Fig 2: Distribution and Morphology of ER and Lysosomes(A) Panel of immunofluorescent images of 6 month astrocytes stained for GFAP (red) and the ER marker ERp57 (green). Scale bars, 50 µm.(B) Electron micrographs comparing morphology of ER (red arrows) between groups. Scale bars, 800 nm.(C) Immunostaining of lysosomes via LAMP2 (green) on 6 month astrocytes. Scale bars, 10 µm.(D) Average signal of ERp57 in astrocyte processes between groups (n = 3).(E) Average LAMP2 signal area between groups.*p = 0.05 and **p = 0.001–0.01 Data are represented as mean ± SD.

Fig 3: Clinical features and genetics of LAMP-2–deficient patients. (a) Pedigree and LAMP-2 mutations in the first family. The affected son (II-2) was hemizygous for c.1009_1010 delGT: p.Val337Phe fs*12 (delGT), while the mother (I-2) was heterozygous. Arrowheads in the chromatograms indicate the position of the deletion. wt, wild-type. (b,c) Brain MRA images in the affected mother at the age of 47 years (B, axial view; C, coronal view) are shown. She exhibited a bilateral diffuse narrowing of the middle cerebral artery (arrowheads). There was also mild, irregular stenosis in the proximal portion of the right internal carotid artery (asterisks). (d) Western blot analysis of skeletal muscle in the affected son (II-2). LAMP-2 is abundantly expressed in control muscle, whereas only trace levels were found in the patient’s muscle. (e) Pedigree of the second family. A splice-donor site mutation (c.864 + 5 G >C, in intron 6) causes an in-frame exon-6 skipping (+5 G > C); genotypes are shown below the symbols. Light microscopy showed thickened media in muscular arteries of both the mother (f) and her son (g). Brain autopsy was not performed in the mother. Azan staining was used for the coronary arteries, whereas H&E staining was used for the renal and cerebral arteries. Scale bars: 50 µm.

Fig 4: Alterations in mitochondrial dynamics and function of VSMC under LAMP-2 deficiency. (a) Morphology of mitochondria in live VSMC. Mitotracker staining (red) revealed more marked fragmentation of mitochondria in LAMP-2 siRNA human brain VSMC, compared with control cells. The nuclei and cytoplasmic LAMP-2 protein were stained by DAPI (blue) and LAMP-2 (green), respectively. Scale bars: 10 µm. The graphs show that LAMP-2 deficiency induced significantly decreased individual mitochondrial area and significantly increased number of mitochondria in ROI per cell. ***P < 0.001, compared with control cells. (b) Double immunostaining of DRP-1 (turquoise) and Mitotracker (red). The nuclei were visualized by DAPI (blue). The colocalization of DRP-1 protein and mitochondria is indicated by white. Scale bars: 10 µm. Colocalization of DRP-1 with MitoTracker is more obvious in VSMC under LAMP-2 deficiency, compared with control cells. ***P < 0.001. (c) 3D time-lapse reconstructed images of mitochondria in live VSMC. Time-lapse tracking during 3 minutes revealed that mitochondria in LAMP-2–deficient VSMC changed their shape to become smaller, swollen, and more spherical, thereby resulting in a fragmented pattern (arrowheads). In contrast, control cells maintained normal mitochondrial dynamics, with fine tubular networks. Scale bars: 5 µm. (d) Immunocytochemical detection of H2O2 in LAMP-2 siRNA human brain VSMC. Green fluorescence indicates DCF production. Nuclei were visualized by DAPI (blue). Bars: 50 µm. (e) H2O2 content was evaluated in flow cytometry analysis by quantification of DCF. Histograms showed enhanced DCF expression in LAMP-2 siRNA-treated human brain VSMC, compared with control cells. Quantified data from 3 independent experiments are shown. *P < 0.05. (f) Mitochondrial respiration assay. Oligomycin, FCCP, and a mix of rotenone/antimycin A were sequentially added to measure basal respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration. (g) Representative line graphs of mitochondrial respiration assays are shown. (h) LAMP-2 siRNA human brain VSMC showed a significant increase in mitochondrial respiration. *P < 0.05, **P < 0.01, ***P < 0.001, and ns, not significant, compared with control cells. A two-tailed Student’s t-test was used for all panels. Ctrl, control; MFI, mean fluorescence intensity.

Fig 5: Localization of TMEM135 to the mitochondria.(A) Mitochondrial localization of TMEM135 in MFs co-transfected with GFP tagged TMEM135 vector (green) and DsRed2 tagged mitochondria vector (red). GFP-TMEM135 signals were detected as puncta to the mitochondria as well as in the cytoplasm. Colocalization of TMEM135 and mitochondria in MFs transfected with AcGFP1 tagged mitochondria vector (green) and immunostained with anti-TMEM135 antibody (red). Scale bar = 10 µm. (B) Colocalization of TMEM135 (anti-TMEM135 antibody, green) and mitochondira (MitoTracker, red) in wild-type and FUN025 mouse fibroblasts. Scale bar = 10 µm. (C) Immuno-EM revealed localization of GFP-tagged TMEM135 to the mitochondria. (D–E) Colocalization of TMEM135 (anti-TMEM135 antibody, green) and mitochondria (MitoTracker and TOMM20, red) in Cos-7 cells and primary mouse hippocampal neuron. Scale bar = 10 µm. (F) The mitochondrial fraction isolated from the WT mouse brain show TMEM135 signals by immunoblotting. Following proteins were used as organelle markers: MFN2–mitochondria; LAMP2–lysosome; Lamin B1– nucleus; PDI– endoplasmic reticulum (ER). (G) Strong TMEM135 signals (green) in GCL, IPL, OPL, inner segments of photoreceptor cells, and RPE from wild-type and FUN025 mouse retina. Throughout the retina, TMEM135 is colocalized with mitochondria (anti-TOMM20 antibody, red). Scale bar = 10 µm. (H) Colocalization of TMEM135 (anti-TMEM135 antibody, green) and mitochondria (MitoTracker, red) in wild-type and FUN025 primary mouse RPE cell culture. Scale bar = 5 µm.DOI: http://dx.doi.org/10.7554/eLife.19264.008