Mitochondrial dysfunctions in leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL)

Abstract

Several inherited human diseases have been linked to mitochondrial aminoacyl-tRNA synthetases (mtARSs). Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a leukodystrophy caused by mutations in the DARS2 gene which encodes mitochondrial aspartyl-tRNA synthetase. As mitochondrial ARSs are key components of the mitochondrial translation apparatus, we investigated the effects of DARS2 mutations on mitochondrial functions and mitochondrial morphology in an LBSL patient. In fibroblasts from the patient with LBSL, biosynthesis of respiratory chain complex proteins encoded by mitochondrial DNA was decreased, while those encoded by nuclear DNA were not. Cellular oxygen consumption rates and respiratory control ratio were decreased in the LBSL patient; in addition, fragmentation of mitochondria was increased, while their tubular elongation and interconnectivity were decreased. Taken together, these findings suggest that DARS2 mutations impair translations of mitochondrial DNA-encoded respiratory chain complex proteins, consequently causing dysfunction of cellular respiration and impediment of mitochondrial dynamics, which highlights the role of mtARSs in the maintenance of normal mitochondrial bioenergetics and dynamics.

Fig 1. Genetic study of the LBSL patient.

A DARS2 mutation c.228–16 C>A (upper panel, indicated by arrow) in intron 2 was identified, which causing a DARS2 mRNA transcript lacking exon 3 (lower panel); (B) TaqMan copy number assay (vertical bars indicating the ranges of values) for DARS2 exon 12 showed loss of a copy of exon 12.

Fig 2. DARS2 defect leads to reduced mitochondrial translation.

(A) Representative image of immunoblotting. Nuclear and mitochondria DNA-encoded proteins of mitochondrial respiratory complexes in fibroblasts derived from normal human, LBSL and MERRF patient were used for assay. (B) Quantitative histogram of protein expression. *p<0.05 between indicated groups.

Fig 3. DARS2 defect causes respiratory dysfunction of mitochondria.

(A, B and C) Representative results of polarographic data. Cells resuspended in oxymeter were sequentially loaded with indicated drugs to dissect respiratory capacity. G/M, glutamate and malate. A, ADP. O, oligomycin, F, FCCP. R, rotenone. (D) Basal, ADP-stimulated and FCCP-activated maximal respiration rate. (E) Respiratory control ratio calculated from ration of state 3 to state 4 respiration rate. State 3 and state 4 were defined as oxygen consumption under ADP and oligomycin, respectively. *p<0.05 between indicated groups.

Fig 4. Cells with DARS2 defect present fragmented morphology of mitochondria.

(A) Representative image of mitochondrial morphology. Scale bar, 10 μm. (B-E) Mitochondrial morphology was analyzed using Dagda’s method, as described in Materials and Methods. *p<0.05 between indicated groups.