Mitochondrial diseases are a large family of extremely heterogeneous disorders genetically determined by mutations in either the nuclear genome or the mitochondrial DNA. Most of the mitochondrial disease genes are expressed in all cell types. However, in many conditions, some cell types are more affected than others. However, the reasons for this tissue-specificity remain poorly understood. To investigate the functional basis of the striking tissue-specificity in mitochondrial diseases, we analyzed several bioenergetic parameters, including oxygen consumption rates, Q redox poise, and reactive oxygen species production in mouse brain and liver mitochondria fueled by different substrates. In addition, we determined how these functional parameters are affected by electron transport chain impairment in a tissue-specific manner using pathologically relevant mouse models lacking either Ndufs4 or Ttc19, leading to complex I or III defects, respectively. No cure is currently available for most of the mitochondrial diseases. We previously showed that the coordinated activation of autophagy, lysosomal biogenesis, and mitochondrial biogenesis by rapamycin, ameliorated the myopathic phenotype of a muscle-specific knockout mouse for Cox15 (Cox15sm), encoding an enzyme involved in heme A biosynthesis. However, the role of mitophagy has been poorly investigated. We found that urolithin A, a direct mitophagy inducer, improved motor performance and myopathy in the Cox15sm mice, without increasing the activity of the respiratory chain complexes in a 10 week-treatment. These results indicate that activation of mitophagy can be a suitable treatment to ameliorate mitochondrial myopathies.

Our recent observations show that both resistant and stem-like cancer cells predominantly responsible for metastasis differ from chemotherapy-sensitive cells. We have shown bioinformatically and experimentally that mitochondria of such cells are much more prone to oxidative phosphorylation (OXPHOS) than radio- or chemotherapy-sensitive cancer cells from which they evolved during therapeutic interventions. Specifically, in triple-negative breast cancer models, we observed that such resistant cells exhibit higher mitochondrial membrane potential, higher OXPHOS and respiration, and increased resistance to oxidative stress, allowing them to survive chemo-radiotherapy.  These findings of increased expression of OXPHOS-associated genes and proteins in chemoresistant cells and biopsies of relapsed tumors suggest an alternative druggable target. Our in vitro and in vivo (nude mice and Artemia salina) data suggest that certain antibiotics, inducers of mitochondrial dysfunction, create additive oxidative stress and can reduce the growth rate of tumors developed from resistant or stem-like cancer cells. Such repurposed drugs, selected from a chemical library, are also able to resensitize resistant tumors, allowing reuse of chemotherapeutic agents. In addition, their modification with a specific moiety (TPP) allows for increased delivery to mitochondria to reduce cytotoxic pressure on normal cells. Thus, research from our laboratory offers an alternative strategy for anticancer therapy of resistant tumors.

Rett syndrome (RTT), a devastating neurodevelopmental disorder, is caused in 95% of the cases by mutations in the X-chromosome-localized MECP2 gene. RTT manifests as a range of multisystem disturbances including altered lipid profile, subclinical inflammation, and overall OxInflammatory status in which mitochondrial dysfunction acts as central player. To decipher the molecular mechanisms underlying the pathophysiological manifestations affecting patients, we investigated whether mitochondria may play a role in the aberrant immune and oxidative responses of RTT. Recent findings from our and other labs unraveled several abnormalities in RTT mitochondria including atypical mitochondrial structure, deregulated expression of genes encoding oxidative phosphorylation factors and mitochondrial organization factors, impaired mitochondrial quality control, depressed energetic profile, and augmented mt-ROS production. In other brain diseases, mitochondrial dysfunction is a vital event during the activation of NLPR3 inflammasome, a multi-protein complex involved in innate immune response, that represents a common denominator in the crosstalk between inflammation and oxidative stress. Interestingly, using primary fibroblasts and lympho-monocytes isolated from RTT patients, we found a constitutive hyperactivation of NLRP3:ASC inflammasome associated with increased levels of nuclear p65 and ASC proteins, and pro-IL-1β mRNA, without the ability to further respond to the LPS + ATP stimuli. Furthermore, increased circulating levels of ASC, interleukin (IL)-18, and 1β were found in RTT individuals, thus corroborating the aforementioned cellular findings. In order to evaluate NLRP3 involvement in the transition from pre-symptomatic to symptomatic phase of RTT, we detected higher serum levels of IL-1β and IL-18 in symptomatic Het mice compared to WT. Of note, increased gene expression of Il-1b, Nlrp3, and ASC was observed in Het brains at the pre-symptomatic stage, suggesting a likely role of NLRP3 impairment in the early stages of the disease. Preliminary data showed that treatment with resveratrol, known to improve mitochondrial function, ameliorated the RTT mouse phenotype by restoring levels of some NLRP3-related components. Furthermore, mitochondrial dysfunction can result in ferroptosis, a form of cell death characterized by iron-dependent lipid peroxidation and accumulation of reactive oxygen species.  After treatment with two ferroptosis inducers, erastin (GPX4 inhibitor) or RSL3 (inhibitor of the cystine/glutamate antiporter), we found changes in GPx and GR activity, alteration in GPX4 protein levels and increased formation of 4HNE protein adducts. Mitochondrial ROS production and lipid peroxidation levels were higher in RTT after ferroptosis induction, while co-treatment with ferrostatin-1, a well-known inhibitor of ferroptosis, significantly prevented these processes. Interestingly, co-treatment with mito-TEMPO, a mitochondria-targeted superoxide dismutase mimetic, mitigated mitochondrial oxidative burden and prevented ferroptosis cell death in RTT cells. Overall, our results demonstrate the decisive role of mitochondrial dysfunction in RTT OxInflammation. Thus, we can speculate that exposure of RTT cells to any condition affecting the already compromised mitochondrial function could not only hyperactivate the inflammatory status but also precipitate ferroptosis cell death. Targeting mitochondria in RTT could represent a strategic coadjuvant therapy to improve the quality of life of the affected patients.