Repeated mild traumatic brain injuries perturb the mitochondrial biogenesis via DNA methylation in the hippocampus of rat

Highlights

• Repeated mild traumatic brain injuries (rMTBI) decreased TFAM levels.

• rMTBI-induced nuclear DNA hypermethylation declined TFAM levels.

• Mitochondrial DNA (mtDNA) hypomethylation after rMTBI attenuated TFAM binding.

• Low TFAM occupancy on hypomethylated mtDNA altered mitochondrial biogenesis.

• Methionine treatment restored rMTBI-induced mitochondrial biogenesis via mtDNA methylation.

Abstract

Mitochondrial biogenesis in the brain is impaired in various neurological disorders including traumatic brain injury (TBI). The long-lasting effects of TBI may be, in part, attributed to epigenetic mechanisms such as DNA methylation. However, the role of DNA methylation on regulatory elements of nuclear and mitochondrial genome in mitochondrial biogenesis is not known. We examined the epigenetic regulation of mitochondrial transcription factor A (TFAM), and further probed its implications in mitochondrial dysfunction in the hippocampus of rats subjected to repeated mild TBI (rMTBI) using weight drop injury paradigm. rMTBI-induced hypermethylation at TFAM promoter resulted in deficits in its protein levels in mitochondria after immediate (48 h) and protracted (30 d) time points. Further, rMTBI also caused hypomethylation of mitochondrial DNA (mtDNA) promoters (HSP1 and HSP2), which further culminated into low binding of TFAM. rMTBI-induced changes weakened mitochondrial biogenesis in terms of reduced mtDNA-encoded rRNA, mRNA, and protein levels leading to shortages of ATP. To verify the potential role of mtDNA methylation in rMTBI-induced persistent mitochondrial dysfunction, rMTBI-induced rats were treated with methionine, a methyl donor. Methionine treatment restored the methylation levels on HSP1 and HSP2 resulting in efficient binding of TFAM and normalized the rRNA, mRNA, and protein levels. These findings suggest the crucial role of DNA methylation at nuclear and mitochondrial promoter regions in mitochondrial gene expression and ATP activity in the hippocampus after rMTBI.

Introduction

The brain is an energy-demanding organ, and disruptions in energy homeostasis produce emotional and cognitive disabilities (Kapogiannis and Mattson, 2011). Abnormalities in the neuronal energy metabolism after traumatic brain injury (TBI) are important instigators of neuropsychological outcomes (Briones et al., 2014, Casey et al., 2008, Sheng et al., 2012, Vagnozzi et al., 2010). Patients suffering from mild TBI (MTBI) often display long-lasting neuropathological changes owing to secondary injuries, despite minimal neuronal damage (Cole and Bailie, 2016, Grandhi et al., 2017). The perpetual neurological outcomes post TBI were attributed to oxidative stress-induced mitochondrial dysfunction in different parts of the brain (Abdul-Muneer et al., 2015, Fehily and Fitzgerald, 2017, Fischer et al., 2016, Hiebert et al., 2015, Mendes Arent et al., 2014). For example, previous studies report persistent alterations in mitochondrial DNA (mtDNA) copy number (mtDNAcn) in the mesocorticolimbic neurocircuitry as a result of repeated MTBI (rMTBI) induced by weight drop head injury (Balasubramanian et al., 2019). Moreover, reductions in superoxide dismutase 2 (SOD2) levels build mitochondrial oxidative stress after rMTBI resulting in substantial neurodegeneration in the hippocampus (Balasubramanian et al., 2021). A similar paradigm of rMTBI also induces anxiety-like behaviors and deficits in learning and memory (Sagarkar et al., 2019, Sagarkar et al., 2017), which are recognized as the most prevalent symptoms of post-traumatic stress disorders (PTSD). These coincident observations help draw the parallelism between mitochondrial dysfunction in the hippocampus and associated phenotypic outcomes.

The mitochondrial bioenergetics function is intricately dependent on local biogenesis processes (Leary et al., 1998) that is orchestrated by proteins encoded by nuclear as well as mitochondrial genomes. One of the most prominent examples is the nuclear-encoded mitochondrial transcription factor A (TFAM), which drives transcription and replication of the mitochondrial genome (Dominy and Puigserver, 2013, Jornayvaz and Shulman, 2010, Ventura-Clapier et al., 2008). TFAM possesses histone-like properties by binding to mtDNA, packaging them into nucleoids, and maintaining their copy number (Alam et al., 2003, Ekstrand et al., 2004, Kaufman et al., 2007). Accumulating evidence reveals a positive correlation between mtDNAcn and TFAM protein levels in neuronal cells (Garrido et al., 2003, Kang et al., 2007, Larsson et al., 1998). TFAM was found to be decreased in patients as well as animal models of Parkinson’s disease (PD) (Grünewald et al., 2016), Alzheimer’s disease (AD) (Pedrós et al., 2014, Petrov et al., 2016), and aging (Thomas et al., 2012). The null mice of TFAM produce embryonic lethality due to aberrations in the neurogenesis plausibly caused by energy depletion and severe respiratory defects (Larsson et al., 1998). It is evident that the reduced ATP generation due to mitochondrial dysfunction significantly contributes to secondary injuries after TBI (Cheng et al., 2012). ATP production is dependent on the activities of electron transport chain (ETC) proteins encoded by both nuclear and mitochondrial genomes (Zhao et al., 2019). Therefore, the regulation of mitochondrial biogenesis by TFAM is vital for mitochondrial function.

Apart from the role of mitochondrial genome compaction, TFAM specifically binds to the promoters of mtDNA identified as heavy strand promoters 1 and 2 (HSP1 and HSP2), and light strand promoter (LSP). The binding of transcription factors to the promoter of a gene is affected by epigenetic alterations such as DNA methylation (Zhu et al., 2016). DNA methylation is associated with gene repression in several neurodegenerative diseases (Lu et al., 2013, Wen et al., 2016) and is also causally linked to MTBI-induced neuropsychological outcomes (Balasubramanian et al., 2021, Haghighi et al., 2015, Nagalakshmi et al., 2018, Sagarkar et al., 2017, Wong and Langley, 2016). A few clinical and preclinical studies reported the significance of mtDNA methylation in the progression of neurodegenerative diseases such as AD, PD, and amyotrophic lateral sclerosis (ALS) (Coppedè and Stoccoro, 2019). While nuclear DNA methylation interferes with the binding of transcription factors at the promoter regions, methylation at mtDNA promoters facilitates the binding of TFAM (Dostal and Churchill, 2019). Although mtDNA methylation is related to neurodegenerative diseases, how it impacts the mitochondrial gene expression via TFAM binding is not known.

The present study probes the possibility that the rMTBI-induced alterations in nuclear as well as mitochondrial DNA methylation may deregulate mitochondrial biogenesis. Herein, we have employed a weight drop (WD) injury model to induce rMTBI in rats and measured the molecular changes at immediate (48 h) and protracted (30 d) time points as reported earlier (Balasubramanian et al., 2021, Balasubramanian et al., 2020, Balasubramanian et al., 2019, Mychasiuk et al., 2014, Sagarkar et al., 2019, 2017). We have examined the DNA methylation levels at TFAM promoter and mtDNA promoters (HSP1 and HSP2) and evaluated the TFAM levels and their binding to mtDNA. The effects of TFAM binding to HSP1 or HSP2 on mtDNA-encoded RNA and protein expression were investigated. The ATP levels were measured to examine if rMTBI results in energy deficits in the hippocampus. To elucidate the causal role of mtDNA methylation in trauma-induced impairment in mitochondrial biogenesis, we examined the effects of methionine, a methyl donor, on the mtDNA methylation levels, TFAM occupancy at the HSPs, and the mitochondrial protein expression in the hippocampus.