Fig. 5

ER stress and Ca²⁺ depletion in LRRK2-GS astrocytes induce mitochondrial dysfunction. a Left: Confocal images of astrocytes isolated from non-Tg or LRRK2-GS mice co-expressing mitochondria-targeted GFP (mito-GFP) and ER-targeted E2-Crimson (ER-Crimson). Right: Statistical analysis of the area of overlap (Pearson’s coefficient) between mito-GFP and ER-Crimson. b Representative electron micrographs of ER–mitochondria associations in non-Tg and LRRK2-GS astrocytes; arrows indicate the ER; M, mitochondria. Bar chart shows the percentage of the mitochondrial surface in close apposition to the ER. Data are presented as means ± SD (error bars). *p < 0.05; n = 50 mitochondria from 10 cells. c Western blot analysis of the indicated proteins in subcellular fractions from HEK293T cells expressing Myc-LRRK2-WT or Myc-LRRK2-GS. C, cytosol; E, ER; Mt., pure mitochondria; M, MAM. d-g Representative time-lapse fluorescence images of G-CEPIA1er-and R-GECO1mito-expressing non-Tg (d) and LRRK2-GS (e) astrocytes, and plots of Ca²⁺ signals in the ER and mitochondria of non-Tg (f) and LRRK2-GS (g). Astrocytes were co-transfected with G-CEPIA1er and R-GECO1mito, and then treated with α-synuclein (α-Syn). After 24 h, cells were stimulated with histamine and imaged by fluorescence microscopy. h Morphology of mitochondria in non-Tg and LRRK2-GS astrocytes stained with MitoTracker Red CMXRos (250 nM). i Quantification of tubular and fragmented mitochondria using a semi-automated system. Images from three independent areas containing 200–300 mitochondria from 5 cells were analyzed from each group. j Mitochondrial membrane potential in astrocytes isolated from non-Tg and LRRK2-GS mice. AU, arbitrary units. k Representative ROS production measured in cells loaded with MitoSOX (5 μM). All graphs represent means ± SD of three independent experiments (*p < 0.05)