Molecular bases, pathogenic mechanisms and possible therapeutic approach in Leber's Hereditary Optic Neuropathy

Leber’s hereditary optic neuropathy (LHON) is a mitochondrial disease characterized by a rapid loss of central vision and optic atrophy, due to the selective degeneration of retinal ganglion cells. The age of onset is around 20, and the degenerative process is fast and usually the second eye becomes affected in weeks or months. Even if this pathology is well known and has been well characterized, there are still open questions on its pathophysiology, such as the male prevalence, the incomplete penetrance and the tissue selectivity. This maternally inherited disease is caused by mutations in mitochondrial encoded genes of NADH ubiquinone oxidoreductase (complex I) of the respiratory chain. The 90% of LHON cases are caused by one of the three common mitochondrial DNA mutations (11778/ND4, 14484/ND6 and 3460/ND1) and the remaining 10% is caused by rare pathogenic mutations, reported in literature in one or few families. Moreover, there is also a small subset of patients reported with new putative pathogenic nucleotide changes, which awaits to be confirmed. We here clarify some molecular aspects of LHON, mainly the incomplete penetrance and the role of rare mtDNA mutations or variants on LHON expression, and attempt a possible therapeutic approach using the cybrids cell model. We generated novel structural models for mitochondrial encoded complex I subunits and a conservation analysis and pathogenicity prediction have been carried out for LHON reported mutations. This in-silico approach allowed us to locate LHON pathogenic mutations in defined and conserved protein domains and can be a useful tool in the analysis of novel mtDNA variants with unclear pathogenic/functional role. Four rare LHON pathogenic mutations have been identified, confirming that the ND1 and ND6 genes are mutational hot spots for LHON. All mutations were previously described at least once and we validated their pathogenic role, suggesting the need for their screening in LHON diagnostic protocols. Two novel mtDNA variants with a possible pathogenic role have been also identified in two independent branches of a large pedigree. Functional studies are necessary to define their contribution to LHON in this family. It also been demonstrated that the combination of mtDNA rare polymorphic variants is relevant in determining the maternal recurrence of myoclonus in unrelated LHON pedigrees. Thus, we suggest that particular mtDNA backgrounds and /or the presence of specific rare mutations may increase the pathogenic potential of the primary LHON mutations, thereby giving rise to the extraocular clinical features characteristic of the LHON “plus” phenotype. We identified the first molecular parameter that clearly discriminates LHON affected individuals from asymptomatic carriers, the mtDNA copy number. This provides a valuable mechanism for future investigations on variable penetrance in LHON. However, the increased mtDNA content in LHON individuals was not correlated to the functional polymorphism G1444A of PGC-1 alpha, the master regulator of mitochondrial biogenesis, but may be due to gene expression of genes involved in this signaling pathway, such as PGC-1 alpha/beta and Tfam. Future studies will be necessary to identify the biochemical effects of rare pathogenic mutations and to validate the novel candidate mutations here described, in terms of cellular bioenergetic characterization of these variants. Moreover, we were not able to induce mitochondrial biogenesis in cybrids cell lines using bezafibrate. However, other cell line models are available, such as fibroblasts harboring LHON mutations, or other approaches can be used to trigger the mitochondrial biogenesis.

Investigating the mechanisms of Moraxella catarrhalis resistance to oxidative stress

Moraxella catarrhalis (Mcat) represents a human pathogen implicated in debilitating diseases, such as Chronic Obstructive Pulmonary Disease (COPD). One of the hallmarks of COPD is the excessive neutrophil oxidative stress mediated by reactive oxygen species (ROS). Mcat shows a higher innate level of resistance to exogenous oxidative stress compared to the co-infecting human airways pathogens such as non-typeable Haemophilus influenzae (NTHi) but the underlying mechanisms are currently not well defined. In this thesis, we demonstrated that, differently from NTHi, Mcat was able to directly interfere with ROS production and ROS-related responses such as neutrophil extracellular traps (NET) and autophagy in differentiated neutrophilic-like dHL-60 cells and primary cells. The underlying mechanisms were shown to be phagocytosis/opsonins-independent but contact-dependent, due to the engagement of the immunosuppressive receptors. Indeed, we identified that through OmpCD porin, Mcat was able to engage Siglec inhibitory receptors suppressing ROS generation by the host cells. Furthermore, Mcat provided a safer niche for the co-infecting NTHi bacterium which was otherwise susceptible to the host antimicrobial arsenal. Subsequently, to deeply characterize the Mcat global transcriptional response to oxidative stress, an RNA-Seq experiment was performed on exponentially growing bacteria exposed to sublethal amounts of H2O2 or CuSO4, stimuli that the pathogens experienced once they are phagocytosed. We unraveled a previously unidentified common transcriptional program following H2O2 and CuSO4 exposure, demonstrating a similar defense mechanism to the stress conditions encountered in neutrophils. We ascertained new crucial factors for this pathogen response and established a novel in vivo Mcat infection model, using the invertebrate Galleria mellonella. Actually, we observed that deletion mutants of genes implicated in oxidative stress resistance exhibited reduced virulence. In conclusion, this work represents an important step in the understanding of Mcat innate resistance mechanisms to oxidative stress and further elucidate the virulence mechanisms during infection.

Characterization of the molecular mechanisms mediated by the insulin-like growth factor-2 mRNA-binding proteins and Ephrin receptor A2 (EPHA2) signaling in the malignancy of CIC-DUX4 sarcomas

Ewing sarcoma (EWS) and CIC-DUX4 sarcoma (CDS) are pediatric fusion gene-driven tumors of mesenchymal origin characterized by an extremely stable genome and limited clinical solutions. Post-transcriptional regulatory mechanisms are crucial for understanding the development of this class of tumors. RNA binding proteins (RBPs) play a crucial role in the aggressiveness of these tumors. Numerous RBP families are dysregulated in cancer, including IGF2BPs. Among these, IGF2BP3 is a negative prognostic factor in EWS because it promotes cell growth, chemoresistence, and induces the metastatic process. Based on preliminary RNA sequencing data from clinical samples of EWS vs CDS patients, three major axes that are more expressed in CDS have been identified, two of which are dissected in this PhD work. The first involves the transcription factor HMGA2, IGF2BP2-3, and IGF2; the other involves the ephrin receptor system, particularly EphA2. EphA2 is involved in numerous cellular functions during embryonic stages, and its increased expression in adult tissues is often associated with pathological conditions. In tumors, its role is controversial because it can be associated with both pro- and anti-tumoral mechanisms. In EWS, it has been shown to play a role in promoting cell migration and neoangiogenesis. Our study has confirmed that the HMGA2/IGF2BPs/IGF2 axis contributes to CDS malignancy, and Akt hyperactivation has a strong impact on migration. Using loss/gain of function models for EphA2, we confirmed that it is a substrate of Akt, and Akt hyperactivation in CDS triggers ligand-independent activation of EphA2 through phosphorylation of S897. Moreover, the combination of Trabectedin and NVP/BEZ235 partially inhibits Akt/mTOR activation, resulting in reduced tumor growth in vivo. Inhibition of EphA2 through ALWII 41_27 significantly reduces migration in vitro. The project aim is the identification of target molecules in CDS that can distinguish it from EWS and thus develop new targeted therapeutic strategies.