Title: Novel mechanisms of gene expression in developing mammalian neurons
Funder: Medical Research Council

Abstract: One of the most complex and challenging problems in biology is to understand how the billions of neurons in the vertebrate nervous system wire up to form the neural circuits that underlie all behaviour. During the early stages of embryonic development, neuronal precursors exit the cell cycle and differentiate into an enormous variety of neurons, each programmed to extend dendrites and an axon that can respond to guidance cues and form synapses with appropriate partner cells. Each of these events requires expression of large numbers of specific genes, which encode proteins that promote differentiation, cell survival, guided cell and growth cone migration, synapse formation, and, later, synaptic plasticity. However, the mechanisms underlying the regulation of gene expression during these critical events remain largely unknown. We have recently discovered a novel epigenetic modification of the chromatin surrounding the promoters of neurotrophin-responsive genes in cortical neurons that influences the transcription of these genes (Riccio et al., Mol Cell, 21,283-94, 2006). Using in vitro and in vivo assays we found that BDNF induced the synthesis of nitric oxide, which regulated histone acetylation by directly nitrosylating nuclear proteins associated with gene promoters. This was the first evidence that an extracellular stimulus can induce epigenetic modifications by means of S-nitrosylation of transcriptional complexes. Furthermore, we now know that histone deacetylase 2 (HDAC2) is one of the targets of BDNF-dependent S-nitrosylation. Structure to function analysis has revealed that two specific cysteines in HDAC2 are S-nitrosylated upon BDNF treatment, resulting in increased chromatin acetylation and gene transcription. These exciting findings offer a wealth of potential directions and I will focus my future research on the following goals: - generate a knock-in transgenic mouse bearing the Cys/Cys HDAC2 mutation - identify genes regulated by NO during neuronal development in vivo - characterize neurotrophin-dependent NO signalling Two additional research projects in my laboratory are centred on epigenetic modifications in developing neurons in vitro and following behavioural stimulation in vivo. Local protein synthesis has emerged as an important mechanism by which gene expression can be regulated in highly polarized cells, such as neurons. In recent years, for example, there has been a great effort to characterize mRNAs localized in dendrites but there has not been a corresponding effort to characterize mRNAs in axons and growth cones. There is a major technical problem in isolating axonal mRNAs, which is that the axons and their terminals must be physically separated from the cell bodies and the surrounding glial cells. We routinely utilize compartmentalized chambers to grow neurons in conditions that allow a complete separation between axon terminals and cell bodies. By combining compartmentalized cultures of sympathetic neurons with Sequence Analysis of Gene Expression (SAGE) assay, we have performed an unbiased screen to identify mRNAs localized in axons. We found several hundred transcripts in axons, and many of them are selectively enriched in axon terminals when compared to cell bodies. A significant number of these encode proteins involved in neuronal growth, axon guidance and regeneration. Unexpectedly, we found that IMPA1, the inositol monophosphatase that is a target of lithium, is the most abundant axonal transcript, and we are now characterizing its role in axonal growth. The screen has been technically extremely challenging but we are now in the position of pursuing several exciting directions: - analyze the role of local protein synthesis for axon growth during development - identify regulatory sequence(s) that influence mRNA targeting in axons - characterize additional mRNAs highly enriched in axons The proper development of the nervous system depends upon the transcription and translation of genes that


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: NEUROLOGICAL