Investigation of the involvement of cholesterol metabolism in the pathogenesis of infantile spinal muscular atrophy.

Summary Infantile spinal muscular atrophy (SMA) is a rare autosomal recessive disease that occurs in childhood and for which no therapy has yet been found to be effective. SMAs are characterized by a specific degeneration of motor neurons leading to critical muscle weakness which, when it reaches the respiratory muscles, causes the death of the patients. The origin of these diseases is the mutation of the Survival of Motor Neuron 1 (Smn1) gene which induces a deficiency of the Survival of Motor Neuron (SMN) protein. All SMA patients have at least 2 copies of the Smn2 gene, which modulates the severity of the disease by producing a low amount of complete SMN protein. Indeed, due to alternative splicing of exon-7 which encodes a stability domain of the SMN protein, the transcripts produced by the expression of Smn2 are in most cases incomplete, and generate unstable, rapidly degraded SMN proteins. Thus, a major therapeutic goal in SMA is to increase SMN protein levels in patients. By deciphering the molecular mechanisms underlying the beneficial effects of exercise in a mouse model of severe SMA, the lab has shown that direct stimulation of NMDA receptors (NMDAR) results in a significant increase in exon 7 inclusion in SMN transcripts generated from Smn2 gene expression in SMA tissues. During my thesis, I was involved in the identification of a second exercise-induced mechanism related to the reduction of expression levels of the IGF-1 receptor (IGF-1R), which is overexpressed in the spinal cord of SMA mice. Reduction of IGF-1R is sufficient to promote SMN expression, both through transcriptional and post-transcriptional effects. Using a transcriptomic approach to identify genes whose expression is comparably altered by modulation of NMDARs or IGF-1R, we identified a set of genes that belong to the cholesterol biosynthetic pathway. RT-qPCR and western blot analyses in the spinal cord of control and SMA model mice revealed that i) cholesterol biosynthetic pathway enzymes are under-expressed in the spinal cord of SMA mice and in a culture of SMA patient fibroblasts and ii) NMDA treatment or IGF-1R reduction reactivates the expression of cholesterol biosynthetic pathway enzymes to levels comparable to controls. Importantly, we demonstrated that specific inhibition of DHCR24, a key enzyme in the terminal pathway of cholesterol biosynthesis, in control fibroblasts induces a significant reduction in SMN expression, both at the mRNA and protein level, and conversely, restoration of DHCR24 expression levels in human SMA fibroblasts induces a significant increase in SMN expression. Moreover, GC-MS quantification of cholesterol, its metabolites, and various oxysterols indicates that the whole cholesterol metabolism is altered in the SMA mouse spinal cord, and enhanced by NMDAR activation. Together, these results demonstrate for the first time that cholesterol homeostasis is disturbed in SMA and that modulating the molecular pathways dependent on cholesterol and its derivatives may constitute a promising therapeutic approach.