THE MBOAT7 AND TM6SF2 GENETIC REPLENISHMENT RESCUES THE MITOCHONDRIAL DYNAMICS IN IN VITRO NAFLD MODELS AND OPENS A SPYHOLE TOWARDS MITOCHONDRIAL BIOMARKERS FOR NON-INVASIVE NAFLD DIAGNOSIS

Background and aims: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disorder worldwide and it could evolve into nonalcoholic steatohepatitis (NASH), which is associated with activation of fibrosis, possibly leading to cirrhosis and hepatocellular carcinoma (HCC). Nowadays, no therapeutic strategies have been approved for NAFLD management, and hepatic biopsy remains the gold standard procedure for its diagnosis. NAFLD is a multifactorial disease therefore several studies underlined the chance to develop a NAFLD risk prediction model based on genetics, biochemical indicators, and metabolic disorders. One of the key events in NAFLD progression is the loss of mitochondrial flexibility that causes the tampering of mitochondrial number, morphology, and activity. Concurrently, the overload of damaged mitochondria prompts the release of circulating mitochondrial DNA fragments (ccf-mtDNA) as damage-associated molecular patterns (DAMPs) signals amplifying the hepatic injury. Increased mt-ccf levels have been associated with advanced NAFLD stages awarding them a promising prognostic power. NAFLD exhibits even a strong hereditable component and the I148M Patatin-like Phospholipase Domain-containing 3 (PNPLA3), the rs641738 in the Membrane bound O-acyltransferase domain containing 7-transmembrane channel-like 4 (MBOAT7-TMC4) locus, and the E167K Transmembrane 6 Superfamily Member 2 (TM6SF2) polymorphisms represent the main predisposing factors to disease onset and progression. Recently, we have demonstrated in 1380 NAFLD patients that the co-presence of PNPLA3, MBOAT7 and TM6SF2 polymorphisms hugely associates with the entire NAFLD spectrum and increases the risk to develop HCC. To translate the human evidence in an in vitro model, we performed the knock-out (KO) of MBOAT7 (MBOAT7-/-) and/or TM6SF2 (TM6SF2-/-, MBOAT7-/-TM6SF2-/-) through CRISPR-Cas9 technology in HepG2 carrying the I148M PNPLA3 variant. We demonstrated that the deletion of MBOAT7, TM6SF2 and mainly both hampers the mitochondrial dynamics resulting in an enrichment of misshapen and failed mitochondria, underlining that genetics seems to achieve an emerging role in the mitochondrial maladaptation. Therefore, in this study we aim to: 1) deepen the mechanisms through which genetics impacts on mitochondrial maladaptation by restoring MBOAT7 and/or TM6SF2 wild-type (WT) genes in KO in vitro models. 2) assess mt-ccf serum levels in 824 NAFLD patients stratified according to PNPLA3, MBOAT7 and TM6SF2 polymorphisms in order to estimate the mt-ccf diagnostic accuracy. Methods: MBOAT7 and/or TM6SF2 were overexpressed through pLenti-C-mGFP-P2A-Puro lentiviral vectors in KO cells. Mitochondrial morphology and number were assessed by confocal microscopy, transmission electron microscopy and D-loop TaqMan Copy Number Assay. Mitochondrial lifecycle and activity were assessed through RT-PCR, Western Blot, Seahorse assay, immunocytochemistry, and transmission electron microscopy. Mt-ccf was quantify in the sera of 824 NAFLD patients through Quantitative RT- PCR. Results: Lipid content was reduced in overexpressed cells compared to KO models, thus improving the steatotic phenotype induced by MBOAT7 and/or TM6SF2 loss-of-functions. The WT upregulated models reduced the levels of PGC1α, master regulator of mitobiogenesis, that was activated in KO cells in response to fusion-fission unbalance. Accordingly, the restore of MBOAT7 and/or TM6SF2 WT proteins prompted the expression of Mfn1, Mfn2 (mitochondrial outer membranes proteins involved in fusion) and OPA1 (inner mitochondrial membranes fusion protein), while decreased Drp1 and FIS1 (fission proteins) levels, re-establishing the mitochondrial lifecycle that is unbalanced in KO cells. Additionally, the mitophagy pathways (PINK/PARKIN/BNIP3/BNIP3-L/LC3/phospho-UBIQUITIN) alongside the presence of intracellular autophagic structures increased after MBOAT7 and/or TM6SF2 WT overexpression, encouraging the disruption of damaged mitochondria. Indeed, the recovery of MBOAT7 and/or TM6SF2 WT genes in KO models decreased the number of damaged-globular mitochondria, while increased the normo-shaped ones, resulting in reduced D-loop levels and emphasizing that the high number of mitochondria could not mirror an enhanced function. The balance of mitochondrial biogenesis is essential for organelles’ homeostasis and activity. Indeed, the overexpressed models augmented the COX-I/SDHA ratio, COX-III, and citrate synthase activity alongside the oxygen consumption rate derived from mitochondria, thus recovering the OXPHOS capacity and Krebs cycle which were impaired in KO cells. Consequently, ROS production, ROS-driven DNA damage, lipid peroxidation and release of mt-ccf as DAMPs signals decreased in WT overexpressed cells, underling the improvement of hepatocellular damage and empowering the role of mt-ccf as mitochondrial failure biomarker in NAFLD. Finally, lactate levels decreased after the upregulation of MBOAT7 and/or TM6SF2 WT genes together with the glycolytic extracellular acidification rate and growth capacity, thereby inhibiting the switch towards anaerobic glycolysis and invasiveness, both hallmarks of tumorigenesis in KO cells. To translate our in vitro findings into clinic, we evaluated the amount of mt-ccf in terms of ccf-COXIII in the sera of 824 NAFLD patients stratified according to PNPLA3, MBOAT7 and TM6SF2 variations. The co-presence of all 3 at-risk variants strongly increases the ccf-COXIII levels independently of disease severity and metabolic confounders, thus supporting our in vitro results and underling the role of mt-ccf as potential non-invasive NAFLD biomarker in genetically predisposed individuals. Conclusions: The rescue of MBOAT7 and/or TM6SF2 wild-type proteins in KO HepG2 cells re-balanced the mitochondrial lifecycle and turnover, thus ensuring the organelles’ function and possibly reversing hepatocellular damage. Therefore, genetics directly impacts on mitochondrial maladaptation during NAFLD. Consistently, the PNPLA3, MBOAT7 and TM6SF2 synergism in NAFLD patients exacerbates the levels of mt-ccf, thus giving them a promising diagnostic value.