Dynamics of reactive astrocytes fosters tissue regeneration after cuprizone-induced demyelination [RNA-seq II]

Demyelination and dysregulated myelination in the CNS are hallmarks of many neurodegenerative diseases such as multiple sclerosis (MS) and leukodystrophies. Here, we studied GFAP+ astrocytes during de- and remyelination in the cuprizone mouse by exploiting the ribosomal tagging (RiboTag) technology. Analyses were performed 5 weeks after cuprizone feeding, at the peak of demyelination in the corpus callosum, and 0.5 and 2 weeks after cuprizone withdrawal, when remyelination and tissue repair is initiated. After 5 weeks of cuprizone feeding, reactive astrocytes showed inflammatory signatures with enhanced expression of genes that modulate leukocyte migration (Tlr2, Cd86, Parp14,Cxcl10). Furthermore, demyelination-induced reactive astrocytes expressed numerous ligands including Cx3cl1, Csf1, Il34, and Gas6 that act on homeostatic as well as activated microglia and thus potentially mediate activation and recruitment of microglia as well as enhancement of their phagocytosis. During early remyelination, region-specific astrocytes displayed reduced inflammatory response signatures as indicated by shut down of CXCL10 production. During late remyelination, the signatures of GFAP+ astrocytes shifted towards resolving inflammation by active suppression of lymphocyte activation and differentiation and support of glia cell differentiation. Astrocytes showed enhanced expression of osteopontin (SPP1) as well as of factors that are relevant for tissue remodelling (Timp1), regeneration and axonal repair. In conclusion, we detected highly dynamic astroglial transcriptomic signatures in the cuprizone model, which reflects excessive communication amongst glia cells and highlights different astrocyte functions during neurodegeneration and regeneration. Overall design: Male GFAP-Cre+/-RPL22HA/wt aged 8-9 weeks fed with 0.2% (w/w) of cuprizone blended into standard rodent chow for a total of 5 weeks to induce demyelination . Animals were fed ad libitum until defined experimental endpoints. After 5 weeks post-cuprizone feeding, mice were put on regular chow for additional 2 weeks (total 7 weeks) to examine remyelination. Natural remyelination occurs starting 0.5 weeks after Cuprizone withdrawal (total 5.5wks). For this study, brains were collected at 5, 5.5 and 7 weeks, which corresponds, to acute demyelination, earlier remyelination and late remyelination respectively. Along with this, aged-matched control mice were kept of a normal diet.At the experimental endpoint, animals were euthanized, brain harvested and corpus callosum was micro-dissected. Thereafter, corpus callosum was homogenized and lysate used to pull down HA-tagged ribosomes and to isolate RNA from the immunoprecipitate (IP) for bulk RNA-sequencing.