Mitochondrial aminoacyl-tRNA synthetases trigger unique compensatory mechanisms in neurons

Abstract

Mitochondrial aminoacyl-tRNA synthetase (mt-ARS) mutations cause severe, progressive, and often lethal diseases with highly heterogeneous and tissue-specific clinical manifestations. This study investigates the molecular mechanisms triggered by three different mt-ARS defects caused by biallelic mutations in AARS2, EARS2, and RARS2, using an in vitro model of human neuronal cells. We report distinct molecular mechanisms of mitochondrial dysfunction among the mt-ARS defects studied. Our findings highlight the ability of proliferating neuronal progenitor cells (iNPCs) to compensate for mitochondrial translation defects and maintain balanced levels of oxidative phosphorylation (OXPHOS) components, which becomes more challenging in mature neurons. Mutant iNPCs exhibit unique compensatory mechanisms, involving specific branches of the integrated stress response, which may be gene-specific or related to the severity of the mitochondrial translation defect. RNA sequencing revealed distinct transcriptomic profiles showing dysregulation of neuronal differentiation and protein translation. This study provides valuable insights into the tissue-specific compensatory mechanisms potentially underlying the phenotypes of patients with mt-ARS defects. Our novel in vitro model may more accurately represent the neurological presentation of patients and offer an improved platform for future investigations and therapeutic development.

Keywords: aminoacyl-tRNA synthetase; mitochondrial biology; neurological disease; protein synthesis.

Figure 1

Protein abundance of OXPHOS components in mt-ARS mutant iNPCs and iNPC-derived neurons. Western blot images of OXPHOS subunits and TOM20 in iNPCs (A), and iNPC-derived neurons (E). Quantification of the relative signal intensities normalized to controls and to TOM20 levels as a loading control in complex I (NDUFB8), complex II (SDHD), complex III (UQCRC2), complex IV (MT-CO2), complex V (ATP5A) of iNPCs (B-D) and neurons (F-H). Data shown as mean ± SEM from at least 3 independent experiments, ^*P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001, ^****P ≤ 0.0001, two-way ANOVA (Bonferroni).

Figure 2

Impact of mt-ARS mutation on mtDNA copy numbers. Relative mtDNA copy numbers in mt-ARS iNPCs vs control (A), and in neurons vs control (B). Data normalized to control mtDNA levels. (C) mtDNA copy numbers during differentiation. Data normalized to control iNPC level. The relative amounts of mtDNA were calculated as mean ΔCt values of the difference in cycle threshold (Ct) of the mitochondrial encoded gene ND1 minus the Ct of the nuclear gene B2M. Data shown as mean ± SEM of 3 independent experiments, ^*P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001, ^****P ≤ 0.0001, one-way ANOVA (Bonferroni).

Figure 3

Mitochondrial respiration rate is altered in mutant mt-ARS iNPCs. Mitochondrial stress test result measured by Seahorse XFe96 analyser (A) oxygen consumption rate (OCR) in iNPCs carrying mt-ARS defects. (B) Extracellular acidification rate (ECAR). (C–G) Parameters relating to mitochondrial respiration calculated from OCR data. (H) Ratio of OCR to ECAR. Data was normalized to total DNA content. Data shown as mean ± SEM, ^*P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001, ^****P ≤ 0.0001, one-way ANOVA (Bonferroni).

Figure 4

Pathway analysis of RNA sequencing data from MT-ARS iNPCs. Gene set enrichment analysis and volcano plot highlighting nuclear-encoded OXPHOS subunits, which increased in AARS2 (A and B), and RARS2 (E and F), but reduced in EARS2 (C and D) iNPCs.

Figure 5

Mt-ARS defect alter protein translation rate and activate ISR in iNPCs. (A) Western blot image of the relative amount of puromycin incorporated into newly synthesized polypeptides during 10-min incubation of iNPCs. Cycloheximide (CX) was used as indicated as a negative control to block translation. H3 was used as a loading control. (B–D) Western blot and quantification of phosphorylated and total GCN2 and eIF2a in iNPCs. Activation of GCN2 and eIF2a shown as ratios of phosphorylated to total levels normalized to H3 as a loading control. (E) Quantification of puromycin blot. Puromycin signal was measured for each lane and normalized to H3 signal. Data shown as mean ± SEM of 3 independent experiments, ^*P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001, ^****P ≤ 0.0001, one-way ANOVA (Bonferroni).

Figure 6

Summary of the proposed processes operating in iNPCs carrying mutations in mt-ARS. (A) Mt-ARS defects impair mitochondrial translation causing mitochondrial dysfunction which may include insufficient ATP production and activation of mitochondrial stress. (B) The integrated stress response may mediate the mitochondrial stress signal to the nucleus activating the expression of stress-induced genes to reach adaptive homeostasis and improve mitochondrial function while also inhibiting cytosolic protein translation. (C) Compensatory mechanisms aimed to improve mitochondrial function may include increasing mtDNA copy numbers or production of OXPHOS components. Affected cells may also shift their metabolism to glycolysis to meet ATP demand (as in AARS2 and EARS2 lines) causing a build-up of lactic acid.