased endogenous LAMP-2A and hsc70 protein levels respectively and resulted in significant α-synuclein accumulation. The analysis of PD brains confirmed that six and two of these miRNAs were significantly increased in substantia nigra compacta and amygdala respectively. These data support the hypothesis that decreased CMA caused by miRNA-induced downregulation of CMA proteins plays an important role in the α-synuclein pathology associated with PD, and opens up a new avenue to investigate PD pathogenesis.     

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.     

Gaucher disease and brucella: just a mere coincidence?

Gaucher disease type I and brucellosis are chronic diseases with similar symptoms and physical signs though the former is the most common lysosomal storage disease and the latter is an infectious disease. The similarities between these diseases make differential diagnosis difficult. Immunodeficiency is a feature of Gaucher disease type I and increases the susceptibility towards infections. A Gaucher disease type I patient with brucellosis is presented with improvement after treatment of brucellosis.     

Lrrk2 interaction with α-synuclein in diffuse Lewy body disease

Mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are the leading cause of genetically inherited Parkinson’s disease (PD) and its more severe variant diffuse Lewy body disease (DLB). Pathological mutations in Lrrk2 are autosomal dominant, suggesting a gain of function. Mutations in α-synuclein also produce autosomal dominant disease. Here we report an interaction between Lrrk2 and α-synuclein in a series of diffuse Lewy body (DLB) cases and in an oxidative stress cell based assay. All five cases of DLB, but none of five controls, showed co-immunoprecipitation of Lrrk2 and α-synuclein in soluble brain extracts. Colocalization was also found in pathological deposits in DLB postmortem brains by double immunostaining. In HEK cells transfected simultaneously with plasmids expressing Lrrk2 and α-synuclein, co-immunoprecipitation of Lrrk2 and α-synuclein was detected when they were exposed to oxidative stress by H₂O₂. Taken together, these results suggest the possibility that in PD and related synucleinopathies, oxidative stress upregulates α-syn and Lrrk2 expression, paving the way for pathological interactions. New therapeutic approaches to PD and the synucleinopathies may result from limiting the interaction between Lrrk2 and α-synuclein.     

A feedforward loop links Gaucher and Parkinson's diseases?

Mutations in the GBA gene that encodes glucocerebrosidase cause the lysosomal storage disorder Gaucher disease but also increase the risk for Parkinson's disease. Mazzulli et?al. (2011) uncover a possible mechanism to explain this connection: loss of glucocerebrosidase creates a positive feedback loop of reduced lysosomal function and α-synuclein accumulation, ultimately leading to neurodegeneration.     

Synthesis and in vitro evaluation of α-synuclein ligands

Accumulation of misfolded α-synuclein in Lewy bodies and Lewy neurites is the pathological hallmark of Parkinson's disease (PD). To identify ligands having high binding potency toward aggregated α-synuclein, we synthesized a series of phenothiazine derivatives and assessed their binding affinity to recombinant α-synuclein fibrils using a fluorescent thioflavin T competition assay. Among 16 new analogues, the in vitro data suggest that compound 11b has high affinity to α-synuclein fibrils (K(i)=32.10 ± 1.25 nM) and compounds 11d, 16a and16b have moderate affinity to α-synuclein fibrils (K(i)≈50-100 nM). Further optimization of the structure of these analogues may yield compounds with high affinity and selectivity for aggregated α-synuclein.     

Inhibition of formation of α-synuclein inclusions by mannosylglycerate in a yeast model of Parkinson's disease

Background

Protein aggregation in the brain is a central hallmark in many neurodegenerative diseases. In Parkinson's disease, α-synuclein (α-Syn) is the major component of the intraneuronal inclusions found in the brains of patients. Current therapeutics is merely symptomatic, and there is a pressing need for developing novel therapies. Previously we showed that mannosylglycerate (MG), a compatible solute typical of marine microorganisms thriving in hot environments, is highly effective in protecting a variety of model proteins against thermal denaturation and aggregation in vitro.

Methods

Saccharomyces cerevisiae cells expressing eGFP-tagged α-Syn, were further engineered to synthesize MG. The number of cells with fluorescent foci was assessed by fluorescence microscopy. Fluorescence spectroscopy and transmission electron microscopy were used to monitor fibril formation in vitro.

Results

We observed a 3.3-fold reduction in the number of cells with α-Syn foci and mild attenuation of α-Syn-induced toxicity. Accordingly, sucrose gradient analysis confirmed a clear reduction in the size-range of α-Syn species in the cells. MG did not affect the expression levels of α-Syn or its degradation rate. Moreover, MG did not induce molecular chaperones (Hsp104, Hsp70 and Hsp40), suggesting the implication of other mechanisms for α-Syn stabilization. MG also inhibited α-Syn fibrillation in vitro.

Conclusions

MG acts as a chemical chaperone and the stabilization mechanism involves direct solute/protein interactions.

General significance

This is the first demonstration of the anti-aggregating ability of MG in the intracellular milieu. The work shows that MG is a good candidate to inspire the development of new drugs for protein-misfolding diseases.     

Resistance to MPTP-neurotoxicity in α-synuclein knockout mice is complemented by human α-synuclein and associated with increased β-synuclein and Akt activation

Genetic and biochemical abnormalities of α-synuclein are associated with the pathogenesis of Parkinson's disease. In the present study we investigated the in vivo interaction of mouse and human α-synuclein with the potent parkinsonian neurotoxin, MPTP. We find that while lack of mouse α-synuclein in mice is associated with reduced vulnerability to MPTP, increased levels of human α-synuclein expression is not associated with obvious changes in the vulnerability of dopaminergic neurons to MPTP. However, expressing human α-synuclein variants (human wild type or A53T) in the α-synuclein null mice completely restores the vulnerability of nigral dopaminergic neurons to MPTP. These results indicate that human α-synuclein can functionally replace mouse α-synuclein in regard to vulnerability of dopaminergic neurons to MPTP-toxicity. Significantly, α-synuclein null mice and wild type mice were equally sensitive to neurodegeneration induced by 2'NH(2)-MPTP, a MPTP analog that is selective for serotoninergic and noradrenergic neurons. These results suggest that effects of α-synuclein on MPTP like compounds are selective for nigral dopaminergic neurons. Immunoblot analysis of β-synuclein and Akt levels in the mice reveals selective increases in β-synuclein and phosphorylated Akt levels in ventral midbrain, but not in other brain regions, of α-synuclein null mice, implicating the α-synuclein-level dependent regulation of β-synuclein expression in modulation of MPTP-toxicity by α-synuclein. Together these findings provide new mechanistic insights on the role α-synuclein in modulating neurodegenerative phenotypes by regulation of Akt-mediated cell survival signaling in vivo.     

Dopamine quinones interact with α-synuclein to form unstructured adducts

α-Synuclein (αsyn) fibril formation is considered a central event in the pathogenesis of Parkinson’s disease (PD). In recent years, it has been proposed that prefibrillar annular oligomeric β-sheet-rich species, called protofibrils, rather than fibrils themselves, may be the neurotoxic species. The oxidation products of dopamine (DAQ) can inhibit αsyn fibril formation supporting the idea that DAQ might stabilize αsyn protofibrils.In the present work, through different biochemical and biophysical techniques, we isolated and structurally characterized αsyn/DAQ adducts. Contrary to protofibrils, we demonstrated that αsyn/DAQ adducts retain an unfolded conformation. We then investigated the nature of the modifications induced on αsyn by DAQ. Our results indicate that only a small fraction of αsyn interacts with DAQ in a covalent way, so that non-covalent interaction appears to be the major modification induced by DAQ on αsyn.     

The Parkinson disease protein α-synuclein inhibits autophagy

Parkinson disease (PD) is the most common movement disorder affecting people. It is characterized by the accumulation of the protein α-synuclein in Lewy body inclusions in vulnerable neurons. α-Synuclein overexpression caused by gene multiplications is sufficient to cause this disease, suggesting that α-synuclein accumulation is toxic. Here we review our recent study showing that α-synuclein inhibits autophagy. We discuss our mechanistic understanding of this phenomenon and also speculate how a deficiency in autophagy may contribute to a range of pleiotropic features of PD biology.     

The Parkinson disease protein α-synuclein inhibits autophagy

Parkinson disease (PD) is the most common movement disorder affecting people. It is characterized by the accumulation of the protein α-synuclein in Lewy body inclusions in vulnerable neurons. α-Synuclein overexpression caused by gene multiplications is sufficient to cause this disease, suggesting that α-synuclein accumulation is toxic. Here we review our recent study showing that α-synuclein inhibits autophagy. We discuss our mechanistic understanding of this phenomenon and also speculate how a deficiency in autophagy may contribute to a range of pleiotropic features of PD biology.