MSTO1 mutations cause mtDNA depletion, manifesting as muscular dystrophy with cerebellar involvement

Abstract

MSTO1 encodes a cytosolic mitochondrial fusion protein, misato homolog 1 or MSTO1. While the full genotype-phenotype spectrum remains to be explored, pathogenic variants in MSTO1 have recently been reported in a small number of patients presenting with a phenotype of cerebellar ataxia, congenital muscle involvement with histologic findings ranging from myopathic to dystrophic and pigmentary retinopathy. The proposed underlying pathogenic mechanism of MSTO1-related disease is suggestive of impaired mitochondrial fusion secondary to a loss of function of MSTO1. Disorders of mitochondrial fusion and fission have been shown to also lead to mitochondrial DNA (mtDNA) depletion, linking them to the mtDNA depletion syndromes, a clinically and genetically diverse class of mitochondrial diseases characterized by a reduction of cellular mtDNA content. However, the consequences of pathogenic variants in MSTO1 on mtDNA maintenance remain poorly understood. We present extensive phenotypic and genetic data from 12 independent families, including 15 new patients harbouring a broad array of bi-allelic MSTO1 pathogenic variants, and we provide functional characterization from seven MSTO1-related disease patient fibroblasts. Bi-allelic loss-of-function variants in MSTO1 manifest clinically with a remarkably consistent phenotype of childhood-onset muscular dystrophy, corticospinal tract dysfunction and early-onset non-progressive cerebellar atrophy. MSTO1 protein was not detectable in the cultured fibroblasts of all seven patients evaluated, suggesting that pathogenic variants result in a loss of protein expression and/or affect protein stability. Consistent with impaired mitochondrial fusion, mitochondrial networks in fibroblasts were found to be fragmented. Furthermore, all fibroblasts were found to have depletion of mtDNA ranging from 30 to 70% along with alterations to mtDNA nucleoids. Our data corroborate the role of MSTO1 as a mitochondrial fusion protein and highlight a previously unrecognized link to mtDNA regulation. As impaired mitochondrial fusion is a recognized cause of mtDNA depletion syndromes, this novel link to mtDNA depletion in patient fibroblasts suggests that MSTO1-deficiency should also be considered a mtDNA depletion syndrome. Thus, we provide mechanistic insight into the disease pathogenesis associated with MSTO1 mutations and further define the clinical spectrum and the natural history of MSTO1-related disease.

Keywords: Cerebellar atrophy; MSTO1; Mitochondrial fusion; MtDNA depletion; Muscular dystrophy.

Fig. 1

Muscle and brain imaging. a Lower extremity muscle MRI of patients P6 [p.(Asp236His); p.(Phe217Leu)], P7 [p.(Leu450Phe); deletion], P8 [p.(Phe217Leu); p.(Asp236His)] and P10 [p.(Asp236Gly); p.(Arg279His)] at ages 37 years, 9 years, 6 years and 16 years, respectively. Abnormal signal intensity of muscles such as the posterior gastrocnemius muscle in patient P6 (white arrow), reflects muscle breakdown and replacement with adipose tissue. b Brain MRI completed in 12 patients consistently demonstrates moderate-to-severe cerebellar volume loss or hypoplasia involving the vermis and both hemispheres. Repeat MRI images were available in patients P4 [p.(Gly420ValfsX2); p.(Arg256Gln)], P8 [p.(Phe217Leu); p.(Asp236His)], P10 [p.(Asp236Gly); p.(Arg279His)] and P12 [p.(Arg345His); p.(Thr324Ile)] demonstrate mild [P12] to no progression [P4, P8 and P10] of cerebellar volume loss over time (second row)

Fig. 2

Muscle biopsy, MSTO1 pathogenic variants and pedigrees. a Histology findings from the vastus lateralis muscle biopsy of P7 [p.(Leu450Phe); deletion] at age 20 months include internalized nuclei on hematoxylin and eosin (H&E) staining (white arrow) (i) and variation in fiber size on nicotinamide dinucleotide (NADH) staining (ii) and whorled fibres evident on Gömöri trichrome (inset) (iii) and COX staining (white arrow) (iv). b Muscle biopsy electron microscopy (EM) findings are notable for aggregates of subsarcolemmal mitochondria in both P9 [p.(Tyr478Cys); missing)] (i and ii) and P10 [p.(Asp236Gly); p.(Arg279His)] (iii and iv) and non-specific mitochondrial morphologic abnormalities (variations in mitochondrial shape and size) in P10. c Schematic of new and reported human MSTO1 pathogenic variants. Shown in numbered light blue squares are cDNA exons (RefSeq isoform NM_018116.3 of MSTO1). Corresponding known protein domains are shown in orange (tubulin 3 domain) and beige (Misato segment II tubulin-like domain). Variants written in black text are recessive; the single mutation in red has been previously reported to cause dominantly inherited MSTO1-related disease. The top half of the figure depicts novel variants reported in this publication; the bottom half of the figure depicts variants which have been previously reported. Bolded variants depict previously reported mutations that were also present in our cohort. The dotted line depicts a large deletion (exons 9-14). d Pedigree of two families consistent with recessive inheritance of MSTO1 pathogenic variants

Fig. 3

Pathogenic variants lead to MSTO1 protein instability. a Western blot analysis of total cell lysates from control and patient fibroblast. As a control, total cell lysates from HeLa cells overexpressing MSTO1-V5 or empty vector were also included. Blots were probed with antibodies against endogenous MSTO1, VDAC1, HSP60 and V5. Black arrow corresponds to endogenous MSTO1 protein further verified in HeLa cell lysates; meanwhile, bands underneath are nonspecific. b Western blot analysis of cell lysates as in a. Blots were probed against fusion proteins (Mfn1, Mfn2 and Opa1) and loading controls

Fig. 4

Characteristics of MSTO1 patient fibroblasts. a Representative confocal microscopy images of control and patient cells. Mitochondrial networks in MSTO1 patient cells are more fragmented and contain fewer but larger mtDNA nucleoids compared to the control cells. Live cells were stained with MitoTracker Red (red, mitochondria) and PicoGreen (green, nuclear and mitochondrial DNA). b Quantification of mitochondrial morphology from control and patient cells performed from three independent replicates. Statistical analysis was performed on the number of cells with partly fragmented mitochondrial morphology in control versus patient cells; Student T test, *p < 0.05, **p < 0.001

Fig. 5

Enlarged lysosomal vacuoles in MSTO1 patient fibroblasts. a Representative confocal images of control and patient cells fixed and stained with antibodies against TOMM20 (red, mitochondria) and LAMP1 (green, lysosomes). Compared to an unaffected control, patient cells contain distinct lysosomal clusters. b Quantification of cells containing enlarged lysosomes in control and patient fibroblasts performed from two independent replicates. Statistical analysis was performed; Student T test, *p <0.05

Fig. 6

Pathogenic variants in MSTO1 are linked to mtDNA depletion. a Relative mtDNA copy number normalized to the nuclear-encoded 18S gene. Data represent at least three independent biological replicates. b Analysis of mtDNA nucleoid counts per cell from 35 cells for each group. c Quantification of nucleoid sizes in control and patient cells. Data represent average nucleoid sizes from the same cells as in b. Average mtDNA nucleoid size is presented in a violin plot. K–S test was performed to determine statistical significance. d Frequency of nucleoids larger than 0.2 µm² in all 35 cells quantified per fibroblast line. Student T test was performed as indicated for a, c and d. *p <0.05, **p <0.01, ***p <0.0001

Fig. 7

Expression of wild-type MSTO1 rescues cellular phenotypes in MSTO1 patient fibroblasts. Control, P4 and P7 fibroblast cells were transfected with MSTO1-P2A-mCherry or the mCherry empty vector control. Representative images of fibroblasts transfected with MSTO1-P2A-mCherry, for a live cells stained with picogreen and MitoTracker Deep Red, or b fixed cells stained with antibodies against TOMM20 (green, mitochondria) and LAMP1 (blue, lysosomes). Scalebars: 10 µm. Transfected fibroblasts, as identified by cytosolic mCherry signal, were characterized as described above for the following cellular phenotypes: c mitochondrial morphology, d lysosome morphology, e average mtDNA nucleoid size, f mtDNA nucleoid counts, and g relative mtDNA copy number. Student T test was performed as indicated for c, d, f, and g. K–S test was performed to determine statistical significance for e. *p < 0.05, **p < 0.01, ***p < 0.0001