Indrieri, Alessia (2010) A medaka model to study the the molecular basis of Microphthalmia with Linear Skin defects (MLS) syndrome. [Tesi di dottorato] (Inedito)

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Abstract

The Microphthalmia with linear skin defects (MLS) syndrome is an X- linked dominant male-lethal neuro-developmental disorder associated to mutations in the holocytochrome c-type synthetase (HCCS) transcript. Female patients display unilateral or bilateral microphthalmia and linear skin defects, additional features include central nervous system (CNS) malformation and mental retardation. HCCS codifies a mitochondrial protein that catalyzes the attachment of heme to both apocytochrome c and c1, necessary for proper functioning of the mitochondrial respiratory chain. Although mutation analysis clearly indicates a role for HCCS in the pathogenesis of this genetic condition, the molecular mechanisms underlying the developmental anomalies in the presence of HCCS dysfunction are still unknown. Previous studies demonstrated the early lethality of mouse embryonic Hccs knock-out stem cells. To overcome the problem of the possible embryonic lethality, we decided to generate an animal model for MLS syndrome in the medaka fish (Oryzia latipes) using a morpholino-based technology. Fish models (zebrafish and medaka) are considered good models to study developmental biology processes and in particular eye developmental defects. Three specific morpholinos directed against different portions of the olhccs transcript have been designed and injected and our data indicated that all morpholinos effectively downregulate the expression of the olhccs gene. The injection of the three different morpholinos resulted in a pathological phenotype, which resembles the human condition. Morphants displayed microphthalmia, coloboma, and microcephaly associated to a severe cardiac pathology. To date, this is the only animal model that recapitulates the phenotype observed in MLS syndrome. Analysis with markers for specific retinal cell types showed defects in differentiation of the ventral neural retina. Characterization of morphants revealed that hccs down-regulation results in impairment of mitochondrial functions, overproduction of reactive oxygen species (ROS) and a strong increase of apoptosis mediated by activation of the mitochondrial-dependent cell death pathway in the CNS and in the eyes. Our results clearly indicate that HCCS plays a critical role in mitochondria and imply that MLS should be considered a mitochondrial disease. It is well established that the intrinsic mitochondrial dependent apoptotic pathway rely on the formation of apoptosomes, which require the presence and/or the activity of cytochrome c, Apaf1, and caspase 9. Detailed studies of the mechanisms that underlie intrinsic apoptosis have shown that the heme group of cytochrome c is necessary for Apaf1 activation, apoptosome formation and activation of caspase 9. Interestingly, our data indicate that, in our model, the mitochondrial dependent apoptosis is triggered by caspase 9 activation and occur in a Bcl-dependent but apoptosome-independent manner suggesting that at least in some tissues the apoptosis can occur in a non-canonical way. Our data support the evidence of an apoptosome-indipendent activation of caspase 9 and suggest the possibility that this event might be tissue specific. Our study shed new light into the functional role of HCCS in the mitochondria. In addition, we provide strong evidences that mitochondrial mediated apoptotic events underlie microphtalmia providing new insights into the mechanisms of this developmental defect.

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