Phd in GENETICS AND MOLECULAR BIOLOGY

Thesis title: Analysis of the pathogenic mechanisms of primary microcephaly using Drosophila as a model system

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by two main features: congenital microcephaly and non-progressive intellectual disability. More than 30 genes are implicated in the manifestation of MCPH, with approximately half involved in centrosome biogenesis, suggesting that disruption in the balance between symmetric and asymmetric cell division or alteration in cell division pattern during brain development play a crucial role in MCPH pathogenesis. However, altered spindle positioning during neurogenesis and imbalances between symmetric and asymmetric division may not be sufficient to induce significant changes in cell fate. This thesis aims to uncover novel pathogenic mechanisms of the centrosome genes Sas4 and asp, associated with MCPH, in relation to chromatin modification and integrity and to mitochondrial function. To address these aims, D. melanogaster was used as model animal. We found that loss of Sas4 and Asp results in a reduction and alteration of H3K9me2/3 and H3K27me3 heterochromatin marks during Drosophila larval brain development. Notably, we observed a significant reduction in both Lamin B and HP1a, associated with nuclear structural deformations, such as Lamin invaginations, dispersed HP1a and LINC complex (Klarsicht). Additionally, depletion of Sas4 and Asp led to endogenous DNA damage accumulation, increased sensitivity to DNA damage and delays in DNA damage repair, suggesting that in Sas4 and asp mutants the altered nuclear architecture impairs DNA damage repair process. Our results also showed a reduction in the mitochondrial proteins ATP5A and Cox IV in Sas4 depleted adult brains suggesting that Sas4, but not Asp, may have a role in ATP synthesis and oxidative phosphorylation processes. Furthermore, loss of Sas4 results in morphological changes in mitochondria in the calyx and cell body regions of the mushroom body. Finally, we found that spd-2 mutations result in reduced heterochromatin marks and increased DNA damage, indicating that spd-2 may be a novel MCPH candidate gene. In conclusion, this study demonstrates that Sas4 and Asp have a critical role in maintaining genomic stability, nuclear architecture, and mitochondrial function. Deficiencies in these proteins during development can contribute to severe microcephaly.