Disease‐specific phenotypes in iPSC ‐derived neural stem cells with POLG mutations

Abstract

Mutations in POLG disrupt mtDNA replication and cause devastating diseases often with neurological phenotypes. Defining disease mechanisms has been hampered by limited access to human tissues, particularly neurons. Using patient cells carrying POLG mutations, we generated iPSCs and then neural stem cells. These neural precursors manifested a phenotype that faithfully replicated the molecular and biochemical changes found in patient post-mortem brain tissue. We confirmed the same loss of mtDNA and complex I in dopaminergic neurons generated from the same stem cells. POLG-driven mitochondrial dysfunction led to neuronal ROS overproduction and increased cellular senescence. Loss of complex I was associated with disturbed NAD + metabolism with increased UCP2 expression and reduced phosphorylated SirT1. In cells with compound heterozygous POLG mutations, we also found activated mitophagy via the BNIP3 pathway. Our studies are the first that show it is possible to recapitulate the neuronal molecular and biochemical defects associated with POLG mutation in a human stem cell model. Further, our data provide insight into how mitochondrial dysfunction and mtDNA alterations influence cellular fate determining processes.

Stem cell derived astrocytes withPOLGmutations and mitochondrial dysfunction including abnormal NAD+ metabolism is toxic for neurons

“…In micro-dissected post-mortem neurons, we showed previously that respiratory chain complex I was lost in POLG affected frontal and cerebellar neurons [2] and we subsequently confirmed this in iPSCderived neural stem cells (NSCs) and iPSC-derived dopaminergic (DA) neurons [19]. We, therefore, investigated the levels of each respiratory chain complex in our astrocytes using flow cytometry, immunohistochemistry, and western blotting.…”

“…4h & i), although WS5A astrocytes did not reach significance. These data indicate that the respiratory chain defect in astrocytes carrying POLG mutations appears more extensive than that seen in post-mortem neurons [23] and neuronal stem cells derived from the same iPSCs [19], where only loss of complex I was identified. A decreased expression of voltagedependent anion channels (VDAC), located in the mitochondrial outer membrane, was also identified in WS5A and CP2A astrocytes compared to controls, although it failed to reach significance ( Fig.…”

“…We generated patient iPSCs (Table S1) as previously described [19] using fibroblasts of two patients, one homozygous for c.2243G>C; p.W748S (WS5A) and one compound heterozygous c.1399G>A/c.2243G>C; p.A467T/W748S (CP2A). Control iPSCs were generated from two normal human fibroblast lines, Detroit 551 and AG05836B.…”

“…To generate patient and control astrocytes ( Fig. 1a), we first generated neural stem cells (NSCs) using a previously published protocol [19]. Within 5 days of induction, iPSC cells (Fig.…”

“…Previous studies, including our own in post-mortem tissue [2] and NSCs [19], have focused on the neuronal consequences of POLG mutations. Here, we show, for the first time, that mitochondrial defects including decreased membrane potential, respiratory chain complex loss, lowered ATP levels and mtDNA depletion also occur in iPSC-derived astrocytes.…”