Parkin Deficiency Impairs Mitochondrial DNA Dynamics and Propagates Inflammation

Abstract

A BS TRACT: Background: Mutations in the E3 ubiquitin ligase parkin cause autosomal recessive Parkinson's disease (PD). Together with PTEN-induced kinase 1 (PINK1), parkin regulates the clearance of dysfunctional mitochondria. New mitochondria are generated through an interplay of nuclear-and mitochondrial-encoded proteins, and recent studies suggest that parkin influences this process at both levels. In addition, parkin was shown to prevent mitochondrial membrane permeability, impeding mitochondrial DNA (mtDNA) escape and subsequent neuroinflammation. However, parkin's regulatory roles independent of mitophagy are not well described in patientderived neurons. Objectives: We sought to investigate parkin's role in preventing neuronal mtDNA dyshomeostasis, release, and glial activation at the endogenous level. Methods: We generated induced pluripotent stem cell (iPSC)-derived midbrain neurons from PD patients with parkin (PRKN) mutations and healthy controls. Live-cell imaging, proteomic, mtDNA integrity, and gene expression analyses were employed to investigate mitochondrial biogenesis and genome maintenance. To assess neuroinflammation, we performed single-nuclei RNA sequencing in postmortem tissue and quantified interleukin expression in mtDNA/lipopolysaccharides (LPS)-treated iPSC-derived neuron-microglia co-cultures. Results: Neurons from patients with PRKN mutations revealed deficits in the mitochondrial biogenesis pathway, resulting in mtDNA dyshomeostasis. Moreover, the energy sensor sirtuin 1, which controls mitochondrial biogenesis and clearance, was downregulated in parkindeficient cells. Linking mtDNA disintegration to neuroinflammation, in postmortem midbrain with PRKN mutations, we confirmed mtDNA dyshomeostasis and detected an upregulation of microglia overexpressing proinflammatory cytokines. Finally, parkin-deficient neuron-microglia co-cultures elicited an enhanced immune response when exposed to mtDNA/LPS. Conclusions: Our findings suggest that parkin coregulates mitophagy, mitochondrial biogenesis, and mtDNA maintenance pathways, thereby protecting midbrain neurons from neuroinflammation and degeneration.

Midbrain on Fire: mtDNA Ignites Neuroinflammation in PRKN‐P

“…For instance, exogenous expression of the water forming NADH dehydrogenase from Lactobacillus brevis (LbNOX) can restore the cellular NAD + /NADH balance and has been used to establish metabolic consequences of a low NAD + /NADH ratio in the setting of OXPHOS deficiency 19 . Expressing the NADH dehydrogenase in Parkin‐deficient cells would test whether the low NAD + /NADH ratio is responsible for the defects in mitochondrial biogenesis and cytosolic mtDNA leak observed by Wasner et al 8 or merely coincident. Likewise, expressing LbNOX in the Parkin KO; mutator mouse could establish whether the low NAD + /NADH ratio is responsible for DA neuronal loss in this model.…”

“…Lowered NAD + suppresses PGC1‐α signaling by decreasing the activity of SIRT1, an NAD + ‐dependent deacetylase 10 . Although Wasner et al 8 do not directly measure NAD + and NADH, a recent study found that the NAD + /NADH ratio is decreased in PRKN KO and patient iDA neurons, 11 and both studies found an increase in the lactate/pyruvate ratio, 8,11 which is metabolically coupled to the NAD + /NADH ratio. Based on these findings, the authors suggest that Parkin deficiency may decrease mitochondrial biogenesis through lowered NAD + , via the SIRT1–PGC1‐α/NRF1–TFAM signaling axis.…”

“…The source of the low NAD + /NADH ratio in PRKN ‐deficient cells is not directly addressed experimentally by Wasner et al, 8 but is plausibly a moderate decrease in OXPHOS resulting from Parkin deficiency. Notably, Wasner et al 8 observed decreased CI activity in PRKN KO SH‐SY5Y cells, consistent with an earlier study that found decreased CI activity in the Prkn KO mouse brain 6 . CI is primarily responsible for oxidizing NADH to NAD + in mitochondria and so maintains the high NAD + /NADH ratio needed to drive metabolic reactions.…”

“…Wasner et al 8 additionally explored a possible connection between mtDNA dyshomeostasis (with lowered TFAM levels and mtDNA expression) and neuroinflammation in PRKN ‐deficient cells. Each mitochondrion contains several copies of mtDNA, which are packaged into bacteria‐like nucleoids by the mtDNA transcriptional factor TFAM.…”

“…Although questions remain, Wasner et al 8 have nonetheless taken strides toward connecting NADH reductive stress, mtDNA dyshomeostasis, and neuroinflammation in PRKN ‐PD cells and tissue and are helping bridge the gap between model systems and patient samples toward a more complete understanding PRKN ‐PD pathogenesis.…”