Modulating microtubule stability via α-tubulin acetylation partially restores Golgi fragmentation in spinal muscular atrophy

Figures

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  Figure 1. Level of α-tubulin acetylation in SMA model organisms. Representative Western blots and densitometric quantifications of α-tubulin acetylation in D. melanogaster (A, B) and mouse models (C, D) of SMA. For the D. melanogaster model, SMA (smnf05960) and WT control flies were collected on the day they emerged from their pupae (Day 0), and on the subsequent days (Days 5, 10, 30, and 40), n=4 (10 flies were pooled for each replicate). For mouse models of SMA, spinal cord tissues of Smn2B/− (whole tissue) and Smn−/−; SMN2+/+ (Taiwanese, T3–T13 segments) at symptomatic stages (P18 for Smn2B/−, P8 for Smn−/−; SMN2+/+) were used to quantify α-tubulin acetylation level and compared with controls. n=4 for Smn2B/− mice, n=6 for Smn−/−;SMN2+/+. All normalizations were performed against total α-tubulin. Bars show the mean with the standard error of the mean (SEM). Statistical significance was tested by using the Mann–Whitney U test (*p < 0.05, ns: not significant).

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  Figure 2. Acetylated α-tubulin and HDAC6 levels in SMA patient-derived fibroblast cells. Representative blots and densitometric quantifications of acetylated α-tubulin levels in SMA fibroblast cells compared with healthy control cells (A, B). Overall acetylated α-tubulin levels shown in SMA and control fibroblast cells (C). Representative immunofluorescence images of control (GM08333) and SMA type I (GM09677) fibroblast cells stained by acetylated α-tubulin antibody (D). Fluorescence intensity analysis of acetylated α-tubulin in whole cells as well as in the perinuclear area, which was determined using the center of the nucleus as a reference point. A 50 μm × 50 μm square was drawn around the center as a region of interest and analyzed in all cells (E). Images of 10 cells were analyzed in a blinded manner in independent culture experiments (n=2). Scale bar, 20 μm. Densitometric analysis of HDAC6 level in SMA patient and control fibroblast cells relative to total α-tubulin (A, F). Overall HDAC6 levels shown in SMA and control fibroblast cells (G). Patient fibroblast cells shown in the blots and bar graphs are SMA Type I: GM09677, ML39, ML16, and ML17, and Type II: GM03813. Control…

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  Figure 3. Relationship between Golgi fragmentation and α-tubulin acetylation in SMA patient-derived fibroblast cells. Immunofluorescence costaining of acetylated α-tubulin (green) and a cis-Golgi marker (GM130, red) in SMA and control fibroblast cells (A). Representative images of nontreated, nocodazole-treated, and tubastatin A-treated SMA and control cells were provided, along with merged images at 20× magnification. The product of the differences from the mean (PDM) images was also given to show the colocalization, with orange representing colocalized pixels and blue indicating segregation. Scale bar, 20 μm. Colocalization analysis of acetylated α-tubulin and Golgi in nontreated, nocodazole-treated, and tubastatin A-treated SMA and control fibroblast cells (B). Fifteen cells were analyzed in a blinded manner in three independent culture experiments, and Pearson’s correlation coefficient was calculated by ImageJ. Analysis of the Golgi area in nontreated and tubastatin A-treated SMA and control fibroblast cells (C). The Golgi area was measured as a readout of fragmentation, since intact Golgi occupies a smaller area compared with fragmented Golgi. A total of >50 cells were used for each…

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  Figure 4. Proposed mechanistic model for reduced α-tubulin acetylation, Golgi fragmentation, and its partial morphological restoration in SMA. TuBA and Ac indicate tubastatin A and acetyl groups, respectively. Created in BioRender2.