mitochondrial DNA leakage promotes inflammation and intervertebral disc degeneration

Intervertebral disc (IVD) degeneration (IDD) is a major cause of low back pain, affecting 39% of American adults over the age of 18 and costing $87.6 billion in estimated healthcare expenditure. IDD is characterized by structural and functional deterioration, extracellular matrix degradation, oxidative stress, sterile inflammation, and impaired biomechanical function in the disc. Mitochondria are endosymbionts derived from bacteria and contain circular, double-stranded DNA with inflammatogenic unmethylated CpG motifs. Due to its prokaryotic nature, mitochondrial DNA (mtDNA) that escapes into the cytosol is recognized as “foreign” by the cellular DNA sensing system, cyclic GMP AMP synthase (cGAS) and stimulator of interferon genes (STING), thereby triggering an inflammatory response. However, the mechanism of mtDNA escape is not fully understood. The mitochondrial permeability transition pore (mPTP), a multiprotein complex, allows the passage of small molecules through the mitochondrial membrane. As mtDNA is large, its escape through the mPTP requires cleavage into smaller fragments by mitochondrial endonucleases. Notably, mtDNA is 10-20-fold more susceptible to ROS-induced DNA damage than nuclear DNA. Based on a rigorous review of published studies on mPTP, cGAS/STING pathway, and mtDNA in chronic inflammation in disc and cartilage, we hypothesize that “oxidative damage to mtDNA under pathological conditions leads to its fragmentation, allowing its escape through the mPTP into the cytosol, to induce sterile chronic inflammation and promote intervertebral disc degeneration.” In this study, we ask the following key questions: (1) Does oxidative damage to mtDNA result in its fragmentation and escape from mitochondria? (2) What is the mechanism of escape of mtDNA? (3) Does mtDNA escape induce inflammation in the IVD? (4) What is the mechanism of mtDNA sensing in IVDs? We will employ in vitro and in vivo approaches using isolated disc cells from human surgical samples and mouse discs, as well as organotypic cultures of functional spine units (FSU) from mice, and multiple mouse models, including mtDNA polymerase gamma (PolgA) proofreading mutant, cyclophilin D (CypD) knockout (KO), and Sting KO mice. We will perform disc needle puncture surgery (NPS) on the caudal discs of mice to induce IDD and investigate the role of mtDNA in sterile chronic inflammation and IDD. We will use both males and females in all our in vivo studies to investigate any sex bias in the disease pathology in these mouse lines. The overall goal of this study is to examine the impact of mtDNA leakage on inflammation and identify novel therapeutic targets for the management of IDD.