Defective NADPH production in mitochondrial disease complex I causes inflammation and cell death

Eduardo Balsa,Mark Jedrychowski,John G Doench,Steven P Gygi,Elizabeth A Perry,Pere Puigserver,Christopher F Bennett

doi:10.1038/s41467-020-16423-1

Abstract

Electron transport chain (ETC) defects occurring from mitochondrial disease mutations compromise ATP synthesis and render cells vulnerable to nutrient and oxidative stress conditions. This bioenergetic failure is thought to underlie pathologies associated with mitochondrial diseases. However, the precise metabolic processes resulting from a defective mitochondrial ETC that compromise cell viability under stress conditions are not entirely understood. We design a whole genome gain-of-function CRISPR activation screen using human mitochondrial disease complex I (CI) mutant cells to identify genes whose increased function rescue glucose restriction-induced cell death. The top hit of the screen is the cytosolic Malic Enzyme (ME1), that is sufficient to enable survival and proliferation of CI mutant cells under nutrient stress conditions. Unexpectedly, this metabolic rescue is independent of increased ATP synthesis through glycolysis or oxidative phosphorylation, but dependent on ME1-produced NADPH and glutathione (GSH). Survival upon nutrient stress or pentose phosphate pathway (PPP) inhibition depends on compensatory NADPH production through the mitochondrial one-carbon metabolism that is severely compromised in CI mutant cells. Importantly, this defective CI-dependent decrease in mitochondrial NADPH production pathway or genetic ablation of SHMT2 causes strong increases in inflammatory cytokine signatures associated with redox dependent induction of ASK1 and activation of stress kinases p38 and JNK. These studies find that a major defect of CI deficiencies is decreased mitochondrial one-carbon NADPH production that is associated with increased inflammation and cell death.

Highlights

Electron transport chain (ETC) defects occurring from mitochondrial disease mutations compromise ATP synthesis and render cells vulnerable to nutrient and oxidative stress conditions
We report that limited glucose availability suppresses phosphate pathway (PPP) activity, which render cells completely dependent on malic enzyme 1 (ME1) and IDH1 for NADPH production
Our results suggest that mitochondrial-produced NADPH can be transferred to the cytosol via isocitrate dehydrogenase 2 (IDH2)/IDH1 shuttle, establishing a functional link between one-carbon metabolism and the IDH isoforms

Summary

Results

Gain-of-function CRISPR/Cas[9] screen identifies ME1. Mitochondrial mutations in genes encoding for OXPHOS components reduce cellular fitness, including survival, which is exacerbated under stress conditions[16,17]. To gain new insights into this metabolic problem, we designed a gain-of-function whole-genome CRISPR-based positive screen to identify genes whose increased expression protected human mitochondrial disease CI mutant ND1 (3796A>G) cybrid cells from galactose-induced cell death. Increasing supplementation of malate, did not result in cell survival rescue suggesting that protein levels or activity of the enzyme rather than substrate availability underlie these beneficial effects (Fig. 2f). These results suggest that increased ME1 expression in glucose-restricted CI mutant cells promoted glutamine flux through the mitochondrial/ cytoplasmic reductive pathway

BCL2A1

NDUFS4

Discussion

Methods