Duchenne muscular dystrophy (DMD) is a lethal X-linked disease characterized by muscular wasting and neurological disturbances due to lack of dystrophin (Dp427), a cytoskeletal protein expressed in muscle and selected brain regions (i.e. hippocampus, cerebellum, prefrontal cortex). Dp427 binds to the Dystrophin Glycoprotein Complex (DGC) that spans the plasma membrane and is implicated in maintaining structural and functional properties of cellular sub-domains. The DGC also binds to several other molecules, some of which drive important intracellular signaling pathways. Among them is the neuronal nitric oxide synthase (nNOS), which synthesizes nitric oxide (NO), a signaling molecule involved in several physiological and pathological events: i.e. neurogenesis, hippocampal long-term potentiation, neurodegeneration, neuronal survival and differentiation. NO signaling implies nitrosylation of target proteins, among which histone deacetylases (HDACs), nuclear enzymes that, by modulating chromatin acetylation, regulate the balance between transcriptional activation and repression. In both skeletal muscles and nervous system, HDAC2 is one of the most represented. S-nitrosylation of HDAC2 results in its release from chromatin and consequent increase in histone acetylation and gene expression. Deregulation of NOS activity determines a dramatic reduction in histone acetylation and, hence, gene expression. This has been well demonstrated in dystrophic skeletal muscles lacking Dp427, but never investigated in neurons. By using mdx mice, a mouse model of DMD, and their wild type, this project aims at identifying: 1) changes in histone acetylation in different brain areas known to be affected in DMD; 2) evaluate the response of both skeletal muscles and neurons to class I HDACs specific inhibitor administration; 3) evaluate behavioral performances, before and after HDAC inhibition, by using tasks directed to specifically determine motor skills and different cognitive abilities.
Structural and functional abnormalities in several brain regions, among which hippocampus, cerebellum and prefrontal cortex have been described in DMD patients and mdx mice, often correlating with a wide range of mild-to-severe cognitive disturbances. This cohort of alterations establishes very soon, during pre- and early post-natal stages, when the development and successive refinement of neural circuits occur. A delicate balance between highly specific and tightly regulated gene expression and protein synthesis characterizes proper neural circuit connectivity and physiology. A similar dependency is observed in cognitive performances, as learning and memory consolidation, throughout life. To date, still too little attention is dedicated to neurological problems associated to DMD, which in some cases might have consequences, on patient lives, as severe as the characteristc muscular damage.
In this project, we focus on the role that a proper histone acetylation has in regulating gene expression, in both skeletal muscles, where large part of the studies in this direction has been made, and central nervous system, where no data on this aspect have yet been reported. A lesson from muscle is that lack of Dp427 impairs nNOS activity and, hence, HDAC nitrosylation. This transition is crucial in order to inhibit the activity of deacetylases, which is determinant for histone acetylation and gene expression.
Therefore, this project will move on two frontlines: muscle and brain. We will: 1) evaluate for the first time how lack of Dp427 impacts nNOS activity and HDAC nitrosylation in neurons; 2) analyze the impact that focused inhibition of class I HDACs, obtained by systemic drug administration, has on skeletal muscle regeneration; 3) study whether class I HDACs inhibition, obtained with a protocol that allows to cross the blood brain barrier, has a beneficial effect on neurons known to be affected by the lack of Dp427.
All these information will be of great importance for advancing in the research of therapeutic treatments of DMD, which should take into account not only muscle regeneration, but also amelioration of neurological disturbances.
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