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

Abstract

The inability to reliably replicate mitochondrial DNA (mtDNA) by mitochondrial DNA polymerase gamma (POLG) leads to a subset of common mitochondrial diseases associated with neuronal death and depletion of neuronal mtDNA. Defining disease mechanisms remains difficult due to the limited access to human tissue. Astrocytes are highly abundant in the brain, playing a crucial role in the support and modulation of neuronal function. Astrocytes also respond to insults affecting the brain. Following damage to the center neural system, which can be hypoxia, inflammation or neurodegeneration, astrocytes become activated, lose their supportive role and gain toxic functions that induce rapid death of neurons and oligodendrocytes. The role of astrocyte reactivation and the consequences this has for neuronal homeostasis in mitochondrial diseases has not been explored. Here, using patient cells carrying POLG mutations, we generated iPSCs and then differentiated into astrocytes. We demonstrated that POLG-astrocytes exhibited both mitochondrial dysfunctions, including loss of mitochondrial membrane potential, energy failure, complex I and IV defects, disturbed NAD+/NADH metabolism, and mtDNA depletion. Further, POLG derived astrocytes presented an A1-like reactive phenotype with increased proliferation, invasion, upregulation of pathways involved in response to stimulus, immune system process, cell proliferation and cell killing. Under direct and indirect co-culture with neurons, POLG-astrocytes exhibited a toxic effect leading to the death of neurons. Our findings demonstrate that mitochondrial dysfunction caused by POLG mutations leads not only to intrinsic defects in energy metabolism affecting both neurons and astrocytes, but also to neurotoxic damage driven by astrocytes. Our studies provide a robust astroglia-neuron interaction model for future investigation of mitochondrial involvement in neurogenesis and neurodegenerative diseases.

Nicotinamide Riboside and Metformin Ameliorate Mitophagy Defect in Induced Pluripotent Stem Cell-Derived Astrocytes With POLG Mutations

“…A previous study in mice links mitochondrial dysfunction in astrocytes with POLG-related disease (Ignatenko et al, 2018). In an earlier study, we showed that POLG-mutant astrocytes exhibited mitochondrial dysfunction including loss of mitochondrial membrane potential, loss of ATP production, loss of complex I, disturbed NAD + /NADH metabolism, and mtDNA depletion (Liang et al, 2020a). However, whether and how POLG mutations affect mitophagy in these cells remains unclear.…”

“…Detroit 551 fibroblasts (ATCC R CCL 110 TM ) and AG05836B fibroblasts (RRID: CVCL_2B58) were used as control lines. The generation and maintenance of iPSCs and their derived NSCs were described previously (Liang et al, 2020a(Liang et al, ,b, 2021. Astrocytes were differentiated from NSCs according to the protocol described previously (Liang et al, 2020a).…”

“…The generation and maintenance of iPSCs and their derived NSCs were described previously (Liang et al, 2020a(Liang et al, ,b, 2021. Astrocytes were differentiated from NSCs according to the protocol described previously (Liang et al, 2020a). In addition, commercial human normal astrocytes (HNA) (Lonza) was applied as a control.…”

“…To generate astrocytes, we generated iPSCs showing positive expression of SOX2, OCT4, and NANOG (Supplementary Figure 1) and then differentiated these into NSCs as described previously (Liang et al, 2020a(Liang et al, ,b, 2021. Two patient lines were used, one homozygous for c.2243G > C; p.W748S (WS5A) and the other a compound heterozygous for c.1399G > A/c.2243G > C; p.A467T/W748S (CP2A).…”

“…In addition, one commercial human primary astrocyte line, HNA, was used as a positive astrocyte lineage control. We differentiated iPSC-derived NSCs to astrocytes as previously described (Liang et al, 2020a(Liang et al, ,b, 2021. The astrocytes from all patients and controls (Supplementary Figure 4) showed typical stellate morphology (Figure 1A).…”