The proper wiring of the mammalian nervous system is a task of unique complexity, which requires specific point-to-point connections between billions of neurons. At the heart of this fundamental biological process lies the ability of extracellular cues, such as neurotrophins, to regulate the expression of specific genes that encode proteins that promote neuronal survival, axon growth, differentiation, synapse formation, and, later, synaptic plasticity. Two major regulatory events influence gene expression: binding and activation of nuclear proteins to cis-acting regulatory elements in gene promoters and epigenetic modifications that alter chromatin packing and thereby access of DNA-binding proteins to their DNA sites. While much work has been published on how neurotrophins and other extracellular signals regulate the nuclear factors that control gene expression during neuronal development, surprisingly little is known about the mechanisms by which extracellular cues induce chromatin modifications in these cells. Interestingly, several chromatin-modifying proteins have been associated with neurodegenerative diseases and mental retardation syndromes. Understanding how neurotrophins and neurotransmitters induce chromatin remodelling is therefore, of great importance and may provide the rationale for novel treatment strategies.
Fig 1: DAF staining of cortical neurons treated with BDNF. Nitric oxide accumulates in both the cytoplasm and the nucleus
Once a gene is transcribed, its mRNA must be translated into a protein, which then has to be transported to where it is needed. This transport can be especially challenging in differentiated neurons because the nucleus can be very far away from the final location of the protein: the growth cone of a sensory neuron in a mouse embryo, for example, can be millimetres, or even centimetres away from the cell body. Moreover, the half-life of a newly translated protein is often much shorter than the time required for it to travel along the axon to the growth cone. Therefore, many mRNAs encoding proteins required for either axon growth during development or axonal regeneration after injury must be transported and locally translated in axons. The targeting and translation of many of these RNAs may well be regulated by neurotrophins. Thus, the identification of axonal mRNAs and the signaling pathways that regulate their local translation should help us understand how neurotrophins promote axon growth during development and facilitate nerve regeneration in adults.
In our laboratory, we are currently following three lines of research:
1) Nitric oxide-dependent regulation of gene expression and chromatin remodelling in developing neurons
Fig 2: CHD3 immunostaining of mouse cortex at the indicated developmental stages.
We have recently identified nitric oxide (NO) as a novel mediator of gene expression in neurons. We have already characterized histone deacetylase 2 (HDAC2) as a target of BDNF-dependent nuclear S-nitrosylation. We are now performing mass spectrometry analysis of nuclear proteins that co-immunoprecipitate with HDAC2. We are also exploring whether S-nitrosylation of HDAC2 influences the expression of genes necessary for neurogenesis and neuronal migration in vivo.
2) Analysis of nuclear architecture and chromatin structure following synaptic activation in vivo and in vitro
Fig 3: DNA FISH showing colocalization of c-fos with RNA POLII transcriptional factories
A second research theme in my laboratory is centred on the identification of neuronal genes that are regulated in vivo following exposure to Novel Enriched Environmental (NEE) conditions, a complex somatosensory mode of stimulation.. Prolonged exposure to NEE protects against neurodegeneration; it also enhances neurogenesis, dendritic arborization and resistance to apoptosis. We initially identify genes regulated following NEE conditions, by using ChIPSeq assay, a technique that combines chromatin immunoprecipitation with large-scale direct ultrahigh-throughput DNA sequencing. Analysis of H3K9/K14 acetylation (a chromatin mark enriched in promoter regions of actively transcribed genes) of somatosensory cortex coupled with microarray analysis revealed that following exposure to NEE conditions neurons undergo a reactivation of a developmental transcriptional program. Moreover, an iperacetylated and highly conserved region was significantly enriched within the promoters of genes induced following NEE stimulation. We have now evidence that this sequence mediates chromatin tethering and recruitment to transcription factories of genes that are co-transcribed following synaptic stimulation.
3) Transport and targeting of mRNAs to axons of developing neurons
Fig 5: Compartmentalized cultures of sympathetic neurons
To identify transcripts localized in axons we have performed an unbiased screen by combining compartmentalized cultures of sympathetic neurons with Sequential Analysis of Gene Expression. More than 11,000 tags matching known transcripts were found in axons. Surprisingly, the most abundant transcript in axons IMPA1, a key enzyme that regulates the inositol cycle and the main target of lithium in neurons. We also found that a novel localization element within the 3’UTR of IMPA1 specifically targeted IMPA1 transcript to sympathetic neuron axons and regulated local IMPA1 translation in response to Nerve Growth Factor (NGF). We are now performing extensive 5’ and 3’ RACE analyses of transcripts that are enriched in axons with the scope of identifying common structural features within the 3’ and 5’UTRs that will allow the biochemical purification of mRNA binding proteins. We are also testing the hypothesis that neurotrophins induce cytoplasmic remodelling of 3’UTR regions that is required for translational de-repression of axonal transcripts.