INVESTIGATING THE ROLE OF DEFECTIVE CELL TO CELL COMMUNICATION MECHANISMS IN RETT SYNDROME PATHOGENESIS
Rett syndrome (RTT) is a devastating neurodevelopmental disorder representing the main cause of severe intellectual disability in girls worldwide. Over 95% of individuals suffering from a classic form of RTT carry sporadic mutations in the X-linked MECP2 gene, encoding for the methyl-GpC-binding protein 2 (MeCP2), responsible for the regulation of the expression of thousands of genes. RTT is characterized by defects in neuronal morphology, activity and synaptic transmission, which are in part caused by the insufficient support from astrocytes. Indeed, astrocytes in RTT not only exhibit alterations in their morphology, metabolism and calcium signaling, but they also fail to adequately sustain neuronal growth and activity. Although understanding the causative mechanisms could guide the development of novel and effective therapeutic strategies, they remain largely unknown to date. This study explores the impact of Mecp2 knock-out (KO) astrocytes on synapses, mainly focusing on the paracrine signals, exploiting two different in vitro culture systems. On one hand, we treated neurons with astrocyte conditioned medium (ACM) obtained from wild type (WT) or Mecp2 KO astrocytes. On the other hand, we used a co-culture system between neurons and astrocytes, in which the latter are cultured on transwell inserts preventing the direct contact with neurons while allowing the exchange of secreted molecules. In both the cases, we observed that Mecp2 KO astrocytes severely compromise synaptogenesis and synaptic maintenance, suggesting that they either release synaptotoxic molecules or fail to secrete necessary synaptogenic factors. A transcriptomic study revealed that molecules secreted by Mecp2 KO astrocytes induce an abnormal inflammatory response in neurons, and coherently we found an increase of Interleukin-6 (IL-6) levels in the medium of the co-culture. The excessive amount of IL-6 was proven to be a critical mediator of synaptic defects as its blockade with a specific neutralizing antibody significantly recovered the synaptic phenotype. However, the persistence of post-synaptic defects implies the involvement of other factors. Thus, in addition to IL-6, our investigation extended to cholesterol, a well-known synaptogenic factor, mainly provided by astrocytes in mature brain. Interestingly, Mecp2 KO astrocytes show the downregulation of many genes involved in cholesterol synthesis and transport. Interestingly, the treatment of Mecp2 KO astrocytes with Trofinetide, the only FDA-approved drug for RTT, rescued the expression of some of the downregulated genes, suggesting that part of the beneficial effects elicited by this drug might be due to the modulation of cholesterol metabolism. Furthermore, cholesterol supplementation improved synaptic deficits in neurons treated with KO ACM, and similar benefits were observed in neurons obtained from heterozygous Mecp2+/- female mice. In conclusion, this study reveals that the overproduction of IL-6 and dysregulation of cholesterol metabolism in Mecp2 KO astrocytes have detrimental effects on synaptic health, offering novel potential therapeutic targets for RTT. These findings support the hypothesis that insufficient release of synaptogenic factors by astrocytes, combined with the release of synaptotoxic factors, contributes to pre- and post-synaptic impairments characterizing the pathology.
Rett syndrome, Interleukin-6, cholesterol, astrocyte, neuron, Settore BIOS-08/A - Biologia molecolare, Settore BIOS-10/A - Biologia cellulare e applicata
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