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Abstract

Brain-derived neurotrophic factor (BDNF) is involved in several neuronal processes such as neuronal growth and differentiation, synapse development, and synaptic plasticity. Alterations in endogenous BDNF levels have been associated with neurological disorders, such as schizophrenia and depression. At the molecular level, stimulating cortical neurons with BDNF activates signalling cascades that culminate in specific transcriptional responses. Although extensive efforts have been made to study BDNF-induced gene regulation of selected loci, little is known about genome-wide chromatin changes triggered by BDNF and how these are regulated. In this PhD thesis, I sought to investigate (1) the molecular response of BDNF stimulation in chromatin regulatory proteins and their cooperation in orchestrating BDNF-dependent transcription and (2) the plasticity of BDNF-induced changes in chromatin upon reiterated stimulation events, in mouse cortical neurons. To address the first aim, I compared the changes in the subcellular proteomes -in cytosol, nucleoplasm, and chromatin- following stimulation with either BDNF or KCl (which triggers membrane depolarisation). The comparison between responses showed a significantly stronger recruitment of the transcription factor Fos to chromatin upon BDNF treatment. BDNF-affected distal regulatory elements were enriched in Fos binding motifs, often accompanied by other TF motifs. I confirmed that one of these TF families, EGR, regulates the expression of the BDNF-responsive synaptic regulator Arc through a novel BDNF-specific enhancer and that this is accomplished through a cooperative effect with Fos. The chromatin response to BDNF led to a prominent activation of distal regulatory elements, half of which remained accessible for 10 hours post-stimulation. I hypothesised that this sustained accessibility could act as a priming event, influencing the transcriptional outcome in future encounters with BDNF. To investigate this, I sought to identify differences in the transcriptional response of mouse primary neurons primed with BDNF compared to naive cells. The gene expression changes in primed and naive neurons revealed six different clusters, of which four showed marked variations between the first and second encounters with BDNF. Primed neurons presented a strong downregulation of lncRNAs, such as Meg3, Malat1 and Ftx, and lower induction of gene targets of the PI3K pathway. Furthermore, the induction of many BDNF-responsive transcriptional regulators (Npas4, Nr4a2, Klf6) was milder in primed neurons, but the expression of neuronal physiology-related genes (Snca, Cav2, Calca) was enhanced upon a second BDNF treatment. Chromatin accessibility profiling did not uncover any priming event, but instead, revealed a highly plastic and reversible behaviour in BDNF-regulated regions. However, at the proteome level, I found macroH2A, a transcriptionally repressive histone variant, to accumulate in primed neurons, pointing out a possible mechanism in conditioning the transcriptional outcome of subsequent stimulations. In conclusion, the results reported in this thesis enhance our understanding of the BDNF-regulated chromatin responses in neurons and shed light on a novel phenomenon of putative transcriptional adaptation upon priming with BDNF. Taken together, a better understanding of the cellular and molecular consequences of BDNF can pave the road for the design of new therapies for treating BDNF-associated brain disorders.