Identification of regional astrocyte heterogeneity associated with cuprizone-induced de- and remyelination using spatial transcriptomics

The cuprizone model is a well-characterized model to study processes of demyelination and remyelination, which are known features of multiple sclerosis. Cuprizone induces oligodendrocyte loss and severe demyelination in the brain, including the corpus callosum, hippocampus, and cortex. Loss of oligodendrocytes and myelin is accompanied by microgliosis and astrogliosis, wherein microglia and astrocytes partially lose their homeostatic functions and acquire a reactive/activated state. Cuprizone-induced demyelination peaks later in grey matter (GM) than in white matter (WM), and remyelination is more efficient in WM areas. Here, we aim to better understand regional diversity in microglia, astrocytes, and oligodendrocytes and their respective role in remyelination efficiency, by characterizing their response to cuprizone across brain regions. We applied spatial transcriptomics (ST) for unbiased gene activity profiling of multiple brain regions in a single tissue section, to identify region-associated changes in gene activity following cuprizone treatment. Gene activity changes were detected in highly abundant cell types, like neurons, oligodendrocytes, and astrocytes, but challenging to detect in low-abundant cell types such as microglia and oligodendrocyte precursor cells. ST revealed a significant increase in the expression of astrocyte markers Clu, Slc1a3, and Gfap during the demyelination phase in the WM fiber tract. In the cortex, the changes in GFAP expression were less prominent, both at the transcriptional and protein level. By mapping genes obtained from scRNAseq of FACS-sorted ACSA2-positive astrocytes onto the ST data, we observed astrocyte heterogeneity beyond the simple classification of WM- and GM-astrocytes in both control and cuprizone-treated mice. In the future, the characterization of these regional astrocyte populations could aid the development of novel strategies to halt the progression of demyelination and support remyelination. Highlights Astrocyte markers Clu, Slc1a3, and Gfap are increased in WM fiber tracts during demyelination Expression dynamics of astrogliosis markers Gfap and Vim during de-and remyelination depend on the brain region Combining scRNAseq with ST data revealed astrocyte heterogeneity beyond WM- and GM-differences scRNAseq-identified gene sets were differently affected by cuprizone treatment across brain regions