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.     

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.     

Oxidized quercetin inhibits α-synuclein fibrillization

Background

α-Synucein is a small (14 kDa), abundant, intrinsically disordered presynaptic protein, whose aggregation is believed to be a critical step in Parkinson's disease (PD). Oxidative stress is reported to be a risk factor for dopamine cell degeneration in PD. Flavonoids are suggested to be important antioxidant against oxidative stress. Flavonoids were reported to inhibit fibrillization and disaggregate the preformed fibrils of α-synucein, but the molecular mechanism was still not clear.

Methods

Quercetin, a well-recognized flavonoid antioxidant, was tested for its inhibition of α-synucein aggregation by thioflavin T assay, light scattering measurement, size-exclusion high performance liquid chromatography, atomic force microscopy, etc.

Results

The pre-incubated quercetin exhibited a noticeably stronger inhibition behavior to the fibril formation than that of the freshly prepared. The inhibition is significant in the presence of ortho- and para-benzenediol isomers and inconsiderable in the presence of meta-isomer. The oxidized quercetin species (i.e., chalcantrione, benzyfuranone, quercetinchinone, and other derivatives) cause stronger inhibition than quercetin does because of the elevated polarity and hydrophilicity. Presence of quercetin disaggregates α-synucein fibrils, rather than oligomers and amorphous aggregations.

Conclusions

Instead of the antioxidant activity, the 1:1 covalent binding of quercetin with α-synucein, and the increased hydophilicity of the covalently modified α-synucein oligomers or monomers, account for the inhibition of α-synucein fibrillation.

General significance

Clarification of the molecular mechanism of the inhibition and disaggregation may help to screen safer and more effective flavonoid therapeutic in combating PD.     

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.     

Mechanistic aspects of Parkinson’s disease: α-synuclein and the biomembrane

A key feature in Parkinson’s disease is the deposition of Lewy bodies. The major protein component of these intracellular deposits is the 140-amino acid protein α-synuclein that is widely distributed throughout the brain. α-synuclein was identified in presynaptic terminals and in synaptosomal preparations. The protein is remarkable for its structural variability. It is almost unstructured as a monomer in aqueous solution. Self-aggregation leads to a variety of β-structures, while membrane association may result in the formation of an amphipathic helical structure. The present article strives to give an overview of what is currently known on the interaction of α-synuclein with lipid membranes, including synthetic lipid bilayers, membraneous cell fractions, synaptic vesicles and intact cells. Manifestations of a functional relevance of the α-synuclein–lipid interaction will be discussed and the potential pathogenicity of oligomeric α-synuclein aggregates will be briefly reviewed.     

Proteolytic cleavage of extracellular α-synuclein by plasmin: implications for Parkinson disease

Parkinson disease (PD) is the second most common neurodegenerative disease characterized by a progressive dopaminergic neuronal loss in association with Lewy body inclusions. Gathering evidence indicates that α-synuclein (α-syn), a major component of the Lewy body, plays an important role in the pathogenesis of PD. Although α-syn is considered to be a cytoplasmic protein, it has been detected in extracellular biological fluids, including human cerebrospinal fluid and blood plasma of healthy and diseased individuals. In addition, a prion-like spread of α-syn aggregates has been recently proposed to contribute to the propagation of Lewy bodies throughout the nervous system during progression of PD, suggesting that the metabolism of extracellular α-syn might play a key role in the pathogenesis of PD. In the present study, we found that plasmin cleaved and degraded extracellular α-syn specifically in a dose- and time- dependent manner. Aggregated forms of α-syn as well as monomeric α-syn were also cleaved by plasmin. Plasmin cleaved mainly the N-terminal region of α-syn and also inhibited the translocation of extracellular α-syn into the neighboring cells in addition to the activation of microglia and astrocytes by extracellular α-syn. Further, extracellular α-syn regulated the plasmin system through up-regulation of plasminogen activator inhibitor-1 (PAI-1) expression. These findings help to understand the molecular mechanism of PD and develop new therapeutic targets for PD.     

Enhanced autophagy from chronic toxicity of iron and mutant A53T α-synuclein: implications for neuronal cell death in Parkinson disease

Parkinson disease (PD), a prevalent neurodegenerative motor disorder, is characterized by the rather selective loss of dopaminergic neurons and the presence of α-synuclein-enriched Lewy body inclusions in the substantia nigra of the midbrain. Although the etiology of PD remains incompletely understood, emerging evidence suggests that dysregulated iron homeostasis may be involved. Notably, nigral dopaminergic neurons are enriched in iron, the uptake of which is facilitated by the divalent metal ion transporter DMT1. To clarify the role of iron in PD, we generated SH-SY5Y cells stably expressing DMT1 either singly or in combination with wild type or mutant α-synuclein. We found that DMT1 overexpression dramatically enhances Fe(2+) uptake, which concomitantly promotes cell death. This Fe(2+)-mediated toxicity is aggravated by the presence of mutant α-synuclein expression, resulting in increased oxidative stress and DNA damage. Curiously, Fe(2+)-mediated cell death does not appear to involve apoptosis. Instead, the phenomenon seems to occur as a result of excessive autophagic activity. Accordingly, pharmacological inhibition of autophagy reverses cell death mediated by Fe(2+) overloading. Taken together, our results suggest a role for iron in PD pathogenesis and provide a mechanism underlying Fe(2+)-mediated cell death.     

The cellular prion protein (PrPC) as neuronal receptor for α-synuclein

The term ‘prion-like’ is used to define some misfolded protein species that propagate intercellularly, triggering protein aggregation in recipient cells. For cell binding, both direct plasma membrane interaction and membrane receptors have been described for particular amyloids. In this respect, emerging evidence demonstrates that several β-sheet enriched proteins can bind to the cellular prion protein (PrP^C). Among other interactions, the physiological relevance of the binding between β-amyloid and PrP^C has been a relevant focus of numerous studies. At the molecular level, published data point to the second charged cluster domain of the PrP^C molecule as the relevant binding domain of the β-amyloid/PrP^C interaction. In addition to β-amyloid, participation of PrP^C in binding α-synuclein, responsible for neurodegenerative synucleopathies, has been reported. Although results indicate relevant participation of PrP^C in the spreading of α-synuclein in living mice, the physiological relevance of the interaction remains elusive. In this comment, we focus our attention on summarizing current knowledge of PrP^C as a receptor for amyloid proteins and its physiological significance, with particular focus on α-synuclein.     

The role of the prion protein in the internalization of α-synuclein amyloids

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein amyloids in several regions of the brain. α-Synuclein fibrils are able to spread via cell-to-cell transfer, and once inside the cells, they can template the misfolding and aggregation of the endogenous α-synuclein. Multiple mechanisms have been shown to participate in the process of propagation: endocytosis, tunneling nanotubes and macropinocytosis. Recently, we published a research showing that the cellular form of the prion protein (PrP^C) acts as a receptor for α-synuclein amyloid fibrils, facilitating their internalization through and endocytic pathway. This interaction occurs by a direct interaction between the fibrils and the N-terminal domain of PrP^C. In cell lines expressing the pathological form of PrP (PrP^Sc), the binding between PrP^C and α-synuclein fibrils prevents the formation and accumulation of PrP^Sc, since PrP^C is no longer available as a substrate for the pathological conversion templated by PrP^Sc. On the contrary, PrP^Sc deposits are cleared over passages, probably due to the increased processing of PrP^C into the neuroprotective fragments N1 and C1. Starting from these data, in this work we present new insights into the role of PrP^C in the internalization of protein amyloids and the possible therapeutic applications of these findings.     

Dimethyl α-ketoglutarate inhibits maladaptive autophagy in pressure overload-induced cardiomyopathy

It has been a longstanding problem to identify specific and efficient pharmacological modulators of autophagy. Recently, we found that depletion of acetyl-coenzyme A (AcCoA) induced autophagic flux, while manipulations designed to increase cytosolic AcCoA efficiently inhibited autophagy. Thus, the cell permeant ester dimethyl α-ketoglutarate (DMKG) increased the cytosolic concentration of α-ketoglutarate, which was converted into AcCoA through a pathway relying on either of the 2 isocitrate dehydrogenase isoforms (IDH1 or IDH2), as well as on ACLY (ATP citrate lyase). DMKG inhibited autophagy in an IDH1-, IDH2- and ACLY-dependent fashion in vitro, in cultured human cells. Moreover, DMKG efficiently prevented autophagy induced by starvation in vivo, in mice. Autophagy plays a maladaptive role in the dilated cardiomyopathy induced by pressure overload, meaning that genetic inhibition of autophagy by heterozygous knockout of Becn1 suppresses the pathological remodeling of heart muscle responding to hemodynamic stress. Repeated administration of DMKG prevents autophagy in heart muscle responding to thoracic aortic constriction (TAC) and simultaneously abolishes all pathological and functional correlates of dilated cardiomyopathy: hypertrophy of cardiomyocytes, fibrosis, dilation of the left ventricle, and reduced contractile performance. These findings indicate that DMKG may be used for therapeutic autophagy inhibition.     

Dimethyl α-ketoglutarate inhibits maladaptive autophagy in pressure overload-induced cardiomyopathy

It has been a longstanding problem to identify specific and efficient pharmacological modulators of autophagy. Recently, we found that depletion of acetyl-coenzyme A (AcCoA) induced autophagic flux, while manipulations designed to increase cytosolic AcCoA efficiently inhibited autophagy. Thus, the cell permeant ester dimethyl α-ketoglutarate (DMKG) increased the cytosolic concentration of α-ketoglutarate, which was converted into AcCoA through a pathway relying on either of the 2 isocitrate dehydrogenase isoforms (IDH1 or IDH2), as well as on ACLY (ATP citrate lyase). DMKG inhibited autophagy in an IDH1-, IDH2- and ACLY-dependent fashion in vitro, in cultured human cells. Moreover, DMKG efficiently prevented autophagy induced by starvation in vivo, in mice. Autophagy plays a maladaptive role in the dilated cardiomyopathy induced by pressure overload, meaning that genetic inhibition of autophagy by heterozygous knockout of Becn1 suppresses the pathological remodeling of heart muscle responding to hemodynamic stress. Repeated administration of DMKG prevents autophagy in heart muscle responding to thoracic aortic constriction (TAC) and simultaneously abolishes all pathological and functional correlates of dilated cardiomyopathy: hypertrophy of cardiomyocytes, fibrosis, dilation of the left ventricle, and reduced contractile performance. These findings indicate that DMKG may be used for therapeutic autophagy inhibition.     

Direct interaction of α-synuclein and AKT regulates IGF-1 signaling: implication of Parkinson disease

Genetic mutation of α-synuclein (α-SYN) is clearly verified as the causal factor of human and mouse Parkinson's disease. However, biological function of α-SYN has not been clearly demonstrated until now. In this investigation, we reveal that α-SYN is a co-regulator of growth factor-induced AKT activation. Elimination of SYN reduces the IGF-1-mediated AKT activation. Similarly, mutant SYN suppresses the IGF-1-induced AKT activation. Wild-type SYN can interact with AKT and enhance the solubility and plasma localization of AKT in response to IGF-1, whereas mutant α-SYNs do not interact with AKT. In addition, elevated expression of SYN blocks the AKT activation. We also find that si-RNA against α-SYN abolished the protective effect of IGF-1 against DNA damage-induced apoptosis. Our result strongly indicates that Parkinson's disease, induced by α-SYN mutation, is evoked by deregulation of the AKT-signaling cascade.     

The role of α-synuclein and tau hyperphosphorylation-mediated autophagy and apoptosis in lead-induced learning and memory injury

Lead (Pb) is a well-known heavy metal in nature. Pb can cause pathophysiological changes in several organ systems including central nervous system. Especially, Pb can affect intelligence development and the ability of learning and memory of children. However, the toxic effects and mechanisms of Pb on learning and memory are still unclear. To clarify the mechanisms of Pb-induced neurotoxicity in hippocampus, and its effect on learning and memory, we chose Sprague-Dawley rats (SD-rats) as experimental subjects. We used Morris water maze to verify the ability of learning and memory after Pb treatment. We used immunohistofluorescence and Western blotting to detect the level of tau phosphorylation, accumulation of α-synuclein, autophagy and related signaling molecules in hippocampus. We demonstrated that Pb can cause abnormally hyperphosphorylation of tau and accumulation of α-synuclein, and these can induce hippocampal injury and the ability of learning and memory damage. To provide the new insight into the underlying mechanisms, we showed that Grp78, ATF4, caspase-3, autophagy-related proteins were induced and highly expressed following Pb-exposure. But mTOR signaling pathway was suppressed in Pb-exposed groups. Our results showed that Pb could cause hyperphosphorylation of tau and accumulation of α-synuclein, which could induce ER stress and suppress mTOR signal pathway. These can enhance type II program death (autophgy) and type I program death (apoptosis) in hippocampus, and impair the ability of learning and memory of rats. This is the first evidence showing the novel role of autophagy in the neurotoxicity of Pb.     

The novel mechanism of rotenone-induced α-synuclein phosphorylation via reduced protein phosphatase 2A activity

Rotenone has been shown to induce many parkinsonian features and has been widely used in chemical models of Parkinson’s disease (PD). Its use is closely associated with α-synuclein (α-syn) phosphorylation both in vivo and in vitro. However, the mechanisms whereby rotenone regulates α-syn phosphorylation remain unknown. Protein phosphatase 2A (PP2A) has been shown to play an important role in α-syn dephosphorylation. We therefore investigated if rotenone caused α-syn phosphorylation by down-regulation of PP2A activity in mice. Rotenone increased the phosphorylation of α-syn at Ser129, consistent with the inhibition of PP2A activity by increased phosphorylation of tyrosine 307 at the catalytic subunit of PP2A (pTyr307 PP2Ac). We further explored the interactions among rotenone, PP2A, and α-syn in SK-N-SH cells and primary rat cortical neurons. Rotenone inhibited PP2A activity via phosphorylation of PP2Ac at Tyr307. The reduction in PP2A activity and rotenone cytotoxicity were reversed by treatment with the PP2A agonist, C2 ceramide, and the Src kinase inhibitor, SKI606. Immunoprecipitation experiments showed that rotenone induced an increase in calmodulin–Src complex in SK-N-SH cells, thus activating Src kinase, which in turn phosphorylated PP2A at Tyr307 and inhibited its activity. C2 ceramide and SKI606 significantly reversed the rotenone-induced phosphorylation and aggregation of α-syn by increasing PP2A activity. These results demonstrate that rotenone-reduced PP2A activity via Src kinase is involved in the phosphorylation of α-syn. These findings clarify the novel mechanisms whereby rotenone can induce PD.     

Calbindin 1, fibroblast growth factor 20, and α-synuclein in sporadic Parkinson’s disease

Parkinson’s disease (PD), one of the most common human neurodegenerative disorders, is characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain. Our recent case-control association study of 268 SNPs in 121 candidate genes identified α-synuclein (SNCA) as a susceptibility gene for sporadic PD (P = 1.7 × 10⁻¹¹). We also replicated the association of fibroblast growth factor 20 (FGF20) with PD (P = 0.0089). To find other susceptibility genes, we added 34 SNPs to the previous screen. Of 302 SNPs in a total 137 genes, but excluding SNCA, SNPs in NDUFV2, FGF2, CALB1 and B2M showed significant association (P < 0.01; 882 cases and 938 control subjects). We replicated the association analysis for these SNPs in a second independent sample set (521 cases and 1,003 control subjects). One SNP, rs1805874 in calbindin 1 (CALB1), showed significance in both analyses (P = 7.1 × 10⁻⁵; recessive model). When the analysis was stratified relative to the SNCA genotype, the odds ratio of CALB1 tended to increase according to the number of protective alleles in SNCA. In contrast, FGF20 was significant only in the subgroup of SNCA homozygote of risk allele. CALB1 is a calcium-binding protein that widely is expressed in neurons. A relative sparing of CALB1-positive dopaminergic neurons is observed in PD brains, compared with CALB1-negative neurons. Our genetic analysis suggests that CALB1 is associated with PD independently of SNCA, and that FGF20 is associated with PD synergistically with SNCA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The neurotransmitter serotonin interrupts α-synuclein amyloid maturation

Indolic derivatives can affect fibril growth of amyloid forming proteins. The neurotransmitter serotonin (5-HT) is of particular interest, as it is an endogenous molecule with a possible link to neuropsychiatric symptoms of Parkinson disease. A key pathomolecular mechanism of Parkinson disease is the misfolding and aggregation of the intrinsically unstructured protein α-synuclein. We performed a biophysical study to investigate an influence between these two molecules. In an isolated in vitro system, 5-HT interfered with α-synuclein amyloid fiber maturation, resulting in the formation of partially structured, SDS-resistant intermediate aggregates. The C-terminal region of α-synuclein was essential for this interaction, which was driven mainly by electrostatic forces. 5-HT did not bind directly to monomeric α-synuclein molecules and we propose a model where 5-HT interacts with early intermediates of α-synuclein amyloidogenesis, which disfavors their further conversion into amyloid fibrils.     

The N-terminus of the intrinsically disordered protein α-synuclein triggers membrane binding and helix folding

Alpha-synuclein (αS) is a 140-amino-acid protein that is involved in a number of neurodegenerative diseases. In Parkinson's disease, the protein is typically encountered in intracellular, high-molecular-weight aggregates. Although αS is abundant in the presynaptic terminals of the central nervous system, its physiological function is still unknown. There is strong evidence for the membrane affinity of the protein. One hypothesis is that lipid-induced binding and helix folding may modulate the fusion of synaptic vesicles with the presynaptic membrane and the ensuing transmitter release. Here we show that membrane recognition of the N-terminus is essential for the cooperative formation of helical domains in the protein. We used circular dichroism spectroscopy and isothermal titration calorimetry to investigate synthetic peptide fragments from different domains of the full-length αS protein. Site-specific truncation and partial cleavage of the full-length protein were employed to further characterize the structural motifs responsible for helix formation and lipid-protein interaction. Unilamellar vesicles of varying net charge and lipid compositions undergoing lateral phase separation or chain melting phase transitions in the vicinity of physiological temperatures served as model membranes. The results suggest that the membrane-induced helical folding of the first 25 residues may be driven simultaneously by electrostatic attraction and by a change in lipid ordering. Our findings highlight the significance of the αS N-terminus for folding nucleation, and provide a framework for elucidating the role of lipid-induced conformational transitions of the protein within its intracellular milieu.