alpha-synuclein and Glioma

α-synuclein (aSyn) is a major player in Parkinson's disease and a group of other disorders collectively known as synucleinopathies, but the precise molecular mechanisms involved are still unclear. aSyn, as virtually all proteins, undergoes a series of posttranslational modifications during its lifetime, which can affect its biology and pathobiology. We recently showed that glycation of aSyn by methylglyoxal (MGO) potentiates its oligomerization and toxicity, induces dopaminergic neuronal cell loss in mice, and affects motor performance in flies. Small heat-shock proteins (sHsps) are molecular chaperones that facilitate the folding of proteins or target misfolded proteins for clearance. Importantly, sHsps were shown to prevent aSyn aggregation and cytotoxicity. Upon treating cells with increasing amounts of methylglyoxal, we found that the levels of Hsp27 decreased in a dose-dependent manner. Therefore, we hypothesized that restoring the levels of Hsp27 in glycating environments could alleviate the pathogenicity of aSyn. Consistently, we found that Hsp27 reduced MGO-induced aSyn aggregation in cells, leading to the formation of nontoxic aSyn species. Remarkably, increasing the levels of Hsp27 suppressed the deleterious effects induced by MGO. Our findings suggest that in glycating environments, the levels of Hsp27 are important for modulating the glycation-associated cellular pathologies in synucleinopathies.

Topics: alpha-Synuclein; Brain Neoplasms; Glioma; Glycosylation; Heat-Shock Proteins; Humans; Molecular Chaperones; Protein Aggregates; Pyruvaldehyde; Tumor Cells, Cultured

Cell-to-cell propagation of aggregated alpha synuclein (aSyn) has been suggested to play an important role in the progression of alpha synucleinopathies. A critical step for the propagation process is the accumulation of extracellular aSyn within recipient cells. Here, we investigated the trafficking of distinct exogenous aSyn forms and addressed the mechanisms influencing their accumulation in recipient cells. The aggregated aSyn species (oligomers and fibrils) exhibited more pronounced accumulation within recipient cells than aSyn monomers. In particular, internalized extracellular aSyn in the aggregated forms was able to seed the aggregation of endogenous aSyn. Following uptake, aSyn was detected along endosome-to-lysosome and autophagosome-to-lysosome routes. Intriguingly, aggregated aSyn resulted in lysosomal activity impairment, accompanied by the accumulation of dilated lysosomes. Moreover, analysis of autophagy-related protein markers suggested decreased autophagosome clearance. In contrast, the endocytic pathway, proteasome activity, and mitochondrial homeostasis were not substantially affected in recipient cells. Our data suggests that extracellularly added aggregated aSyn primarily impairs lysosomal activity, consequently leading to aSyn accumulation within recipient cells. Importantly, the autophagy inducer trehalose prevented lysosomal alterations and attenuated aSyn accumulation within aSyn-exposed cells. Our study underscores the importance of lysosomes for the propagation of aSyn pathology, thereby proposing these organelles as interventional targets.

Topics: alpha-Synuclein; Animals; Autophagy; Cell Line, Tumor; Escherichia coli; Glioma; Humans; Lysosomes; Neurons; Parkinson Disease; Protein Aggregation, Pathological; Rats; Rats, Wistar; Recombinant Proteins; Sirolimus; Trehalose

α-Synuclein (aSyn) toxicity is associated with cell cycle alterations, activation of DNA damage responses (DDR), and deregulation of autophagy. However, the relationships between these phenomena remain largely unknown. Here, we demonstrate that in a yeast model of aSyn toxicity and aging, aSyn expression induces Ras2-dependent growth signaling, cell cycle re-entry, DDR activation, autophagy, and autophagic degradation of ribonucleotide reductase 1 (Rnr1), a protein required for the activity of ribonucleotide reductase and dNTP synthesis. These events lead to cell death and aging, which are abrogated by deleting RAS2, inhibiting DDR or autophagy, or overexpressing RNR1. aSyn expression in human H4 neuroglioma cells also induces cell cycle re-entry and S-phase arrest, autophagy, and degradation of RRM1, the human homologue of RNR1, and inhibiting autophagic degradation of RRM1 rescues cells from cell death. Our findings represent a model for aSyn toxicity that has important implications for understanding synucleinopathies and other age-related neurodegenerative diseases.

Topics: alpha-Synuclein; Autophagy; Cell Death; Cell Line, Tumor; Cellular Senescence; DNA Damage; Genetic Vectors; Glioma; Humans; Parkinson Disease; Proteolysis; Ribonucleotide Reductases; S Phase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transfection

Alpha-synuclein (aSyn) plays a crucial role in Parkinson's disease (PD) and other synucleinopathies, since it misfolds and accumulates in typical proteinaceous inclusions. While the function of aSyn is thought to be related to vesicle binding and trafficking, the precise molecular mechanisms linking aSyn with synucleinopathies are still obscure. aSyn can spread in a prion-like manner between interconnected neurons, contributing to the propagation of the pathology and to the progressive nature of synucleinopathies. Here, we investigated the interaction of aSyn with membranes and trafficking machinery pathways using cellular models of PD that are amenable to detailed molecular analyses. We found that different species of aSyn can enter cells and form high molecular weight species, and that membrane binding properties are important for the internalization of aSyn. Once internalized, aSyn accumulates in intracellular inclusions. Interestingly, we found that internalization is blocked in the presence of dynamin inhibitors (blocked membrane scission), suggesting the involvement of the endocytic pathway in the internalization of aSyn. By screening a pool of small Rab-GTPase proteins (Rabs) which regulate membrane trafficking, we found that internalized aSyn partially colocalized with Rab5A and Rab7. Initially, aSyn accumulated in Rab4A-labelled vesicles and, at later stages, it reached the autophagy-lysosomal pathway (ALP) where it gets degraded. In total, our study emphasizes the importance of membrane binding, not only as part of the normal function but also as an important step in the internalization and subsequent accumulation of aSyn. Importantly, we identified a fundamental role for Rab proteins in the modulation of aSyn processing, clearance and spreading, suggesting that targeting Rab proteins may hold important therapeutic value in PD and other synucleinopathies.

Topics: alpha-Synuclein; Biotinylation; Cell Fractionation; Cell Line, Tumor; Cell Membrane; Dynamins; Endocytosis; Glioma; Green Fluorescent Proteins; Humans; Lysosomes; Molecular Imaging; Mutation; Protein Transport; rab GTP-Binding Proteins; rab5 GTP-Binding Proteins; rab7 GTP-Binding Proteins; Transfection

Aggregation of α-synuclein (α-syn) and α-syn cytotoxicity are hallmarks of sporadic and familial Parkinson disease (PD), with accumulating evidence that prefibrillar oligomers and protofibrils are the pathogenic species in PD and related synucleinopathies. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a key regulator of mitochondrial biogenesis and cellular energy metabolism, has recently been associated with the pathophysiology of PD. Despite extensive effort on studying the function of PGC-1α in mitochondria, no studies have addressed whether PGC-1α directly influences oligomerization of α-syn or whether α-syn oligomers impact PGC-1α expression.. We tested whether pharmacological or genetic activation of PGC-1α or PGC-11α knockdown could modulate the oligomerization of α-syn in vitro by using an α-syn -fragment complementation assay.. In this study, we found that both PGC-1α reference gene (RG-PGC-1α) and the central nervous system (CNS)-specific PGC-1α (CNS-PGC-1α) are downregulated in human PD brain, in A30P α-syn transgenic animals, and in a cell culture model for α-syn oligomerization. Importantly, downregulation of both RG-PGC-1α and CNS-PGC-1α in cell culture or neurons from RG-PGC-1α-deficient mice leads to a strong induction of α-syn oligomerization and toxicity. In contrast, pharmacological activation or genetic overexpression of RG-PGC-1α reduced α-syn oligomerization and rescued α-syn-mediated toxicity.. Based on our results, we propose that PGC-1α downregulation and α-syn oligomerization form a vicious circle, thereby influencing and/or potentiating each other. Our data indicate that restoration of PGC-1α is a promising approach for development of effective drugs for the treatment of PD and related synucleinopathies.

Topics: Aged; Aged, 80 and over; alpha-Synuclein; Animals; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Embryo, Mammalian; Enzyme Inhibitors; Female; Gene Expression Regulation; Glioma; Humans; Macrolides; Male; Mice; Mice, Transgenic; Middle Aged; Neurons; Parkinson Disease; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; PPAR gamma; Resveratrol; RNA Polymerase II; Stilbenes; Substantia Nigra; TATA-Box Binding Protein; Transcription Factors

Recent research implicates soluble aggregated forms of α-synuclein as neurotoxic species with a central role in the pathogenesis of Parkinson's disease and related disorders. The pathway by which α-synuclein aggregates is believed to follow a step-wise pattern, in which dimers and smaller oligomers are initially formed. Here, we used H4 neuroglioma cells expressing α-synuclein fused to hemi:GFP constructs to study the effects of α-synuclein monoclonal antibodies on the early stages of aggregation, as quantified by Bimolecular Fluorescence Complementation assay. Widefield and confocal microscopy revealed that cells treated for 48 h with monoclonal antibodies internalized antibodies to various degrees. C-terminal and oligomer-selective α-synuclein antibodies reduced the extent of α-synuclein dimerization/oligomerization, as indicated by decreased GFP fluorescence signal. Furthermore, ELISA measurements on lysates and conditioned media from antibody treated cells displayed lower α-synuclein levels compared to untreated cells, suggesting increased protein turnover. Taken together, our results propose that extracellular administration of monoclonal antibodies can modify or inhibit early steps in the aggregation process of α-synuclein, thus providing further support for passive immunization against diseases with α-synuclein pathology.

Topics: alpha-Synuclein; Animals; Antibodies, Monoclonal; Blotting, Western; Culture Media, Conditioned; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Glioma; Green Fluorescent Proteins; Humans; Immunoenzyme Techniques; Mice; Mice, Inbred BALB C; Neurons; Protein Multimerization; Tumor Cells, Cultured

Mutations in the PSEN1 gene encoding presenilin 1 (PS1) are linked to a vast majority of pedigrees with early-onset, autosomal dominant forms of familial Alzheimer's disease (FAD). Lewy body (LB) pathology is frequently found in the brains of FAD patients harboring PSEN1 mutations. We recently reported on a novel PS1 mutation with the deletion of threonine at codon 440 (deltaT440) in a familial case diagnosed as having the neocortical type of dementia with LBs (DLB) and variant AD. In this report, we investigated the possible involvement of PS1 deltaT440 mutation in aberrant alpha-synuclein accumulation. We established cell lines that stably express either wild-type (WT) PS1 or the FAD-linked PS1 H163R, E280A, deltaE9, and PS1 deltaT440 mutants and now demonstrate that the expression of the PS1 deltaT440 mutant led to a marked elevation in the ratio of beta-amyloid (Abeta) 42/40 peptides in a conditioned medium. More importantly, we report here that the levels of phosphorylated alpha-synuclein increase in neuronal and non-neuronal cells expressing the PS1 deltaT440 mutant compared with cells that express WT PS1 or the PS1 H163R and E280A variants that are not associated with LB pathology. This finding is consistent with our demonstration of elevated levels of phosphorylated alpha-synuclein in the detergent-resistant fraction prepared from a patient's brain with PS1 deltaT440 mutation. These observations raise the intriguing suggestion that the mechanism(s) by which the PS1 deltaT440 mutant causes DLB and variant AD are by enhancing the phosphorylation of alpha-synuclein and the ratio of Abeta(42/40) peptides, respectively, in the brain.

Topics: Aged; alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Animals; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Glioma; Humans; Lewy Body Disease; Mice; Middle Aged; Mutation; Neuroblastoma; Phosphorylation; Presenilin-1; Rats; Receptors, Notch; Threonine; Transfection

Oligomerization and aggregation of alpha-synuclein molecules are believed to play a major role in neuronal dysfunction and loss in Parkinson's disease (PD) and dementia with Lewy bodies. However, alpha-synuclein oligomerization and aggregation have been detected only indirectly in cells using detergent extraction methods. Here, we show for the first time intracellular alpha-synuclein oligomerization using fluorescence lifetime imaging (FLIM). Two forms of alpha-synuclein homomeric interactions were detected: an antiparallel amino terminus-carboxyl terminus interaction between alpha-synuclein molecules, and a close amino terminus-carboxy terminus interaction within single alpha-synuclein molecules. Coexpression of the chaperone protein Hsp70, which can block alpha-synuclein toxicity in several systems, causes alpha-synuclein to adopt a different, open conformation, but Hsp70 does not alter alpha-synuclein-alpha-synuclein interactions. Thus, the neuroprotective effect of Hsp70 can be explained by its chaperone activity on alpha-synuclein molecules, rather than alteration of alpha-synuclein-alpha-synuclein interactions.

Topics: alpha-Synuclein; Cell Line, Tumor; Dimerization; Glioma; Half-Life; HSP70 Heat-Shock Proteins; Humans; Lasers; Microscopy, Fluorescence; Molecular Chaperones; Protein Binding; Protein Conformation; Transfection

Parkinson's disease (PD) is one of the most prevalent neurodegenerative diseases but its etiology is unclear. Alpha-synuclein (alpha-SN) is a major component of Lewy bodies and Lewy neurites, and its missense mutations, A30P and A53T, cause familial PD. In PD, alpha-SN-positive glial inclusions are distributed mainly in the dorso-medial region of the substantia nigra, which contains most of the surviving dopaminergic neurons, suggesting that alpha-SN expression might have a neuroprotective function in glial cells. To investigate this hypothesis, we established alpha-SN transfected C6 glioma cell line clones and evaluated the expression of neurotrophins using semi-quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Brain-derived neurotrophic factor (BDNF) was induced by overexpression of wild-type alpha-SN but not by that of A30P and A53T. These data suggest that the pathogenic alpha-SN mutations, A30P or A53T, are linked to the loss of BDNF production in glial cells.

Topics: alpha-Synuclein; Amino Acid Substitution; Animals; Brain-Derived Neurotrophic Factor; Calcium; Cell Line, Tumor; Chelating Agents; Clone Cells; Dactinomycin; Egtazic Acid; Glioma; Humans; Immunoblotting; Immunohistochemistry; Lac Operon; Nerve Growth Factor; Nerve Tissue Proteins; Protein Synthesis Inhibitors; Rats; Recombinant Proteins; RNA, Messenger; Synucleins; Thapsigargin; Transfection

alpha-, beta- and gamma-synuclein are highly homologous proteins that are found predominantly in neurons. Abnormal accumulation of synucleins has been associated with diseases of the central nervous system particularly Parkinson's disease. Immunoreactivity of alpha-synuclein is demonstrated in brain tumors with neuronal differentiation and in schwannomas, whereas gamma-synuclein has been demonstrated in breast and ovarian carcinomas. The immunoreactivity of synucleins has not been described in glial tumors. Immunoreactivity of synucleins in glial cells in culture and in pathological conditions, however, suggests that synucleins may be expressed by glial tumors. We studied the expression of alpha-, beta-, and gamma-synuclein in 84 human brain tumors (24 ependymomas, 31 astrocytomas, 8 oligodendrogliomas, and 21 medulloblastomas) by immunohistochemistry. Our study demonstrates immunoreactivity for gamma-synuclein in high-grade glial tumors; immunoreactivity is found in all anaplastic ependymomas but in only 33% of ependymomas and 16% of myxopapillary ependymomas. Immunoreactivity for gamma-synuclein is noted in 63% of glioblastomas but not in other astrocytic tumors. Of medulloblastomas, 76% have immunoreactivity for either alpha- or beta-synuclein or both; no immunoreactivity for gamma-synuclein is seen in medulloblastomas.

Topics: alpha-Synuclein; Astrocytes; beta-Synuclein; Brain Neoplasms; Cerebellar Neoplasms; Child, Preschool; Ependymoma; gamma-Synuclein; Glioma; Humans; Immunohistochemistry; Medulloblastoma; Nerve Tissue Proteins; Neuroectodermal Tumors, Primitive; Oligodendroglia; Synucleins

alpha-Synuclein is a major component of Lewy bodies, a neuropathological feature of Parkinson's disease. Two alpha-synuclein mutations, Ala53Thr and Ala30Pro, are associated with early onset, familial forms of the disease. Recently, synphilin-1, a protein found to interact with alpha-synuclein by yeast two hybrid techniques, was detected in Lewy bodies. In this study we report the interaction of alpha-synuclein and synphilin-1 in human neuroglioma cells using a sensitive fluorescence resonance energy transfer technique. We demonstrate that the C-terminus of alpha-synuclein is closely associated with the C-terminus of synphilin-1. A weak interaction occurs between the N-terminus of alpha-synuclein and synphilin-1. The familial Parkinson's disease associated mutations of alpha-synuclein (Ala53Thr and Ala30Pro) also demonstrate a strong interaction between their C-terminal regions and synphilin-1. However, compared with wild-type alpha-synuclein, significantly less energy transfer occurs between the C-terminus of Ala53Thr alpha-synuclein and synphilin-1, suggesting that the Ala53Thr mutation alters the conformation of alpha-synuclein in relation to synphilin-1.

Topics: alpha-Synuclein; Blotting, Western; Carrier Proteins; Energy Transfer; Fluorescent Antibody Technique, Indirect; Gene Expression; Glioma; Humans; Immunohistochemistry; Microscopy, Confocal; Mutagenesis, Site-Directed; Mutation; Nerve Tissue Proteins; Parkinson Disease; Protein Conformation; Structure-Activity Relationship; Subcellular Fractions; Synucleins; Transfection; Tumor Cells, Cultured