Huntingtin in Mitosis – HUNIM

Huntingtin (htt) is a large protein of 350 kDa whose function is not well defined. A major interest in this protein comes from the discovery that htt, when mutated, causes Huntington's disease (HD) a devastating neurodegenerative disorder that affects 1 in 10000 individuals of european origin. The neuropathology of HD is a marked neuronal death in the striatum. The mutation that causes the disease is an abnormal expansion of a polyglutamine (polyQ) stretch in the N-terminus of the protein. The mechanisms by which huntingtin kills striatal neurons are not fully understood but there is growing evidence that loss of the normal function(s) of wild-type htt could act concomitantly and/or synergistically with the gain of new toxic functions of polyQ-htt. However, very little is known about the normal function(s) of wild-type huntingtin. Given the predominant neurological signs developped by HD patients, most of the studies on huntingtin function have focused on post-mitotic neurons and no studies have investigated the possible role of huntingtin in dividing cells. Huntingtin expression is not restricted to differentiated neurons and is found at significant levels in neuronal progenitors and in non-neuronal epithelial dividing cells. Huntingtin binds to microtubules in neurons and non-neuronal cells and associates to the centrosomal region in dividing cells. In addition, wild-type huntingtin interacts with the dynein/dynactin complex and kinesin, to stimulate the MT-dependent transport of organelles. In the laboratory, we have characterized the localization of huntingtin in dividing cells and found huntingtin specifically located at the spindle poles from prophase to late anaphase. We have assessed the physiological consequence of such localization and have shown that huntingtin controls proper spindle orientation by targeting p150Glued subunit of dynactin to the cell cortex (manuscript under preparation). The focus of the current proposal is to further establish the importance of huntingtin during mitosis. To achieve this aim, we now propose to study huntingtin and mitosis in vivo. We will study huntingtin and spindle orientation of neural progenitor cells in the developping mammalian brain. We will perform in utero electroporation of mouse embryos with constructs of interest and analyze the spindle orientation in huntingtin-depleted cells. We will also use Drosophila as a model system and analyze spindle orientation in Drosophila larval neuroblasts. Finally, we propose to analyze spindle orientation in vivo in non-neuronal epithelia in Drosophila and mouse models. We will focus on spermatogenesis as huntingtin is expressed at high levels in testis and conditional inactivation of huntingtin at adulhood in the testis leads to sterility. In particular, analyzing male germline stem cells (GSCs) in Drosophila is relevant to our study as these cells undergo asymmetric divisions that have been well described. We also aim to identify signalling pathways that modulate huntingtin function in mitosis. We have already identified various phosphorylation sites on huntingtin that could be of particular importance in the regulation of huntingtin function at the spindle poles. We will further characterize the regulatory role of these post-translational modifications during mitosis. We will then identify the kinases and phosphatases that are modifying these sites and regulating huntingtin function in spindle orientation. In addition to the candidate approach on specific sites and kinases, we propose to identify new sites on huntingtin protein by systematic mutagenesis of putative phosphorylation sites on huntingtin and analysis of the effects on spindle orientation in vitro and in vivo in Drosophila.