Fluorescence microscopy and 3D image reconstruction of cytokine initiated disruption of the Parkinson disease associated proteins alpha-synuclein, tau and ubiquitin in cultured glial cells.
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].
Dinh
Kha
K
Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, 6431 Fannin Street, MSB 2.288, Houston, TX 77030, USA. kha.dinh@uth.tmc.edu
Poindexter
Brian J
BJ
Barnes
Jennifer L
JL
Schiess
Mya C
MC
Bick
Roger J
RJ
D002478:Cells, Cultured; D005909:Glioblastoma / Q000378:metabolism*; D006801:Humans; D021621:Imaging, Three-Dimensional; D007371:Interferon-gamma / Q000494:pharmacology*; D015850:Interleukin-6 / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D010300:Parkinson Disease / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D025801:Ubiquitin / Q000187:drug effects / Q000378:metabolism*; D051844:alpha-Synuclein / Q000187:drug effects / Q000378:metabolism*; D016875:tau Proteins / Q000187:drug effects / Q000378:metabolism*
D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha; D025801:Ubiquitin; D051844:alpha-Synuclein; D016875:tau Proteins; D007371:Interferon-gamma