Author response: Mouse Tmem135 mutation reveals a mechanism involving mitochondrial dynamics that leads to age-dependent retinal pathologies (2016)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract While the aging process is central to the pathogenesis of age-dependent diseases, it is poorly understood at the molecular level. We identified a mouse mutant with accelerated aging in the retina as well as pathologies observed in age-dependent retinal diseases, suggesting that the responsible gene regulates retinal aging, and its impairment results in age-dependent disease. We determined that a mutation in the transmembrane 135 (Tmem135) is responsible for these phenotypes. We observed localization of TMEM135 on mitochondria, and imbalance of mitochondrial fission and fusion in mutant Tmem135 as well as Tmem135 overexpressing cells, indicating that TMEM135 is involved in the regulation of mitochondrial dynamics. Additionally, mutant retina showed higher sensitivity to oxidative stress. These results suggest that the regulation of mitochondrial dynamics through TMEM135 is critical for protection from environmental stress and controlling the progression of retinal aging. Our study identified TMEM135 as a critical link between aging and age-dependent diseases. https://doi.org/10.7554/eLife.19264.001 eLife digest Older people have an increased risk of developing many diseases, such as diabetes and age-related macular degeneration (which is often shortened to AMD). This suggests that changes that occur during normal aging may some how be linked to how such diseases develop. However, the molecular mechanisms responsible for these links are not clear. AMD causes damage to the retina of the eye, which can lead to visual loss in older people. To investigate the link between aging and age-dependent diseases, Lee et al. used mutant mice whose retina of the eye ages more quickly than normal mice and are prone to developing an eye condition that is similar to AMD. The experiments show that these mice have a mutation in a gene called Tmem135 that is responsible for these visual problems. Tmem135 regulates the size of cell compartments called mitochondria, which produce energy for the cell. This affects the ability of the mitochondria to work properly and makes the cells more sensitive to environmental stress, which in turn makes the retina age more quickly. The findings of Lee et al. show that Tmem135 is a critical link between aging and an AMD-like condition in mice. Furthermore, the experiments suggest that defects in mitochondria may accelerate the normal pace of aging and lead to AMD and other age-dependent diseases. Further studies are needed to find out exactly what role Tmem135 plays in mitochondria and whether it also contributes to the aging of other parts of the body. https://doi.org/10.7554/eLife.19264.002 Introduction One explanation for why age-dependent diseases manifest themselves in an age-dependent manner is that disease-causing mechanisms interact with age-dependent cellular changes that normally occur in aging. The common phenomena observed in both aging and age-dependent diseases may provide clues to this interaction. For example, one of the major age-dependent changes that generally occur in the tissue is accumulation of damages caused by oxidative stress (Harman, 1956, 1972a, 1972b). As by-products of normal cellular respiration, reactive oxygen species (ROS) are constantly generated in cells mainly in the mitochondria. When cellular production of ROS overwhelms its antioxidant capacity (state referred to as 'oxidative stress'), ROS damages cellular macromolecules such as lipids, protein, and DNA/RNA. In the course of aging, such damages caused by ROS are thought to accumulate and contribute to the development of age-dependent tissue dysfunctions. Increase in oxidative damage has been observed in a number of age-dependent diseases as well, and its involvement in the pathogenesis of these diseases has been widely suggested (Davies, 1995). Related to the oxidative damage, another phenomenon that is observed in both aging and age-dependent diseases is the decline in mitochondrial function (Lenaz, 1998). Mitochondria are the organelle that consumes over 90% of cellular oxygen and generates ROS (Harman, 1981; Murphy, 2009). Due to its proximity to the site of ROS generation, mitochondrial components are particularly susceptible to ROS-mediated oxidative damage (Cadenas and Davies, 2000). Prolonged exposure to ROS during aging is thought to result in mitochondrial dysfunctions and significantly contribute to the development of pathologies associated with aging. There is also strong evidence that mitochondrial dysfunction occurs early and acts causally in the pathogenesis of age-dependent neurodegenerative diseases (Lin and Beal, 2006). While these phenomena indicate some of the common aspects between aging and age-dependent diseases, the mechanisms linking these two processes have not been elucidated at the molecular level. Given the complexity in both the aging process and age-dependent diseases, as well as countless variables (including genetic an environmental variables) that exist among human population, it is extremely challenging to study the mechanisms underlying the aging process and how they relate to the disease-causing mechanism in humans. An animal model that shows accelerated aging as well as age-dependent disease symptoms could provide a useful experimental system for this purpose. Furthermore, a forward genetics approach starting with an animal model with these symptoms offers a potential of identifying a responsible gene that is not previously known to be associated with the aging process nor age-dependent diseases. We isolated an N-ethy-N-nitrosourea (ENU)-induced mutant mouse line, FUN025, that exhibits age-dependent retinal abnormalities with a trajectory similar to that found with retinal aging observed in wild-type (WT) mice (Higuchi et al., 2015) but with an early onset and faster progression. In addition, we found that the FUN025 mutation leads to pathologies observed in age-dependent retinal diseases such as age-related macular degeneration (AMD). These phenotypes in FUN025 mice suggest that the responsible gene is involved in regulating the rate of aging in the retina, and that its impairment leads to development of age-dependent disease. In this study, we identify a gene mutation that is responsible for retinal abnormalities in FUN025 mice and characterize the novel molecular functions of this gene/protein associated with regulation of mitochondria as well as sensitivity to oxidative stress. Our findings reveal a molecular link between the aging process and age-dependent diseases, and a molecular mechanisms leading to age-dependent disease pathologies. Results Early-onset and accelerated progression of aging-associated changes in the FUN025 retina FUN025 mice were isolated through fundus examination in an ENU mouse mutagenesis project (Pinto et al., 2004; Vitaterna et al., 2006), and were found to exhibit retinal abnormalities similar to those observed in aged WT mice (Higuchi et al., 2015). It has been shown that, in the WT retina, age-dependent abnormalities including retinal degeneration, increased number of ectopic synapses and increased retinal stress all start from the peripheral retina by 8 months of age on an aging-susceptible A/J background but later on a less susceptible B6 background, which progress to the central retina with age (Higuchi et al., 2015). Histological analysis of homozygous FUN025 mutant and C57BL/6J (B6) WT retina at two and seven months of age revealed that a decrease in the ONLT Index [the thickness of outer nuclear layer (ONL) normalized by the thickness of inner nuclear layer (INL)] in FUN025 mice compared to WT mice is observed by two months of age and becomes more pronounced by seven months of age, indicating progressive loss of photoreceptor cells in the FUN025 retina (Figure 1A,B). Immunohistochemical analysis demonstrated that, while most of the presynaptic photoreceptor terminals (PSD95, green) line up in the outer plexiform layer (OPL) in close opposition to the bipolar cell postsynaptic structures (PKC, red) in the WT retina, ectopic localization of presynaptic terminals and abnormal extension of bipolar cell dendrites into the ONL were observed in the FUN025 retina (Figure 1C,D). Quantification of ectopic synapses indicated that a significantly increased number of ectopic synapses are observed in the peripheral retina of FUN025 mice by two months of age, and in the central retina by seven months of age, indicating that this phenotype progresses from the peripheral to the central retina (Figure 1D). We also observed that the sign of retinal stress, up-regulation of glial fibrillary acidic protein (GFAP) (Higuchi et al., 2015; Lewis and Fisher, 2003), was significantly increased in the peripheral retina of FUN025 mice compared to the WT mice by two months of age, which later progresses toward the central retina (Figure 1E). Thus, FUN025 retina exhibits age-dependent retinal abnormalities with a trajectory similar to that found with retinal aging observed in WT mice (Higuchi et al., 2015) but with an early onset and faster progression. Figure 1 Download asset Open asset Age-dependent retinal abnormalities in FUN025 mice. (A–B) 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. (C–D) 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. https://doi.org/10.7554/eLife.19264.003 FUN025 mice exhibit age-dependent disease pathologies We further investigated whether the early-onset and accelerated aging process in the FUN025 retina is accompanied by pathologies observed in age-dependent diseases. Punctate light deposits were found in the fundus photography of the eyes from FUN025 mice (Figure 2A), which may be due to the accumulation of autofluorescent cells and aggregates observed between photoreceptors and retinal pigment epithelium (RPE) (Figure 2B). These cells/aggregates and RPE exhibit autofluorescence detected using a DPS 561 laser (Figure 2B,C), which resembles that of lipofuscin, protein and lipid rich aggregates known to accumulate in aging tissues and suggested to be involved in age-dependent diseases such as AMD and Alzheimer's disease (Giaccone et al., 2011; Nowotny et al., 2014). The hyper-autofluorescent aggregates (Figure 2B, arrowhead) resemble subretinal drusenoid deposits (SDD) (Rudolf et al., 2008), a type of extracellular lesion between the photoreceptors and RPE often observed in AMD patients (Curcio et al., 2013; Zweifel et al., 2010). To determine the cell type of these autofluorescent cells, immunostaining was performed in 7-month old retinal sections. The subretinal autofluorescent cells were positive for a microglia marker, Iba1, and a macrophage marker, F4/80, in the FUN025 retina (Figure 2C). The Iba1+ cells near the apical surface of the RPE have very few processes and show immunoreactivity to F4/80, suggesting that they have transformed from microglia to macrophages (Figure 2C). These data also suggest that the RPE layer is the origin of stress, toward which the microglia migrate (Jonas et al., 2012; Mcglade-Mcculloh et al., 1989). Long-term, low-grade inflammation (innate immunity) has been widely postulated as a part of the aging process, and is also enhanced in many age-dependent diseases (Salminen et al., 2012). To test if innate immunity is increased in FUN025 mice, immunostaining was performed with an inflammasome marker, NLR Family, Pyrin Domain Containing 3 (NLRP3) and a functional caspase-1 subunit marker, caspase-1 p10 on retinal sections from seven-month old FUN025 and WT mice. Immunoreactivity to both makers was increased in the neural retina as well as RPE of FUN025 mice compared to WT mice (Figure 2D), indicating elevated innate immunity (Franchi et al., 2009). In addition, by seven months, the RPE (highlighted by anti-CRALBP antibody) is uniformly thickened in FUN025 mice compared to that of controls while each RPE cell is still intact (the nuclei of RPE highlighted by anti-Otx1+Otx2 antibody) (Figure 2E). Similar increases in innate immunity markers (Tarallo et al., 2012) and thickness of RPE (Acton et al., 2012; Karampelas et al., 2013; Zhao and Vollrath, 2011) have been observed in AMD patients. To determine if early-onset of retinal aging as well as retinal pathologies observed in FUN025 mice affect the ability of the retina to respond to light, we performed transcorneal electroretinograms (ERG) recordings from seven-month-old FUN025 mice and their WT littermates. ERG data revealed significantly reduced scotopic (dark-adapted) a-wave and b-wave from the rod pathway, and a modest reduction in photopic (light-adapted) b-wave from the cone pathway in FUN025 mice compared to WT controls (Figure 2F) indicating impaired visual function in FUN025 mice. In conclusion, FUN025 mice provide a mouse model with accelerated aging process and age-dependent disease pathologies in the retina. Figure 2 Download asset Open asset 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.). https://doi.org/10.7554/eLife.19264.004 Identification of a Tmem135 mutation in FUN025 mice To identify the causative gene/mutation for retinal phenotypes in FUN025 mice, we performed a genome-wide linkage analysis. Genetic mapping of the FUN025 mutation was performed using F2 intercrosses (C57BL6-FUN025 x C57BR/cdJ and C57BL6-FUN025 x 129S1/SvImJ) and genetic markers to distinguish between the alleles of FUN025 (C57BL/6J) and C57BR/cdJ or 129S1/SvImJ (Figure 3A). The F2 mice were phenotyped by fundus photography and histological analysis at 3 months. Genotype and phenotype data from 70 F2 mice generated a minimal genetic region flanked by SNPOlf294T>C (93764448) and Rs31011252, containing 25 genes. Using a SureSelectXT custom library (Agilent Technologies), we performed a sequence capture array on FUN025 genomic DNA followed by paired end sequencing. Standard bioinformatic analyses of our sequencing data revealed a point mutation (T>C) in the splice-donor site adjacent to exon 12 of Tmem135 in FUN025 mice (Figure 3B,C). The consensus sequence of mouse splice donor sites depicts the necessity of the GT sequence at positions 1 and 2 downstream of the exon boundary for the functionality of the site (Carmel et al., 2004). The mutation disrupts the splice donor site (Figure 3C), resulting in skipping of exon 12 and a frame shift creating an early stop codon in FUN025 mice (Figure 3D,F). The probability of forming transmembrane helices predicted by the program TMHMM (v. 1.0) (http://www.cbs.dtu.dk/) suggested that WT TMEM135 contains five transmembrane helices (Figure 3G and Figure 3—figure supplement 1), while the 4th and 5th transmembrane helices are abolished and the orientation of the remaining 3 transmembrane helices in the membrane is reversed in mutant TMEM135 compared to WT TMEM135 (Figure 3G and Figure 3—figure supplement 1). The c-terminal region of the mutant TMEM135 is also shorter due to the early stop codon (Figure 3E,G). Thus, the FUN025 mutation in Tmem135 which results in the shorter c-terminal region with amino acid sequence changes, predicted loss of two transmembrane helices and reversed orientation within the membrane likely impairs the normal functions of the TMEM135 protein. The TMEM135 antibody recognizing the N-terminus of TMEM135 protein detected the mutant TMEM135 protein in the FUN025 brains as well as the WT TMEM135 protein in WT brains by western blot analysis (Figure 3H). Figure 3 with 2 supplements see all Download asset Open asset Identification of a Tmem135 mutation in FUN025 mice. (A) Minimal genetic region of FUN025 on chromosome 7 determined by genetic mapping. (B) A point mutation (T > C) in the splice-donor site adjacent to exon 12 of Tmem135 in FUN025 mice. (C) The consensus sequence of mouse splice donor sites, depicting the necessity of the GT sequence at positions 1 and 2 downstream of the exon boundary for the functionality of the site. The C57BL/6J and FUN025 sequences are shown below, demonstrating the disrupted site in FUN025 mice. (D) RT-PCR spanning exon 12 and sequencing the product revealed the absence of this exon in the FUN025 retina. (E) Amino acid sequences of the C-terminus of TMEM135 in C57BL/6J and FUN025 mice. The WT protein is 458 amino acids long, whereas the truncated mutant protein is 406. The change in amino acid sequence is highlighted in red. (F) Consequences of the mutation in the genomic sequence of Tmem135. The mutation adjacent to exon 12 of Tmem135 (red star) results in a non-functional splice donor site, causing skipping of exon 12. This results in a frameshift and an early stop codon (chr7: 96,290,429, NCBI build 37). Locations of the stop codons are highlighted in red. (G) A predicted structure of TMEM135 having five transmembrane domains. The FUN025 mutation is predicted to result in a protein with only three transmembrane domains, whose orientation in the membrane is reversed. The rest of the c-terminal region is absent due to the early stop codon (asterisk). (H) Western blot for TMEM135 in WT and FUN025 brains. GAPDH was used as a loading control. https://doi.org/10.7554/eLife.19264.005 To confirm that this mutation, rather than any other unknown mutations that occurred in the ENU-induced FUN025 mutant line, is responsible for the retinal phenotypes in FUN025 mice, a complementation test was performed. We introduced the same point mutation (T > C) as observed in FUN025 mice in the intron 12 of Tmem135 (Chr7:96,296,478) in C57BL/6J mice using the CRISPR/Cas9 system (T > C mice). The T > C heterozygous mice were crossed with the FUN025 homozygous mice to produce F1 (T > C/FUN025 compound heterozygous) mice, which were analyzed for retinal phenotypes. The F1 mice exhibited retinal phenotypes similar to FUN025 homozygous mice, indicating non-complementation (Figure 3—figure supplement 2). This result demonstrates that the point mutation (T > C) in Tmem135 is indeed the FUN025 causative mutation. Therefore, we now designate homozygous FUN025 mice as Tmem135FUN025/FUN025. Localization of TMEM135 TMEM135 protein was suggested to be involved in fat storage and the regulation of longevity in C. elegans (Exil et al., 2010), but the function of TMEM135 is not yet clearly characterized. In order to elucidate the mechanistic role of TMEM135, we characterized the localization of the TMEM135 protein in cultured cells and mouse retina in vivo. Primary WT mouse fibroblast cells (MFs) were co-transfected with the green fluorescent protein (GFP)-tagged vector containing Tmem135 and DsRed2-tagged mitochondria vector (pDsRed2-Mito Vector, Clontech, Mountain View, CA). Immunofluorescence data showed that the GFP-TMEM135 protein displays an intracellular vesicular expression pattern in the cytoplasm, and a proportion is found in punctate structures on mitochondria (Figure 4A). Colocalization of TMEM135 to mitochondria was further confirmed in WT MFs using an antibody and an mitochondria vector Vector, Clontech, Mountain View, (Figure as well as in WT MFs with antibody and a that mitochondria (Figure the proportion of TMEM135 to to the surface of mitochondria, at mitochondrial sites, and at the of mitochondria (Figure and to be less of TMEM135 on mitochondria in FUN025 MFs (Figure revealed that GFP-TMEM135 on the surface of mitochondria (Figure Furthermore, of TMEM135 to mitochondria was observed in cells (Figure and mouse (Figure The mitochondria isolated from mouse brains showed TMEM135 by also indicating that TMEM135 is associated with mitochondria (Figure In the retina from WT and FUN025 mice, TMEM135 were detected in the cell layer inner plexiform layer outer plexiform layer inner of photoreceptor cells, and which with mitochondria with antibody (Figure The of TMEM135 with mitochondria was also observed in the mouse RPE cell (Figure Similar to the in (Figure less of TMEM135 with mitochondria was observed in FUN025 RPE cells compared to WT RPE cells (Figure In conclusion, a proportion of TMEM135 is associated with mitochondria in other intracellular and may be also associated with Figure 4 Download asset Open asset Localization of TMEM135 to the mitochondria. (A) localization of TMEM135 in MFs co-transfected with TMEM135 vector (green) and mitochondria vector GFP-TMEM135 were detected as to the mitochondria as well as in the Colocalization of TMEM135 and mitochondria in MFs with mitochondria vector (green) and with antibody Scale bar = 10 μm. (B) Colocalization of TMEM135 green) and red) in wild-type and FUN025 mouse Scale bar = 10 μm. (C) revealed localization of TMEM135 to the mitochondria. Colocalization of TMEM135 green) and mitochondria and red) in cells and mouse Scale bar = 10 μm. (F) The mitochondrial isolated from the WT mouse show TMEM135 by were used as organelle (G) TMEM135 (green) in inner of photoreceptor cells, and RPE from wild-type and FUN025 mouse retina. the retina, TMEM135 is with mitochondria Scale bar = 10 μm. (H) Colocalization of TMEM135 green) and mitochondria red) in wild-type and FUN025 mouse RPE cell Scale bar = 5 μm. TMEM135 plays a role in mitochondrial dynamics We investigated what TMEM135 in mitochondria. TMEM135 punctate structures were often observed at mitochondrial sites and at the of mitochondria. This localization pattern suggests a role of TMEM135 in the regulation of mitochondrial We isolated MFs from and mice overexpressing WT Tmem135 (Figure supplement and the mitochondria with to WT cells with mitochondria of all and cells showed mitochondrial whereas cells exhibited mitochondrial (Figure We used a et al., in which each cell was as having or mitochondria. more cells were found to have mitochondria to cells isolated from WT mice, indicating that more fusion occurs compared to fission in cells (Figure In among more cells were found to have mitochondria to WT cells, suggesting that more fission than fusion in cells (Figure we compared the number and size of mitochondria between and WT MFs (Figure The size of mitochondria increases and its number in cells indicating that more mitochondrial fusion occurs than fission leading to mitochondria. In the size and of mitochondria decrease in cells indicating that more fission than fusion mitochondria. that mitochondrial size increases in cells was further confirmed by Tmem135 using of Tmem135 in WT MFs using a Tmem135 in a significant increase in mitochondrial size compared to WT MFs with (Figure We analyzed the retinal mitochondrial in the RPE as well as inner of photoreceptor cells from WT and FUN025 mice using (Figure We observed mitochondria in both the RPE and inner of photoreceptor cells from FUN025 mice compared to those from WT mice (Figure these results indicate that TMEM135 is involved in the regulation of the between mitochondrial fission and fusion Figure 5 with 3 supplements see all Download asset Open asset TMEM135 is involved in the of mitochondrial fission and (A) of mitochondria red) in and Scale bar = 10 μm. (B) of mitochondrial in FUN025 and Data from n = n = FUN025, and n = 3 mice per genotype. Quantification of number and of mitochondria in FUN025 and Data from n = n = FUN025, and n = 3 mice per genotype. (F) Tmem135 by Tmem135 results in increased mitochondrial size in WT Data from n = cells with n = cells with Tmem135 Student’s t-test. (G) revealed the of mitochondria in RPE and inner of photoreceptor cells from wild-type and FUN025 mice. = = n = = outer = inner Scale bar = 1 μm. (H) Quantification of mitochondria size in RPE and inner of photoreceptor cells from wild-type and FUN025 mice. RPE data from mitochondria from three wild-type mice and mitochondria from FUN025 mice. data from mitochondria from wild-type mice and mitochondria from FUN025 mice. Immunofluorescence of green) and mitochondria red) in WT and FUN025 Scale bar = 10 μm. Quantification of and on mitochondria. as in Materials and of a cell and with at the indicated TMEM135 that are close to the mitochondrial