Fig. 1: Ttyh1 localizes to endolysosomes and mediates autophagic flux in astrocytes.

a Ttyh1 is primarily expressed in mouse astrocytes. Representative (n = 2 mouse brains; 16-weeks-old female) immunofluorescence image of a mouse hippocampal sagittal section shows nucleus (lavender), Ttyh1 (magenta), and Gfap (green). Insets show magnification of boxed region. b, c Ttyh1 localizes to endolysosomes in astrocytes. Confocal images of primary mouse cortical astrocytes show nucleus (blue), Ttyh1 (magenta), Lamp1 (green) (c) (n = 4 astrocytes), and LC3 (green) (d) (n = 7 astrocytes). d Schematic representations of mouse Ttyh1^flox allele and strategy to delete Ttyh1 in cortical astrocytes isolated from Aldh1l1-cre/ERT2; Ttyh1^fl/fl conditional knockout (cKO) mice. e Loss of Ttyh1 leads to accumulation of autophagosome markers upon autophagy induction. Shown are representative immunoblot images and quantifications of autophagosome markers LC3 (three independent experiments) and p62 (four independent experiment) in primary cortical astrocytes of the cKO mice. Treatment with culture medium was used as control. Nutrient deprivation for 2 h in artificial cerebrospinal fluid (ACSF) was used to induce autophagy. Bafilomycin A1 (BafA1; 100 nM) inhibits lysosomal acidification and blocks autophagic flux. High LC3-II/I ratio and normalized p62 levels indicate autophagic flux blockage. Values are normalized to those of the Ttyh1 WT, medium condition, on the same blot. Data were presented as mean ± SEM. Number of biological replicates (n) is shown in brackets at the bottom of each bar. Unpaired two-tailed t-test (LC3-II/I: t = 5.464, df = 4; p62/αTub: t = 2.877, df = 6). f Ttyh1 mediates autophagic flux. Primary cortical astrocytes from the cKO mice expressing autophagic flux probe GFP-LC3-RFP-LC3ΔG were treated with ACSF for 0, 30, and 90 min. The ratio between GFP and RFP fluorescence inversely correlates with autophagic flux. Shown are representative fluorescence images and quantification of the GFP/RFP ratio of individual astrocytes. All GFP/RFP ratios are normalized to the mean of those at 0 min. Each datapoint represents one astrocyte. Data were presented as mean ± SEM. Number of astrocytes (n) is shown in brackets at the bottom of each bar. Unpaired two-tailed t-test (Ttyh1 WT: t = 4.135, df = 44; Ttyh1 KO: t = 1.200, df = 43; n.s not significant, P = 0.2366). Source data are provided as a Source Data file.