A mutation in the TMEM65 gene results in mitochondrial myopathy with severe neurological manifestations

Abstract

Recent research has suggested that transmembrane protein 65 (TMEM65) is localized within the inner mitochondrial membrane. Little else is known about its function. In this study we investigated the location and function of TMEM65. Further, we report the functional consequences of a novel homozygous splice variant (c.472+1G>A) in the TMEM65 gene in a patient with mitochondrial encephalomyopathy. Here we investigated the location of TMEM65 by immunofluorescence staining of the protein and by immunoblotting of the isolated mitochondrial fractions in healthy fibroblasts and those from the patient. To study the function of TMEM65 we knocked down mRNA using TMEM65-specific siRNA, and measured mitochondrial function by enzymology, protein abundance and oxygen consumption rate in fibroblasts. Subcellular fractionation confirmed that the TMEM65 protein was present in the inner mitochondrial membrane. Knocking down TMEM65 expression in dermal fibroblasts severely affected mitochondrial content and respiration rate. Further evidence for the essential role of TMEM65 in mitochondrial function came from the demonstration of severe cellular and clinical consequences resulting from the novel TMEM65 gene mutation. In conclusion, these findings suggest that TMEM65, an inner mitochondrial membrane protein, plays a significant role in mitochondrial respiratory chain function. We also provide the first evidence that a mutation in the TMEM65 gene results in mitochondrial dysfunction and a severe mitochondrial encephalomyopathy phenotype.

Figure 1

Identification and functional analysis of a novel variant in TMEM65 gene. (A) Pedigree of the family. (B) Electropherogram identifying the heterozygous variant in parents and homozygous variant in the exon 4/intron 4 boundary (NM_194291.2:c.472+1G>A,chr8:g.124,323,320C>T (GRCh38[hg38])) in patient DNA. (C) Real-time PCR of TMEM65 mRNA expression of exon 2 and exon 6 in control (black bars) and in TMEM65 patient (gray bars). The control showed expression of exon 2 and exon 6 while the subject showed lack of mRNA expression of exon 6. (D) Dermal fibroblast from healthy control and TMEM65 patient was cultured and stained for TMEM65. Punctate TMEM65 staining was observed in healthy fibroblast but fibroblast from the TMEM65 patient showed no staining. (E) TMEM65 protein content in control vs TMEM65 patient cells assessed by immunoblotting whole dermal fibroblast cell lysates. The graph shows densitometry measurement of reduced band intensity ratio of TMEM65 to actin in the patient as compared with a healthy age- and sex-matched control. (F) Ultrastructure of skeletal muscle from the TMEM65 patient. a: Mitochondria showed significant pleomorphism and electron densities. b: Some mitochondria contained prominent lipid droplets (white arrow) with most showing electron densities (black arrows). c and d: Sub-sarcolemmal accumulations of rounded mitochondria were frequently seen.

Figure 2

TMEM65 localizes to the inner mitochondrial membrane and a mutation in TMEM65 effects cellular respiration. (a) Dermal fibroblasts taken from healthy control were stained for TMEM65 protein (green) and mitochondria (red) and images were taken on an inverted confocal microscope (Olympus). Co-localization of TMEM65 protein (green) and Mitotracker deep red was captured and calculated by Olympus Fluoview software Version 4.2 (scale bar=10 μm). The yellow color is reflective of the co-localization. (b) Mitochondria isolated from skeletal muscle were subfractionated into the outer mitochondrial membrane (OMM), intermembrane space (IMS), inner mitochondrial membrane (IMM) and matrix (Mx) fractions, and these fractions were subsequently immunoblotted for the compartment-specific proteins voltage-dependent anion channel (VDAC), apoptosis-inducing factor (AIF), cytochrome c oxidase subunit I (COX I), transmembrane protein 65 (TMEM65) and superoxide dismutase 2 (SOD2). (c) Oxygen consumption rate in primary dermal fibroblasts from a TMEM65 patient (gray bars) and compared with primary dermal fibroblast from three healthy age-matched controls (black bars). Oxygen consumption rate (OCR) was determined using the XFe24 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA). During oxygen consumption rate measurements cells were exposed to metabolic inhibitors including oligomycin, FCCP, rotenone and antimycin. The dermal fibroblast from the TMEM65 patient showed an overall reduced cellular respiration rates. The values are normalized to the total cellular protein per well. Graph represents three individual experiments. **P<0.001, ***P<0.0001.

Figure 3

siRNA-mediated knockdown of TMEM65 in healthy human dermal fibroblast affected mitochondrial function. (a) Oxygen consumption rate was estimated in fibroblasts transfected with control scrambled siRNA (black bars) and TMEM65 siRNA (gray bars). The fibroblasts knocked down for TMEM65 showed significantly lower cellular respiration at baseline, after oligomycin exposure and after FCCP injection when compared with control fibroblasts transfected with scrambled siRNA. Graph represents three individual experiments with different fibroblast cells from three healthy individuals. (b) Patient and TMEM65 siRNA knocked down fibroblasts showed significantly reduced mitochondrial DNA copy number and reduced enzyme activity as shown in representative graph. ***P<0.0001.

Figure 4

TMEM65 complementation in patient fibroblast improves mitochondrial function. (a) The representative graph of oxygen consumption rate (OCR) in healthy control fibroblast (black bars) with TMEM65 patient (gray bars) and compared with patient fibroblast transfected TMEM65 expression vector (open bars). The fibroblast from patient showed significantly reduced cellular respiration at baseline, after Oligomycin exposure and after FCCP injection while the oxygen consumption rate was improved in patient fibroblast transfected with TMEM65 expression plasmid. *P<0.01, **P<0.001, ***P<0.0001. (b) Fibroblasts from control and patient were transfected with TMEM65 expression vector and compared for mtDNA copy number and mitochondrial enzyme activity. Enzyme activity and mtDNA copy number were significantly improved in patient after overexpression of TMEM65. *P<0.01, **P<0.001, ***P<0.0001.