Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1H46R-Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking
Figure 8
Loss of ALS2 results in decreased levels of the lysosome-dependent degradation of LC3 in fibroblasts.
(A) Effect of ALS2 expression on the autophagic flux in fibroblasts. Fibroblasts derived from either wild-type (Als2+/+) or Als2−/− mice were incubated in a starvation medium with or without 0.5 µM chloroquine (CQ) for 6 hr. Equal amount of protein from total lysates were analyzed by immunoblotting using antibodies as indicated. Alpha(α)-tubulin served as control. (B) Effect of ALS2 expression on the autophagic flux in fibroblasts. Fibroblasts derived from either wild-type (Als2+/+) or Als2−/− mice were incubated in a starvation medium with or without 20 µg/ml pepstatin A (pepA) for indicated periods. Equal amount of protein from total lysates were analyzed by immunoblotting using antibodies as indicated. Alpha(α)-tubulin served as control. (C) Quantitative densitometry for the levels of LC3-II immunoreactive signals shown in B. Data were normalized by the levels of α-tubulin (LC3-II/α-tubulin). Values are mean±SEM (n = 4) in an arbitrary unit relative to control. Statistical significance is evaluated by ANOVA with Bonferroni's post hoc test (one-way, compared with respective controls; *p<0.05, ***p<0.001, and two-way, compared between WT and Als2−/−; +p<0.05). (D) Representative images for double immunostaining with LC3 (green) and p62 (red) in fibroblasts. Fibroblasts from wild-type (WT) and Als2−/− mice were either left unstarved (0 hr) (upper) or starved for 6 hr (lower). It is notable that a 6 hr of starvation leads to decreased levels of the LC3- and p62-immunoreactive signals in WT cells, but not in Als2−/− cells. Scale bars = 50 µm.