Induction of neuronal cell death by Rab5A-dependent endocytosis of alpha-synuclein.
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The presynaptic alpha-synuclein is a prime suspect for contributing to Lewy pathology and clinical aspects of diseases, including Parkinson's disease, dementia with Lewy bodies, and a Lewy body variant of Alzheimer's disease. Here we examined the pathogenic mechanism of neuronal cell death induced by alpha-synuclein. The exogenous addition of alpha-synuclein caused a marked decrease of cell viability in primary and immortalized neuronal cells. The neuronal cell death appeared to be correlated with the Rab5A-specific endocytosis of alpha-synuclein that subsequently caused the formation of Lewy body-like intracytoplasmic inclusions. This was further supported by the fact that the expression of GTPase-deficient Rab5A resulted in a significant decrease of its cytotoxicity as a result of incomplete endocytosis of alpha-synuclein. (+info)
Muscarinic receptor stimulation induces translocation of an alpha-synuclein oligomer from plasma membrane to a light vesicle fraction in cytoplasm.
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The close correspondence between the distribution of brain alpha-synuclein and that of muscarinic M1 and M3 receptors suggests a role for this protein in cholinergic transmission. We thus examined the effect of muscarinic stimulation on alpha-synuclein in SH-SY5Y, a human dopaminergic cell line that expresses this protein. Under basal conditions, alpha-synuclein was detected in all subcellular compartments isolated as follows: plasma membrane, cytoplasm, nucleus, and two vesicle fractions. The lipid fractions contained only a 45-kDa alpha-synuclein oligomer, whereas the cytoplasmic and nuclear fractions contained both the oligomer and the monomer. This finding suggests alpha-synuclein exists physiologically as a lipid-bound oligomer and a soluble monomer. Muscarinic stimulation by carbachol reduced the alpha-synuclein oligomer in plasma membrane over a 30-min period, with a concomitant increase of both the oligomer and the monomer in the cytoplasmic fraction. The oligomer was associated with a light vesicle fraction in cytoplasm that contains uncoated endocytotic vesicles. The carbachol-induced alteration of alpha-synuclein was blocked by atropine. Translocation of the alpha-synuclein oligomer in response to carbachol stimulation corresponds closely with the time course of ligand-stimulated muscarinic receptor endocytosis. The data suggest that the muscarine receptor stimulated release of the alpha-synuclein oligomer from plasma membrane, and its subsequent association with the endocytotic vesicle fraction may have a role in muscarine receptor endocytosis. We propose that its function may be a transient release of membrane-bound phospholipase D2 from alpha-synuclein inhibition, thus allowing this lipase to participate in muscarinic receptor endocytosis. (+info)
Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation.
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The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin-proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14-3-3, and alpha-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin-proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders. (+info)
Recombinant AAV vectors containing the foot and mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra neurons.
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Recombinant adeno-associated viruses (rAAVs) are promising vectors for gene therapy since they efficiently and stably transduce a variety of tissues of immunocompetent animals. The major disadvantage of rAAVs is their limited capacity to package foreign DNA (< or =5 kb). Often, co-expression of two or more genes from a single viral vector is desirable to achieve maximal therapeutic efficacy or to track transduced cells in vivo by suitable reporter genes. The internal ribosome entry site (IRES) sequence of encephalomyocarditis virus has been widely used to construct bicistronic viral vectors. However, the IRES is rather long and IRES-mediated translation can be relatively inefficient when compared with cap-dependent translation. As an alternative to the IRES for in vivo gene expression, we studied the 16 amino-acid long 2A peptide of foot and mouth disease virus (FMDV). The 2A peptide mediates the primary cis-'cleavage' of the FMDV polyprotein in a cascade of processing events that ultimately generate the mature FMDV proteins. We have generated several different rAAV genomes in which two coding regions are fused in-frame via the FMDV 2A sequence. We show that FMDV 2A efficiently mediates the generation of the expected cleavage products from the artificial fusion proteins in cells. Furthermore, we find that both EGFP and alpha- synuclein are expressed at substantially higher levels from 2A vectors than from the corresponding IRES-based vectors, while SOD-1 is expressed at comparable or slightly higher levels. Finally, we demonstrate for the first time, that the 2A sequence results in effective bicistronic gene expression in vivo after injection of 2A-dependent rAAVs into the rat substantia nigra. We conclude that 2A-containing rAAVs may represent an attractive alternative to IRES-dependent vectors for ex vivo and in vivo gene expression and gene therapy. (+info)
Ubiquitination of a new form of alpha-synuclein by parkin from human brain: implications for Parkinson's disease.
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Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive accumulation in selected neurons of protein inclusions containing alpha-synuclein and ubiquitin. Rare inherited forms of PD are caused by autosomal dominant mutations in alpha-synuclein or by autosomal recessive mutations in parkin, an E3 ubiquitin ligase. We hypothesized that these two gene products interact functionally, namely, that parkin ubiquitinates alpha-synuclein normally and that this process is altered in autosomal recessive PD. We have now identified a protein complex in normal human brain that includes parkin as the E3 ubiquitin ligase, UbcH7 as its associated E2 ubiquitin conjugating enzyme, and a new 22-kilodalton glycosylated form of alpha-synuclein (alphaSp22) as its substrate. In contrast to normal parkin, mutant parkin associated with autosomal recessive PD failed to bind alphaSp22. In an in vitro ubiquitination assay, alphaSp22 was modified by normal but not mutant parkin into polyubiquitinated, high molecular weight species. Accordingly, alphaSp22 accumulated in a non-ubiquitinated form in parkin-deficient PD brains. We conclude that alphaSp22 is a substrate for parkin's ubiquitin ligase activity in normal human brain and that loss of parkin function causes pathological alphaSp22 accumulation. These findings demonstrate a critical biochemical reaction between the two PD-linked gene products and suggest that this reaction underlies the accumulation of ubiquitinated alpha-synuclein in conventional PD. (+info)
A close association of torsinA and alpha-synuclein in Lewy bodies: a fluorescence resonance energy transfer study.
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TorsinA, a novel protein in which a mutation causes dominant, early onset torsion dystonia, may serve as a chaperone for misfolded proteins that require refolding or degradation. It has been hypothesized that misfolded alpha-synuclein, a protein in which two mutations cause autosomal dominantly inherited Parkinson's disease, serves as a nidus for the development of a Lewy body. We hypothesized that torsinA plays a role in the cellular processing of alpha-synuclein. We demonstrate that anti-torsin antibodies stain Lewy bodies and Lewy neurites in the substantia nigra and cortex. Using sensitive fluorescent resonance energy transfer (FRET) techniques, we find evidence of a close association between torsinA and alpha-synuclein in Lewy bodies. (+info)
Pesticides directly accelerate the rate of alpha-synuclein fibril formation: a possible factor in Parkinson's disease.
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Parkinson's disease involves intracellular deposits of alpha-synuclein in the form of Lewy bodies and Lewy neurites. The etiology of the disease is unknown, however, several epidemiological studies have implicated environmental factors, especially pesticides. Here we show that several pesticides, including rotenone, dieldrin and paraquat, induce a conformational change in alpha-synuclein and significantly accelerate the rate of formation of alpha-synuclein fibrils in vitro. We propose that the relatively hydrophobic pesticides preferentially bind to a partially folded intermediate conformation of alpha-synuclein, accounting for the observed conformational changes, and leading to association and subsequent fibrillation. These observations suggest one possible underlying molecular basis for Parkinson's disease. (+info)
alpha-Synuclein occurs in lipid-rich high molecular weight complexes, binds fatty acids, and shows homology to the fatty acid-binding proteins.
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alpha-Synuclein (alphaS) is a 140-residue neuronal protein that forms insoluble cytoplasmic aggregates in Parkinson's disease (PD) and several other neurodegenerative disorders. Two missense mutations (A53T and A30P) are linked to rare forms of familial PD. The normal function of alphaS is unknown, and cultured cell systems that model its modification from soluble monomers to aggregated forms have not been reported. Through a systematic centrifugal fractionation of mesencephalic neuronal cell lines and transgenic mouse brains expressing wild-type or A53T human alphaS, we observed unusual, previously unrecognized species of alphaS that migrate well above the 17-kDa monomeric form in denaturing gels. Incubation at 65 degrees C of high-speed cytosols from cells or brains revealed a modified alphaS species migrating at approximately 36 kDa and an extensive higher molecular mass alphaS-reactive smear. Extraction of the cytosols with chloroform/methanol or with a resin (Lipidex 1000) that binds fatty acids resulted in a similar pattern of higher molecular mass alphaS forms. On the basis of this effect of delipidation, we reexamined the primary structure of alphaS and detected a motif at the N and C termini that is homologous to a fatty acid-binding protein signature. In accord, we found that purified human alphaS binds oleic acid, with an apparent K(d) of 12.5 microM. We also observed an enhanced association of A53T alphaS with microsomal membranes in both mesencephalic cells and transgenic mouse brains. We conclude that alphaS has biochemical properties and a structural motif that suggest it is a novel member of the fatty acid-binding protein family and may thus transport fatty acids between the aqueous and membrane phospholipid compartments of the neuronal cytoplasm. (+info)