The human <i>NGB</i> gene for neuroglobin alters A30P and αSyn aggregation in yeast and mammalian cells.

Abstract

<p>(A) Spotting analysis of <i>YHB1</i> and Δ<i>yhb1</i> yeast cells co-expressing αSyn and GFP (control) with either empty vector as control or <i>YHB1</i> and <i>NGB</i>, respectively, on non-inducing and galactose-inducing SC-Ura medium after 3 days. (B) Quantification of the percentage of cells displaying αSyn aggregates after 6 h induction in galactose-containing medium (n = 3). (C) Spotting analysis of <i>YHB1</i> and Δ<i>yhb1</i> yeast cells co-expressing A30P and GFP (control) with either empty vector (pME2788) as control or <i>YHB1</i> and <i>NGB</i>, respectively, on non-inducing and galactose-inducing SC-Ura medium after 3 days. (D) Quantification of the percentage of cells displaying A30P aggregates after 6 h induction in galactose-containing medium. Significance of differences was calculated with t-test (**, <i>p</i> < 0.01, n = 3). (E) Fluorescence microscopy of H4 cells co-expressing SynT, synphilin-1 and pcDNA (control) or NGB-mCherry. Nuclei are stained with Hoechst dye (blue). Scale bar = 30 μm. (F) Quantification of the percentage of H4 cells displaying αSyn inclusions after 48 h after transfection. Cells were classified into three groups according to the number of αSyn-immunoreactive inclusions observed: cells with 10 inclusions, cells with less than 10 inclusions and cells without inclusions. Significance of differences was calculated with t-test (*, <i>p</i> < 0.05, n = 3). (G) Lactate dehydrogenase (LDH) activity measurements support that <i>NGB</i> is non-toxic for H4 cells. H4 cells transfected with empty mammalian expression vector pcDNA3.1, with empty pcDNA3.1 or pcDNA3.1 encoding neuroglobin-mCherry (<i>NGB</i>) together with SynT and synphilin-1 (SynT+Synphilin-1) were analyzed. Media from indicated H4 cells were collected and the secretion of lactate LDH was determined as a measure of cytotoxicity. Significance of differences was calculated with t-test (not significant (n.s.); n = 3).</p

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