Pathogenic mechanisms in mitochondrial optic neuropathies

Leber’s hereditary optic neuropathy (LHON) and Autosomal Dominant Optic Atrophy (ADOA) are the two most common inherited optic neuropathies and both are the result of mitochondrial dysfunctions. Despite the primary mutations causing these disorders are different, being an mtDNA mutation in subunits of complex I in LHON and defects in the nuclear gene encoding the mitochondrial protein OPA1 in ADOA, both pathologies share some peculiar features, such a variable penetrance and tissue-specificity of the pathological processes. Probably, one of the most interesting and unclear aspect of LHON is the variable penetrance. This phenomenon is common in LHON families, most of them being homoplasmic mutant. Inter-family variability of penetrance may be caused by nuclear or mitochondrial ‘secondary’ genetic determinants or other predisposing triggering factors. We identified a compensatory mechanism in LHON patients, able to distinguish affected individuals from unaffected mutation carriers. In fact, carrier individuals resulted more efficient than affected subjects in increasing the mitochondrial biogenesis to compensate for the energetic defect. Thus, the activation of the mitochondrial biogenesis may be a crucial factor in modulating penetrance, determining the fate of subjects harbouring LHON mutations. Furthermore, mtDNA content can be used as a molecular biomarker which, for the first time, clearly differentiates LHON affected from LHON carrier individuals, providing a valid mechanism that may be exploited for development of therapeutic strategies. Although the mitochondrial biogenesis gained a relevant role in LHON pathogenesis, we failed to identify a genetic modifying factor for the variable penetrance in a set of candidate genes involved in the regulation of this process. A more systematic high-throughput approach will be necessary to select the genetic variants responsible for the different efficiency in activating mitochondrial biogenesis. A genetic modifying factor was instead identified in the MnSOD gene. The SNP Ala16Val in this gene seems to modulate LHON penetrance, since the Ala allele in this position significantly predisposes to be affected. Thus, we propose that high MnSOD activity in mitochondria of LHON subjects may produce an overload of H2O2 for the antioxidant machinery, leading to release from mitochondria of this radical and promoting a severe cell damage and death ADOA is due to mutation in the OPA1 gene in the large majority of cases. The causative nuclear defects in the remaining families with DOA have not been identified yet, but a small number of families have been mapped to other chromosomal loci (OPA3, OPA4, OPA5, OPA7, OPA8). Recently, a form of DOA and premature cataract (ADOAC) has been associated to pathogenic mutations of the OPA3 gene, encoding a mitochondrial protein. In the last year OPA3 has been investigated by two different groups, but a clear function for this protein and the pathogenic mechanism leading to ADOAC are still unclear. Our study on OPA3 provides new information about the pattern of expression of the two isoforms OPA3V1 and OPA3V2, and, moreover, suggests that OPA3 may have a different function in mitochondria from OPA1, the major site for ADOA mutations. In fact, based on our results, we propose that OPA3 is not involved in the mitochondrial fusion process, but, on the contrary, it may regulate mitochondrial fission. Furthermore, at difference from OPA1, we excluded a role for OPA3 in mtDNA maintenance and we failed to identify a direct interaction between OPA3 and OPA1. Considering the results from overexpression and silencing of OPA3, we can conclude that the overexpression has more drastic consequences on the cells than silencing, suggesting that OPA3 may cause optic atrophy via a gain-of-function mechanism. These data provide a new starting point for future investigations aimed at identifying the exact function of OPA3 and the pathogenic mechanism causing ADOAC.

A molecular phylogeny of bivalve mollusks: ancient radiations and divergences as revealed by mitochondrial genes

The main scope of my PhD is the reconstruction of the large-scale bivalve phylogeny on the basis of four mitochondrial genes, with samples taken from all major groups of the class. To my knowledge, it is the first attempt of such a breadth in Bivalvia. I decided to focus on both ribosomal and protein coding DNA sequences (two ribosomal encoding genes -12s and 16s -, and two protein coding ones - cytochrome c oxidase I and cytochrome b), since either bibliography and my preliminary results confirmed the importance of combined gene signals in improving evolutionary pathways of the group. Moreover, I wanted to propose a methodological pipeline that proved to be useful to obtain robust results in bivalves phylogeny. Actually, best-performing taxon sampling and alignment strategies were tested, and several data partitioning and molecular evolution models were analyzed, thus demonstrating the importance of molding and implementing non-trivial evolutionary models. In the line of a more rigorous approach to data analysis, I also proposed a new method to assess taxon sampling, by developing Clarke and Warwick statistics: taxon sampling is a major concern in phylogenetic studies, and incomplete, biased, or improper taxon assemblies can lead to misleading results in reconstructing evolutionary trees. Theoretical methods are already available to optimize taxon choice in phylogenetic analyses, but most involve some knowledge about genetic relationships of the group of interest, or even a well-established phylogeny itself; these data are not always available in general phylogenetic applications. The method I proposed measures the "phylogenetic representativeness" of a given sample or set of samples and it is based entirely on the pre-existing available taxonomy of the ingroup, which is commonly known to investigators. Moreover, it also accounts for instability and discordance in taxonomies. A Python-based script suite, called PhyRe, has been developed to implement all analyses.

Characterization of mitochondrial genomes in bivalve species with doubly uniparental inheritance of mitochondria

Many bivalve species possess two distinct mtDNA lineages, called F and M, respectively inherited maternally and paternally: this system is called doubly uniparental inheritance (DUI). The main experimental project of my PhD was the quantification of the two mtDNAs during the development of the DUI species Ruditapes philippinarum, from early embryos to sub-adults, using Real-Time qPCR. I identified the time interval in which M mtDNA is lost from female individuals, while it is retained in males (which are heteroplasmic through all of their life cycle). The results also suggested absence of mtDNA replication during early embryogenesis, a process constituting a bottleneck that highly reduces the copy number of mtDNA molecules in cells of developing larvae. In males this bottleneck may produce cells homoplasmic for M mtDNA, and could be considered as a first step of the segregation of M in the male germ line. Another finding was the characterization, in young clams approaching the first reproductive season, of a significant boost in copy number of F mtDNA in females and of M in males. Given the age of animals in which this mtDNA-specific growth was observed, the finding could probably be the outcome of the first round of gonads and gametes production. Other lines of research included the characterization of the unassigned regions in mt genomes of DUI bivalves. These regions can harbor signals involved in the control of replication and/or transcription of the mtDNA molecule, as well as additional open reading frames (ORFs) not related to oxidative phosphorylation. These features in DUI species could be associated to the maintenance of separate inheritance routes for the two mtDNAs. Additional ORFs are also found in other animal mt genomes: I summarized the presence of gene duplications as a co-author in a review focusing on animal mt genomes with unusual gene content.

The environmental DNA in the risk assessment and decision making processes for the invasive species management in agri-food sector, hydraulic security and biodiversity conservation

The use of environmental DNA (eDNA) analysis as a monitoring tool is becoming more and more widespread. The eDNA metabarcoding methods allow rapid community assessments of different target taxa. This work is focused on the validation of the environmental DNA metabarcoding protocol for biodiversity assessment of freshwater habitats. Scolo Dosolo was chosen as study area and three sampling points were defined for traditional and eDNA analyses. The gutter is a 205 m long anthropic canal located in Sala Bolognese (Bologna, Italy). Fish community and freshwater invertebrate metazoans were the target groups for the analysis. After a preliminary study in summer 2019, 2020 was devoted to the sampling campaign with winter (January), spring (May), summer (July) and autumn (October) surveys. Alongside with the water samplings for the eDNA study, also traditional fish surveys using the electrofishing technique were performed to assess fish community composition; census on invertebrates was performed using an entomological net and a surber sampler. After in silico analysis, the MiFish primer set amplifying a fragment of the 12s rRNA gene was selected for bony fishes. For invertebrates the FWHF2 + FWHR2N primer combination, that amplifies a region of the mitochondrial coi gene, was chosen. Raw reads were analyzed through a bioinformatic pipeline based on OBITools metabarcoding programs package and QIIME2. The OBITools pipeline retrieved seven fish taxa and 54 invertebrate taxa belonging to six different phyla, while QIIME2 recovered eight fish taxa and 45 invertebrate taxa belonging to the same six phyla as the OBITools pipeline. The metabarcoding results were then compared with the traditional surveys data and bibliographic records. Overall, the validated protocol provides a reliable picture of the biodiversity of the study area and an efficient support to the traditional methods.

Neuro-ophthalmological and electrophysiological characterization of mitochondrial diseases.

Aims and methods: 1) characterization of patients with Dominant Optic Atrophy (DOA) associated with mutations in AFG3L2 and ACO2 genes in comparison with classical OPA1-DOA; 2) characterization of patients with mtDNA mutations causing MELAS and MERRF syndromes and correlation with heteroplasmy; 3) longitudinal evaluation of subacute m.11778G>A/MTND4 Leber’s Hereditary Optic Neuropathy (LHON) patients co-treated with rAAV2/2-ND4 gene therapy and idebenone. We performed a comprehensive neuro-ophthalmological assessment coupled with electrophysiological examination. Results: 1) We described and compared 23 ACO2 and 13 AFG3L2 patients with 72 OPA1 patients. All patients presented temporally predominant optic atrophy, with ACO2 showing higher RNFL and GCL thicknesses at OCT, while AFG3L2 was virtually-indistinguishable from OPA1. 2) Retinopathy was the most common manifestation in 17/33 MELAS patients, conversely, optic atrophy was the most common finding in 7/8 MERRF patients. Correlation of heteroplasmy with neuro-ophthalmological parameters failed to disclose any significance in MELAS, while it negatively correlated with OCT parameters in MERRF. 3) We compared modifications in visual acuity, OCT and electrophysiological parameters at 3 timepoints in 9 LHON patients. We observed significant decrease of RNFL thickness and reduction of PhNR amplitude. Visual acuity improved of about -0.37 LogMAR, correlating significantly with time from onset and from injection, but not with idebenone therapy duration. Discussion: 1) ACO2 seems associated to better preservation of retinal ganglion cells, depending on a different pathogenic mechanism involving mtDNA maintenance, as opposed to AFG3L2 which is involved in OPA1 processing. 2) MELAS and MERRF patients presented with a clearly distinct ocular phenotype, possibly reflecting a selective susceptibility of different retinal cell types to global energy defect or oxidative stress. 3) Follow up of LHON patients treated with gene therapy confirmed the deterioration in OCT and electrophysiological parameters, while the amount of visual improvement was similar to the one observed in recent clinical trials.