Characterization of Mitochondrial Alterations in Aicardi–Goutières Patients Mutated in RNASEH2A and RNASEH2B Genes

Abstract

Aicardi–Goutières syndrome (AGS) is a rare encephalopathy characterized by neurological and immunological features. Mitochondrial dysfunctions may lead to mitochondrial DNA (mtDNA) release and consequent immune system activation. We investigated the role of mitochondria and mtDNA in AGS pathogenesis by studying patients mutated in RNASEH2B and RNASEH2A genes. Lymphoblastoid cell lines (LCLs) from RNASEH2A- and RNASEH2B-mutated patients and healthy control were used. Transmission Electron Microscopy (TEM) and flow cytometry were used to assess morphological alterations, reactive oxygen species (ROS) production and mitochondrial membrane potential variations. Seahorse Analyzer was used to investigate metabolic alterations, and mtDNA oxidation and VDAC1 oligomerization were assessed by immunofluorescence. Western blot and RT-qPCR were used to quantify mtTFA protein and mtDNA release. Morphological alterations of mitochondria were observed in both mutated LCLs, and loss of physiological membrane potential was mainly identified in RNASEH2A LCLs. ROS production and 8-oxoGuanine levels were increased in RNASEH2B LCLs. Additionally, the VDAC1 signal was increased, suggesting a mitochondrial pore formation possibly determining mtDNA release. Indeed, higher cytoplasmic mtDNA levels were found in RNASEH2B LCLs. Metabolic alterations confirmed mitochondrial damage in both LCLs. Data highlighted mitochondrial alterations in AGS patients’ LCLs suggesting a pivotal role in AGS pathogenesis.

Comparison between D-loop methylation and mtDNA copy number in patients with Aicardi-Goutières Syndrome

“…These differences were associated prevalently with the RNASEH2B mutations, the most commonly altered gene in the pathology ( 4 , 5 ). These results are in accordance with our previously published data which underlined an increase in the mtDNA copy number particularly in RNASEH2B -mutated LCLs ( 30 ), but further investigations are needed to better understand how the mtDNA methylation influences the mtDNA copy number increase. Nothing was previously established or described in the literature in relation to this condition, so we explored this feature as well as the pattern of methylation in mtDNA for the first time.…”

“…According to our hypothesis, the amount of mtDNA may be higher in AGS patients and may be released from the abnormal mitochondria, activating immune systems, and producing the elevated levels of IFN-a that are typical of the illness. Interferon stimulated genes (ISGs) are more significantly expressed in the LCLs of AGS patients, as described in another study of ours (30) which also demonstrated the overexpression of the TLR9 gene. Moreover, although we are aware that the age difference between healthy controls and AGS patients, may be a limitation in the correlation, we also observed that the mtDNA copy number, but not the D-loop methylation levels, increased with increasing age at sampling in AGS patients but there was no correlation with sex.…”

“…To date, it is known that changes in methylation distribution can affect the same area in different diseases or cell types ( 39 , 40 ). Additionally, in our previous research ( 30 ), we demonstrated that the rates of oxidative phosphorylation and ROS production are elevated in AGS patients, which suggests that they may be a contributing factor in the oxidative stress condition we demonstrated to be present, particularly in RNASEH2B -mutated patients. More research is required to determine how these factors, along with an increase in mtDNA copy number and the activation of inflammatory pathways, may contribute to some of the symptoms experienced by AGS patients.…”

“…Since we demonstrated the presence of mitochondrial alterations in RNASEH2B and RNASEH2A -mutated Lymphoblastoid Cell Lines (LCLs) derived from AGS patients, highlighting alterations in the mtDNA content ( 30 ), the aim of this study was to assess the possible presence of methylation changes in the D-loop regulatory region of mitochondria and its relation with the mtDNA copy number in peripheral blood cells of AGS patients carrying mutations in different AGS genes and healthy controls as a possible pathomechanism in AGS.…”

Enhanced inflammatory signaling driven by metabolic switch in Aicardi-Goutières syndrome

“…Of possible relevance, we note that AGS has been suggested to involve a disturbance of mitochondria activity in some patients 46, 47 . Notably, a recent report revealed alteration of mitochondrial integrity associated with uncontrolled oxidative phosphorylation, increased ROS activity and mitochondrial stress in AGS patients mutated in RNASEH2A or RNASEH2B 48 . We propose here, that HIF-1εξ loss of activity could be a driver of mitochondrial dysfunction in AGS patients.…”