• Figure 1.
    Download figureOpen in new tabFigure 1. Age-dependent retinal abnormalities in FUN025 mice.

    (AB) A significant decrease of the ONLT index occurred by two months of age in FUN025 retina. Mo = months. Data from n = 10 WT (2 Mo), n = 4 FUN025 (2 Mo), n = 20 WT (7 Mo), n = 8 FUN025 (7 Mo) mice. Scale bar = 20 μm. (CD) Ectopic synapses were observed as bipolar cell neurites (PKC, red) and photoreceptor synaptic terminals (PSD95, green) extending into the ONL indicated by asterisks (C). Scale bar = 10 μm. Significant increase of ectopic synapses were found earlier in the peripheral retina, and later in the central retina of FUN025 compared to WT mice. Data for central retina from n = 3 WT (2 Mo), n = 3 FUN025 (2 Mo), n = 3 WT (7 Mo), n = 3 FUN025 (7 Mo) mice; data for peripheral retina from n = 5 WT (2 Mo), n = 6 FUN025 (2 Mo), n = 6 WT (7 Mo), n = 6 FUN025 (7 Mo) mice. (E) GFAP (green) upregulation was progressively observed in the FUN025 retina. ONL: outer nuclear layer. INL: inner nuclear layer. Outer nuclear layer thickness (ONLT) index = ONL thickness/INL thickness. *p<0.05, Student’s t-test. All data are mean ± s.e.m. Scale bar = 50 μm.

    DOI: http://dx.doi.org/10.7554/eLife.19264.003

    Figure 2.
    Download figureOpen in new tabFigure 2. FUN025 mice show AMD-like pathologies.

    (A) Punctate light deposits were found in the fundus photography of the eyes from WT and FUN025 mice. (B) Autofluorescent cells/aggregates (indicated by arrows) and lipofuscin-like autofluorescence were observed in proximity to the apical surface of the RPE in FUN025 mice. Scale bar = 60 μm. (C) Iba1 (microglia/ macrophage marker) and F4/80 (macrophage marker) positive cells were found in the FUN025 retina at seven months, whereas very few Iba1 positive cells were found in the WT retina at seven months. Scale bar = 20 μm. (D) Signals for inflammasome markers, NLRP3 and caspase1, increased in the RPE in both peripheral and central retina from FUN025 mice compared to WT control. (E) At seven months of age, the RPE (highlighted by CRALBP staining) thickness is significantly increased in both central and peripheral retina of FUN025 mice compared to control mice. The RPE nuclei were highlighted with Otx1+Otx2. Data from n = 4 mice per genotype. (F) Transcorneal electroretinograms (ERG) recordings from seven-month-old FUN025 mice and their WT littermates. Both scotopic (dark-adapted) ERG a- and b-waves from the rod pathway were markedly reduced in FUN025 mice. A reduction was also observed in photopic (light-adapted) ERG b-wave from the cone pathway with higher flash intensity, while no difference between FUN025 and WT was observed in the photopic a-wave, majority of which is postreceptoral in origin. Data from n = 5 mice per genotype. *p<0.05, two-way analysis of variance (ANOVA). All data are mean ± standard error of the mean (s.e.m.).

    DOI: http://dx.doi.org/10.7554/eLife.19264.004

    Figure 4.
    Download figureOpen in new tabFigure 4. 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. (DE) 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

    Figure 7.
    Download figureOpen in new tabFigure 7. Tmem135FUN025/FUN025mice are more sensitive to hyperoxic condition.

    (AB) Tmem135FUN025/FUN025 (FUN025) mice raised in 75% O2 for two weeks show significant decrease of ONLT and increase of TUNEL positive cells, indicating accelerated photoreceptor cell death by apoptosis. Data from n = 4 WT mice in control air, n = 3 WT mice in 75% O2, n = 3 FUN025 mice in control air, and n = 3 FUN025 mice in 75% O2. Scale bar = 20 μm. (C) Upregulation of GFAP (green) indicating retinal stress is observed in FUN025 mice raised in the normal air, as well as WT mice and FUN025 mice raised in 75% O2 for two weeks. FUN025 mice raised in 75% O2 have the highest increase of GFAP signals in the retina. Scale bar = 20 μm. (D) Western blotting showing that hyperoxia results in upregulation of 4-HNE in both WT and FUN025 retina, and an increase of TMEM135 in FUN025 retina but not in WT retina. Data from n = 3 WT mice in control air, n = 3 WT mice in 75% O2, n = 3 FUN025 mice in control air, and n = 3 FUN025 mice in 75% O2. *p<0.05, **p<0.01, two-way ANOVA. All data are mean ± s.e.m.

    DOI: http://dx.doi.org/10.7554/eLife.19264.018

  • Video 1. Live imaging of HT22 cells expressing TMEM135-GFP.

    Live imaging of a HT22 cell expressing TMEM135-GFP and labeled with MitoTracker Red. TMEM135 puncta that are located relatively close to the mitochondrial surface are shown in magenta, whereas those relatively far from the mitochondrial surface are shown in turquoise. Mitochondria (gray) was made partial transparent in order to see TMEM135 puncta located on the back of mitochondria.

    DOI: http://dx.doi.org/10.7554/eLife.19264.013

  • Video 2. Live imaging of WT fibroblasts expressing TMEM135-eGFP.

    Live imaging of fibroblasts derived from B6 (WT) mice transfected with TMEM135-eGFP plasmid and labeled with MitoTracker Red. TMEM135-eGFP are shown in green and mitochondria are shown in red.

    DOI: http://dx.doi.org/10.7554/eLife.19264.014