Investigation Of Valproic Acid-induced Hepatotoxicity In Alpers Syndrome Using An IPSC Disease Model

Valproic acid (VPA) is widely used to treat epilepsy, migraine, chronic headache, bipolar disorder, and as adjuvant chemotherapy, but potentially causes idiosyncratic liver injury. Alpers syndrome (AHS), a neurometabolic disorder caused by mutations in the mitochondrial DNA polymerase gamma (POLG), is associated with an increased risk of developing fatal VPA hepatotoxicity. However, the mechanistic link of this clinical mystery remains unknown. Here, fibroblasts from two AHS patients were reprogrammed to induced pluripotent stem cells, and then differentiated to hepatocyte-like cells (AHS iPSCs-Hep). Both AHS iPSCs-Hep are more sensitive to VPA-induced mitochondrial dependent apoptosis than controls, showing more activated caspase-9and cytochrome c release. Strikingly, levels of both soluble and oligomeric OPA1, which together keep cristae junctions tight, are reduced in AHS iPSCs-Hep. Furthermore, POLG mutation cells show reduced POLG expression, mitochondrial DNA (mtDNA) amount, mitochondrial ATP production as well as abnormal mitochondrial ultrastructure following differentiation to hepatocyte-like cells. Superoxide flashes, spontaneous bursts of superoxide generation, caused by opening of the mitochondrial permeability transition pore (mPTP), occur more frequently in AHS iPSCs-Hep. Moreover, the mPTP inhibitor, cyclosporine A (CsA), rescues VPA-induced apoptotic sensitivity in AHS iPSCs-Hep. This result suggests that targeting mPTP opening could be an effective method to prevent hepatotoxicity by VPA in AHS patients. In addition, carnitine or N-acetylcysteine (NAC), which has been used in the treatment of VPA-induced hepatotoxicity, is able to rescue VPA-induced apoptotic sensitivity in AHS iPSCs-Hep. As an conclusion, AHS iPSCs-Hep are more sensitive to the VPA-induced mitochondrial dependent apoptotic pathway, and this effect is mediated by mPTP opening. This is the first toxicity model in genetic diseases using iPSCs, which also will enable the evaluation of drugs for the therapeutic target.