Activation of Neurotoxic Astrocytes Due to Mitochondrial Dysfunction Triggered by POLG Mutation.

Mitochondrial diseases are associated with neuronal death and mtDNA depletion. Astrocytes respond to injury or stimuli and damage to the central nervous system. Neurodegeneration can cause astrocytes to activate and acquire toxic functions that induce neuronal death. However, astrocyte activation and its impact on neuronal homeostasis in mitochondrial disease remain to be explored. Using patient cells carrying POLG mutations, we generated iPSCs and then differentiated these into astrocytes. POLG astrocytes exhibited mitochondrial dysfunction including loss of mitochondrial membrane potential, energy failure, loss of complex I and IV, 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 manifested a toxic effect leading to the death of neurons. We 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. These findings reveal a novel role for dysfunctional astrocytes that contribute to the pathogenesis of POLG diseases.

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

Mitophagy specifically recognizes and removes damaged or superfluous mitochondria to maintain mitochondrial homeostasis and proper neuronal function. Defective mitophagy and the resulting accumulation of damaged mitochondria occur in several neurodegenerative diseases. Previously, we showed mitochondrial dysfunction in astrocytes with POLG mutations, and here, we examined how POLG mutations affect mitophagy in astrocytes and how this can be ameliorated pharmacologically. Using induced pluripotent stem cell (iPSC)-derived astrocytes carrying POLG mutations, we found downregulation of mitophagy/autophagy-related genes using RNA sequencing-based KEGG metabolic pathway analysis. We confirmed a deficit in mitochondrial autophagosome formation under exogenous stress conditions and downregulation of the mitophagy receptor p62, reduced lipidation of LC3B-II, and decreased expression of lysosome protein lysosomal-associated membrane protein 2A (LAMP2A). These changes were regulated by the PINK1/Parkin pathway and AKT/mTOR/AMPK/ULK1 signaling pathways. Importantly, we found that double treatment with nicotinamide riboside (NR) and metformin rescued mitophagy defects and mitochondrial dysfunction in POLG-mutant astrocytes. Our findings reveal that impaired mitophagy is involved in the observed mitochondrial dysfunction caused by POLG mutations in astrocytes, potentially contributing to the phenotype in POLG-related diseases. This study also demonstrates the therapeutic potential of NR and metformin in these incurable mitochondrial diseases.

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

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.