Earlier work indicates an important role of TMEM135 in “mitochondrial dynamics” (11), which is the collective term for biogenesis, fusion, fission, and mitophagy events required to preserve mitochondrial integrity within cells during times of cellular and nutritional stress (27). Fibroblasts from mice with the Tmem135^FUN025 mutation that causes the loss of TMEM135 function had overly elongated mitochondrial networks that manifested in decreased number and increased size of mitochondria (11), while fibroblasts with overexpression of wild-type Tmem135 had fragmented mitochondria that were more abundant and smaller than wild-type controls (11). Colocalization between TMEM135 and a mitochondrial fission factor, dynamin-related protein 1 (DRP1), was observed at sites of mitochondrial fission (11), suggesting that TMEM135 may regulate mitochondrial fission through interaction with DRP1.

A recent study further elucidated the molecular function of TMEM135 as a regulator of DRP1, and thus, mitochondrial fission and its importance in the interaction between peroxisomes and mitochondria (25). Hu et al. observed that mitochondria appear overly fused in brown adipose tissue cultured from mice with the adipose tissue specific peroxisome deficiency, leading to impaired thermogenesis (25). Proteomic analysis of the mitochondria isolated from the peroxisome deficient brown adipocytes after cold exposure revealed TMEM135 as the most decreased protein (25), suggesting its involvement in the peroxisomal regulation of mitochondrial fission. The absence of TMEM135 on the mitochondria after cold exposure of the peroxisome deficient brown adipocytes indicated the prerequisite of TMEM135 to translocate from peroxisomes to mitochondrial membranes for the initiation of mitochondrial fission (Figure 1) (25). Investigation of the DRP1 phosphorylation state indicated that TMEM135 promotes protein kinase A (PKA)-dependent phosphorylation of DRP1 and its recruitment to mitochondria (25), defining the mechanism through which TMEM135 promotes mitochondrial fission. It was also shown that the translocation of TMEM135 from peroxisomes to mitochondria depends on plasmalogens (25), a class of glycerophospholipids containing a vinyl-ether and ester bond that are dependent on peroxisomes for their production (28). These findings add to the growing substantiation of an intimate relationship between peroxisomes and mitochondria that is needed for proper mitochondrial dynamics and homeostasis (29–33).

A role of TMEM135 in lipid homeostasis was first indicated by transcriptomic profiling of retinal tissues isolated from mice with the Tmem135^FUN025 mutation. The retinal phenotypes of Tmem135^{FUN025/FUN025} mutant mice correlated with increased expression of genes involved in fatty acid metabolism, cholesterol metabolism, and steroid metabolic processes (16), suggesting that the function of TMEM135 is important for the regulation of lipid synthesis. In support of this notion, age-dependent progression of neutral lipid and cholesterol accumulation was observed in the eyecups of Tmem135^{FUN025/FUN025} mutant mice (16).

The relationship between TMEM135 and lipid metabolism was further defined through a high-throughput and semi-quantitative lipidomics analysis of Tmem135^{FUN025/FUN025} mutant tissues. Untargeted profiling of intact lipid species using liquid chromatography with tandem mass spectrometry (LC-MS/MS) in the livers, retinas, hearts, and plasmas of Tmem135^{FUN025/FUN025} mutant mice showed that each tissue had robust decreases in lipids containing Docosahexaenoic acid (DHA or C22:6n3) compared to wild-type control mice (34). Since all lipid classes that are known to harbor DHA were affected by the Tmem135^FUN025 mutation, which was confirmed by gas chromatography mass spectrometry (GC-MS) (34), it became apparent that TMEM135 has a major task in cellular DHA homeostasis.

DHA is an omega-3 polyunsaturated fatty acid (PUFA) important for neuronal development and function as well as an important mediator of inflammation and disease (35). The concentration of DHA within tissues results from the contribution of this omega-3 PUFA from dietary sources and endogenous production within cells (36). Since the diet consumed by Tmem135^{FUN025/FUN025} mutant mice did not contain DHA (34), it must originate from the endogenous production from the ‘Sprecher pathway’ of DHA synthesis that takes place in the ER and completes in peroxisomes in these animals (37). The ER possesses desaturases [fatty acid desaturase 1 (Fads1) and 2 (Fads2)] and elongases [elongation of very long chain fatty acids-like 2 (Elovl2) and 5 (Elovl5)] needed for the desaturation and elongation of dietary essential fatty acid 18:3n3 to generate C24:6n3 (38). Then, C24:6n3 is imported into peroxisomes for retroconversion to C22:6n3 by their beta-oxidation enzymes (39). The ER and peroxisomal components of the ‘Sprecher pathway’ were evaluated in the livers of Tmem135^{FUN025/FUN025} mutant mice to determine the molecular basis of the diminished DHA concentrations due to the Tmem135^FUN025 mutation (34). Remarkably, there were no decreases in any of the components, and rather there were increases in the peroxisomal beta-oxidation enzymes required to produce DHA (34) indicating that reduced DHA in Tmem135^{FUN025/FUN025} mutant mice does not result from a defect in the ‘Sprecher pathway’ of DHA synthesis. The remaining step where TMEM135 may play a role in the generation of DHA within cells is the export of DHA from peroxisomes (Figure 2). While the exact molecular mechanism is unknown, it is thought that there is a protein on peroxisomes capable of exporting DHA from these organelles (40). The results of the lipid and pathway investigation of Tmem135^{FUN025/FUN025} mutant mice strongly suggested that TMEM135 has a critical function in exporting DHA from peroxisomes to deliver DHA to the ER for esterification into lipids (Figure 2) (34). This is consistent with the postulated function of TMEM135 involving the transport of metabolites between organelles (18, 41).

Changes caused by Tmem135 perturbations affect the number of peroxisomes, organelles that have chief responsibilities connected with cellular metabolism through its interactions with mitochondria, lipid droplets, lysosomes, and ER (42). In cultured fibroblasts with the Tmem135^FUN025 mutation, there was an increase of peroxisomes, while fibroblasts overexpressing Tmem135 showed reductions in peroxisomal number (34). It is known that peroxisome proliferation is in part mediated by the actions of the peroxisome proliferator activated receptor (PPAR) family of transcription factors (43). Peroxisomes and their protein contents were decreased in the livers of Tmem135^{FUN025/FUN025} mutant mice upon genetic ablation of PPAR alpha (Ppara) (34), indicating that activation of PPARa signaling is involved in increasing peroxisome proliferation in these mice. While it is unclear what drives the changes in PPAR signaling due to the changes in TMEM135 function, it is possible that impaired DHA export from peroxisomes results in the generation of peroxisome-derived metabolites that interact with PPARs such as ether phosphatidylethanolamines (EtherPEs) known to activate the PPAR signaling (44) which is increased in Tmem135^{FUN025/FUN025} mutant mice (34). More work is required to discern the molecular mechanism underlying the peroxisome proliferation changes observed in Tmem135 mutant and overexpressing cells, and its relationship with the TMEM135 molecular function.

TMEM135 has been implicated to have a role in the distribution of intracellular cholesterol by two different studies (45, 46). First, Tmem135 was identified in a shRNA screen for genes involved in trafficking of cholesterol from low-density lipoprotein (LDL) to the plasma membrane of HeLa cells (45). TMEM135 was further validated as a protein involved in intracellular cholesterol trafficking by knocking it down in HeLa cells, which resulted in fewer contacts between lysosomes and peroxisomes as well as decreased cholesterol in the plasma membrane (45). These results suggested that lysosome-peroxisome trafficking of cholesterol mediated by contacts between these organelles is impaired in Tmem135 knockdown cells. This result was further confirmed in another study using RPE1 cells, an immortalized RPE cell line often utilized in cilia-focused research (46). The authors observed fewer lysosome-peroxisome contacts (46) and an increased accumulation of cholesterol in the lysosomes of cells with decreased Tmem135 expression (18). After treatment with LDL, the knockdown of Tmem135 expression impaired the ability of cholesterol from LDL particles to reach the ER. Accumulation of cholesterol was observed in the eyecups of Tmem135^{FUN025/FUN025} mutant mice (16), which may occur due to defective cholesterol transport in these mice. Interestingly, accumulation of cholesterol in Tmem135^{FUN025/FUN025} mutant eyecups coincided with upregulation of sterol regulatory element binding transcription factor 2 (SREBP2)-targeted genes that are involved in cholesterol metabolism including hydroxymethylglutaryl-CoA synthase (Hmgcs1), sterol O-acyltransferase 1 (Soat1), ATP-binding cassette subfamily A member 1 (Abca1), and ATP-binding cassette subfamily A member 1 (Abcg1) (16). It is worth exploring whether these biochemical and expression manifestations result from defective lysosome-peroxisome interactions in Tmem135^{FUN025/FUN025} mutant mice. Moreover, based on the TMEM135 function in DHA export (34), it would be interesting to investigate whether DHA-esterified lipids influence membrane fluidity and interactions of these organelles.

Tmem135^{FUN025/FUN025} mutant mice develop an age-dependent photoreceptor cell degeneration (11, 16), which coincided with visual loss, ectopic synapse development, and neuroinflammation consisting of Müller glia activation and immune cell infiltration into the subretinal space (11, 16). Tmem135^{FUN025/FUN025} mutant mice also showed changes in the RPE such as autofluorescence, increased thickness, increased density, decreased electroretinogram c-wave amplitudes, and lipid accumulation (11, 16, 47) (Table 1).

It is possible that the retinal phenotypes of the Tmem135^{FUN025/FUN025} mutant mice may be occurring due to their deficiency of DHA (34). DHA has an important role in membrane fluidity of rod photoreceptor outer segments that is required for phototransduction (51). There are reports of other mouse models with retinal DHA deficiency including elongation of very-long-chain fatty acids-like 2 (Elovl2) mutant (52), acyl-CoA synthetase 6 (Ascl6) knockout (53), major facilitator superfamily domain containing 2A (Mfsd2a) knockout (54, 55), and adiponectin receptor 1 (Adipor1) knockout mice (56, 57) that show similar retinal pathologies to those observed in Tmem135^{FUN025/FUN025} mutant mice (11, 16, 47).

It is also possible that the retinal phenotypes of Tmem135^{FUN025/FUN025} mutant mice result from enhancement of mitochondrial fusion triggered by the Tmem135 mutation (11). Boosting mitochondrial fusion in the retinas of Tmem135^{FUN025/FUN025} mutant mice may lead to increased nutrient intake and metabolic stress as detected in other tissues caused by excessive mitochondrial fusion (58, 59). Supporting this idea, a NMR-based metabolomics study revealed an accumulation of metabolites from glucose, amino acid and lipid metabolic pathways in primary-cultured Tmem135^{FUN025/FUN025} mutant RPE cells compared to wild-type RPE cells (50). In addition, mice with retinal metabolic stress such as RPE-specific vascular endothelial growth factor A (Vegfa) or superoxide dismutase 2 (Sod2) have thicker RPE like the Tmem135^{FUN025/FUN025} mutant mice (11, 47). Future studies will need to be undertaken to determine the roles of the reduced DHA and overly fused mitochondria in Tmem135^{FUN025/FUN025} mutant mice in regard to the development of their retinal pathologies.

In contrast to Tmem135^{FUN025/FUN025} mutant mice, Tmem135 TG mice exhibit progressive RPE degenerative phenotypes including migration, vacuolization, dysmorphia, and thinning (47). Additionally, Tmem135 TG mice displayed thinner myelin sheaths of axons in the optic nerve (49). However, unlike Tmem135^{FUN025/FUN025} mutant mice, there were no signs of photoreceptor cell dysfunction or degeneration in Tmem135 TG mice at least until one year of age (47).

The retinal phenotypes of Tmem135 TG mice may result from their excess mitochondrial fragmentation (11, 47) and/or decreased peroxisome proliferation (34). It is believed that mitochondrial fission is important for the removal of damaged mitochondrial membranes in order to maintain mitochondrial function (58, 60). However, excessive mitochondrial fragmentation initiated by Tmem135 overexpression could cause mitochondrial dysfunction in the RPE and lead to degeneration of this cell type. Similar to Tmem135 TG mice, mice with RPE-specific ablation of transcription factor A, mitochondrial (Tfam) or PPARG coactivator 1 alpha (Pgc-1α) show attenuated mitochondrial function and degenerated RPE (61, 62). It is plausible that mitochondrial fragmentation in Tmem135 TG could stem from their decreased peroxisomal proliferation (34). Inhibition of proper peroxisome biogenesis by eliminating peroxisomal biogenesis factor 3 (Pex3) or peroxisomal biogenesis factor 5 (Pex5) expression promoted mitochondrial fragmentation in mouse embryonic fibroblasts (33). Recent work has shown that genetic ablation of the multifunctional protein 2 (Mfp2; also known as hydroxysteroid (17-beta) dehydrogenase 4 or Hsd17b4) gene encoding D-bifunctional protein (DBP), which is a critical enzyme required for peroxisomal beta-oxidation (34, 63), specifically in RPE cells can lead to RPE degenerative changes (64). Interestingly, expression of Mfp2 is decreased in the eyecups of Tmem135 TG mice (34). Dissecting out the roles for mitochondrial fragmentation and decreased peroxisomal proliferation will be critical to determine the contributions of these organelles to the RPE degeneration observed in Tmem135 TG mice.

Studies from cell culture and animal experiments signal a substantial role of TMEM135 in energy homeostasis in aging. To date, there has been no direct association between TMEM135 and age-related retinal diseases including AMD. However, there are multiple levels of similarities between Tmem135 mouse models and AMD including ocular pathologies and molecular and cellular changes (11, 16). Here, we will discuss potential involvement of TMEM135 in AMD pathogenesis.

There have been published associations with TMEM135 and other human medical conditions such as osteoporosis (122–127), breast cancer (128, 129), prostate cancer (130, 131), melanoma (132, 133), non-small lung cancer (134), glioblastoma multiforme (134), non-alcoholic fatty liver disease (135), cognitive disorders (136), and metabolic disease (25). Significance of TMEM135 functions have been also indicated by phenotypes in other tissues due to Tmem135 mutation and overexpression. While readers are encouraged to refer to individual studies for details, we will summarize the phenotypes in other mouse tissues caused by modulation of Tmem135.

Overexpression of Tmem135 impacts the heart along with the RPE (48). The hearts of Tmem135 TG mice on a mixed C57BL/6J and FVB/NJ background show hypertrophy with increased fibrosis (48). Ultrastructural abnormalities such as large vacuoles co-occupying the space between myofibrils with mitochondria were observed in Tmem135 TG hearts at varying severities (48). Cardiac phenotypes of Tmem135 TG mice most likely derive from their mitochondrial fragmentation. Other mouse models with heart-specific conditional ablation of mitochondrial fusion display cardiac phenotypes comprising dilated cardiomyopathy and cardiac hypertrophy (137, 138). It remains unknown if mitochondrial fragmentation in Tmem135 TG originates from mitochondria or peroxisomes as TMEM135 can translocate from peroxisomes to mitochondria for fission events (11, 25, 34).

While livers of Tmem135^{FUN025/FUN025} mutant mice appear and function normally regardless of the remarkable changes in their hepatic peroxisomes and lipidome (34), physiological significance of these cellular changes could be observed when the Tmem135^FUN025 mutation was combined with the leptin mutation (Lep^ob ), which causes metabolic disease with significant hepatic lipid adjustments and dependency on functional peroxisomes in mice (139–141). Both male and female mice that are homozygous for Tmem135 and leptin mutations (Tmem135^{FUN025/FUN025}/Lep^ob/ob ) had lower body, liver, and gonadal fat pad weights compared to their Lep^ob/ob counterparts (34). The Tmem135^FUN025 mutation also decreased the amount of plasma cholesterol by impairing the secretion of very low-density lipoprotein (VLDL) and LDL (142). Modifications of the classic obesity and dyslipidemia phenotype in Lep^ob/ob mice by the Tmem135^FUN025 mutation correlated with attenuation of their non-alcoholic fatty liver disease (NAFLD) phenotypes. There were less severe NAFLD pathologies and hepatic lipid accumulation in Tmem135^{FUN025/FUN025}/Lep^ob/ob mice compared to Lep^ob/ob mice (34). Together, these phenotypic changes suggest that impairment of TMEM135 function affects molecular pathways involved in the pathogenesis of metabolic disease with dysregulated lipid metabolism, which may include activation of PPAR signaling and increased peroxisome proliferation (34).

Most recently, adipose-specific deletion of Tmem135 was shown to result in impaired thermogenesis and increased diet-induced obesity and insulin resistance in mice, revealing significant roles of TMEM135 in the brown fat and energy homeostasis (25). Conversely, Tmem135 overexpression increased thermogenesis and prevented diet-induced obesity and insulin resistance (25). This study revealed aforementioned function of TMEM135 in the regulation of mitochondrial fission and placed TMEM135 as a critical mediator of the peroxisomal regulation of mitochondrial fission and thermogenesis (25). Additionally, the authors identified a single nucleotide polymorphism (SNP) in the human TMEM135 gene associated with increased body mass index (BMI) in a Hispanic population (25). Functional studies indicated that this specific SNP in TMEM135 reduces the function of the protein and may promote the occurrence of human metabolic diseases (25).