Characterization of the C584R variant in the mtDNA depletion syndrome gene FBXL4, reveals a novel role for FBXL4 as a regulator of mitochondrial fusion

https://doi.org/10.1016/j.bbadis.2019.165536Get rights and content
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Highlights

  • FBXL4 protein promotes mitochondrial fusion.

  • The C584R FBXL4 variant cannot promote mitochondrial fusion.

  • mtDNA depletion in patients is most likely caused by impaired mitochondrial fusion.

  • DCA improved lactic acidosis and cardiac hypertrophy in an FBXL4 patient.

Abstract

Mutations in FBXL4 (F-Box and Leucine rich repeat protein 4), a nuclear-encoded mitochondrial protein with an unknown function, cause mitochondrial DNA depletion syndrome. We report two siblings, from consanguineous parents, harbouring a previously uncharacterized homozygous variant in FBXL4 (c.1750 T > C; p.Cys584Arg). Both patients presented with encephalomyopathy, lactic acidosis and cardiac hypertrophy, which are reported features of FBXL4 impairment. Remarkably, dichloroacetate (DCA) administration to the younger sibling improved metabolic acidosis and reversed cardiac hypertrophy. Characterization of FBXL4 patient fibroblasts revealed severe bioenergetic defects, mtDNA depletion, fragmentation of mitochondrial networks, and abnormalities in mtDNA nucleoids. These phenotypes, observed with other pathogenic FBXL4 variants, confirm the pathogenicity of the p.Cys584Arg variant. Although treating FBXL4 fibroblasts with DCA improved extracellular acidification, in line with reduced lactate levels in patients, DCA treatment did not improve any of the other mitochondrial functions. Nonetheless, we highlight DCA as a potentially effective drug for the management of elevated lactate and cardiomyopathy in patients with pathogenic FBXL4 variants. Finally, as the exact mechanism through which FBXL4 mutations lead to mtDNA depletion was unknown, we tested the hypothesis that FBXL4 promotes mitochondrial fusion. Using a photo-activatable GFP fusion assay, we found reduced mitochondrial fusion rates in cells harbouring a pathogenic FBXL4 variant. Meanwhile, overexpression of wildtype FBXL4, but not the p.Cys584Arg variant, promoted mitochondrial hyperfusion. Thus, we have uncovered a novel function for FBXL4 in promoting mitochondrial fusion, providing important mechanistic insights into the pathogenic mechanism underlying FBXL4 dysfunction.

Abbreviations

ATP5A
ATP Synthase Subunit Alpha
COX II
Cytochrome c oxidase subunit 2
DCA
Dichloroacetate
DGUOK
Deoxyguanosine Kinase
DMEM
Dulbecco's Modified Eagle Medium
dNTP
Deoxyribonucleotide triphosphate
DRP1
Dynamin Related Protein 1
ECAR
Extracellular Acidification Rate
FBXL4
F-Box and Leucine rich repeat 4
FCCP
Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone
GFP
Green Fluorescent Protein
HA
Hemagglutinin
HEK
Human Embryonic Kidney
HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
HSP60
Heat Shock Protein 60
IMS
Intermembrane Space
IVSd
Interventricular Septum Thickness
MELAS
Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes
MEM
Minimum Essential Media
MFN1/2
Mitofusin1/2
mtDNA
Mitochondrial deoxyribonucleic acid
MTO1
Mitochondrial Translation Optimization 1
NDUFB8
NADH:Ubiquinone Oxidoreductase Subunit B8
OCR
Oxygen Consumption Rate
OMIM
Online Mendelian Inheritance in Man
OPA1
Optic Atrophy 1
OXPHOS
Oxidative Phosphorylation
PA-GFP
Photoactivatable Green Fluorescent Protein
PBS
Phosphate Buffered Saline
PDHC
Pyruvate Dehydrogenase Complex
POLG
Polymerase Gamma
QPCR
Quantitative Polymerase Chain Reaction
RRM2B
Ribonucleotide Reductase Regulatory TP53 Inducible Subunit M2B
SDHB
Succinate Dehydrogenase Complex Iron Sulfur Subunit B
SUCLA2
Succinate-CoA Ligase ADP-Forming Beta Subunit
SUCLG1
Succinate-CoA Ligase Alpha Subunit
TK2
Thymidine Kinase 2
TMRE
Tetramethylrhodamine, ethyl ester
TWNK
Twinkle
TYMP
Thymidine Phosphorylase
UQCRC2
Ubiquinol-Cytochrome C Reductase Core Protein 2

Keywords

Mitochondria
Mitochondrial DNA depletion
Mitochondrial fusion
FBXL4
Dichloroacetate

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