Neuronal death triggered by Ena/Vasp downregulation

FRANCO DL, REZÁVAL C, HERNÁNDEZ MV, SCHINDER AF AND CERIANI MF

San Pablo

Congreso; Molecular Medicine: A.C. Camargo Global Meeting of Translational Science. 2nd Sao Pablo School of Translational Science. Molecular Medicine: From bench to the bedside.; 2011

We have developed a novel forward genetic screen based on an automated behavioural assay in young and aged Drosophila individuals to identify genes associated with neurodegenerative processes. One of the genes identified through this screen is enabled, a protein involved in actin cytoskeleton remodelling (Rezával et al. 2008). To increase our understanding on the mechanisms leading to neuronal degeneration in this work, we investigate the effect of deregulation of ena ortholog genes in mouse hippocampal neurons. There are three related ENA/VASP proteins in vertebrates: Mena, EVL and VASP. In order to inhibit the function of all family members primary neuronal cultures were transfected with short interfering RNA pools (siRNAs) directed to each individual gene. Transfection of 3-day-old cultures with siRNAs effectively induced a downregulation of the ENA/VASP family, both at the mRNA and protein levels. Strikingly, ENA/VASP downregulation triggered axonal retraction. Downregulation of each protein per se did not result in a morphological phenotype, strengthening the notion of overlapping functions among family members. Such neuritic retraction leads to neuronal cell death through an apoptotic pathway. Remarkably, this retraction initially affected the axonal structure, showing no effect on dendrites. Reduction in ENA/VASP levels blocked the neuritogenic effect of a specific RhoA kinase (ROCK) inhibitor, suggesting that these proteins could participate in the Rho-signaling pathway. Altogether these observations suggest that ENA/VASP proteins are implicated in the establishment and maintenance of neuronal polarity, and that subtle alterations on their expression levels could trigger degeneration (Franco et al. 2010). Since our ultimate goal is to confirm these results in a mouse model of late onset progressive neurodegeneration, we characterized potential dominant negative (DN) versions to identify the most effective one. Preliminary experiments involving over expression in mouse hippocampal neurons of specific domains that potentially act as DNs of ENA/VASP function led to neurite retraction, recreating what was observed with RNAi pools. Interestingly, one of them displayed the most potent inhibition, identifying it as the candidate to generate the inducible transgenic model.