alpha-synuclein and Glioblastoma

α-Synuclein is the major component of Lewy bodies, Lewy neurites, and glial cytoplasmic inclusions. It plays an important role in neurodegenerative diseases such as Parkinson's disease, multiple system atrophy, and other synucleinopathies. However, the pathogenesis and neurodegenerative effects of α-synuclein remain unknown. In this study, we established an α-synuclein and an α-synuclein-EGFP overexpressing U251 cell line. α-Synuclein overexpression increases oxidative stress and alters the cell surface and mitochondrial morphologies. We provide fluorescent-protein tagging, immunofluorescence and ultrastructural evidence showing that α-synuclein accumulations are associated with clusters of cytoplasmic vesicles and the diameter of these vesicles increases by H

Topics: alpha-Synuclein; Brain Neoplasms; Cell Line, Tumor; Cytoplasmic Vesicles; Glioblastoma; Humans; Hydrogen Peroxide; Malondialdehyde; Oxidative Stress; Protein Binding

Glial cytoplasmic inclusions (GCIs) containing alpha-synuclein (AS) are a neuropathologic hallmark of multiple system atrophy (MSA). Oligomerized AS is thought to be the pathogenic form of the protein. Glial cells normally express little AS, but they can take up AS from the extracellular fluid. 3,4-Dihydroxyphenylacetaldehyde (DOPAL), an obligate intermediate in the intra-neuronal metabolism of dopamine (DA), potently oligomerizes AS. In this study we tested whether DOPAL is taken up by human glial cells and augments intracellular oligomerization of AS.. DOPAL (exogenous or endogenous from co-incubation with PC12 cells) and AS (native or A53T mutant form) were added to the incubation medium of glial cells (glioblastoma or MO3.13 oligodendrocytes). Glial cellular contents of DOPAL and its intracellular metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were measured at up to 180 min of incubation. Glial cellular AS oligomers were quantified by Western blotting.. Neither glioblastoma nor MO3.13 cells contained endogenous catecholamines or AS. Co-incubation of the cells with DA-producing PC12 cells produced time-related increases in DOPAL and DOPAC contents. Similarly, glial cellular DOPAL and DOPAC contents increased rapidly after addition of DOPAL to the medium. After addition of native or A53T-AS, intracellular AS also increased. Incubation of glial cells with both DOPAL and AS enhanced the intracellular oligomerization of native and A53T-AS.. DOPAL is transmissible to glial cells and enhances intracellular oligomerization of AS. An interaction of DOPAL with AS might help explain the formation of CGIs in MSA.

Topics: 3,4-Dihydroxyphenylacetic Acid; alpha-Synuclein; Animals; Catecholamines; Cell Line; Dose-Response Relationship, Drug; Glioblastoma; Humans; Mutation; Neuroglia; PC12 Cells; Protein Aggregates; Rats; Subcellular Fractions; Time Factors

Human derived glioblastoma cells were cultured and treated with cytokines interleukin-6 (IL6), tumor necrosis factor alpha (TNF) and interferon-gamma (IFN) and imaged by fluorescence deconvolution microscopy to localize alpha-synuclein, tau and ubiquitin. Exposures were for short (2 h) and prolonged times (up to 96 h), with doses at both low (10 ng/ml), and high (100 ng/ml) concentrations. Further experiments used additive doses up to 200 ng/ml (2 x 100 ng), mimicking a super-infection state. Single, low doses of the cytokines initiated changes in levels of intracellular proteins, but these changes, be they increases or decreases, were not sustained, so we added higher doses of cytokine to the culture medium or fresh aliquots of cytokines over time. Finally, we treated cells with high, single doses of cytokine (200 ng/ml), to try to sustain perturbations of the proteins with cytokines. IFN caused a disruption and reduction of peripheral synuclein, TNF treatment resulted in increased levels of ubiquitin and IL6 disrupted and appeared to fragment tau. Of note, each of the proteins was found in a specific locale, tau being perinuclear, ubiquitin residing in the cytoplasm, and alpha-synuclein occupying the tips of cellular processes, exhibiting the characteristics of an adhesion protein/molecule [Word count=198].

Topics: alpha-Synuclein; Cells, Cultured; Glioblastoma; Humans; Imaging, Three-Dimensional; Interferon-gamma; Interleukin-6; Microscopy, Fluorescence; Parkinson Disease; tau Proteins; Tumor Necrosis Factor-alpha; Ubiquitin

Intracellular alpha-synuclein inclusion formation in glial cells is frequently seen in Parkinson's disease and multiple system atrophy. Microglial activation in these neurodegenerative disorders suggests that neuroinflammatory responses might interact with alpha-synuclein and contribute to the pathogenesis of these disorders. To study the role of tumor necrosis factor-alpha (TNF-alpha), an important proinflammatory cytokine produced by microglia, on cells overexpressing alpha-synuclein we have used the astrocytoma cell line U373 engineered to express C-terminally truncated alpha-synuclein as a fusion protein with red or green fluorescent proteins. We demonstrate that alpha-synuclein overexpression augmented TNF-alpha-induced apoptotic cell death in U373 cells by induction of caspase activation. Furthermore, TNF-alpha exposure was associated with significant cytoskeletal changes characterized by altered inclusion composition with loss of cytoskeletal proteins and elevation of high-molecular-weight alpha-synuclein species. We conclude that alpha-synuclein overexpression significantly increases the vulnerability of U373 cells to apoptosis through TNF-alpha-mediated pathways.

Topics: alpha-Synuclein; Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cytoskeleton; Gene Expression; Glioblastoma; Humans; Nerve Tissue Proteins; Synucleins; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha