Extended Data Fig. 4. Astrocyte and oligodendrocyte cluster analysis and spatial transcriptomics in MS lesions.

(a) Differential spatial expression patterns of astroglial GFAP in subcortical versus cortical demyelination by IHC (left); tSNE plots visualizing astrocyte specific genes corresponding to all (RFX4) protoplasmic (SLC1A2, GPC5) and fibrous/reactive astrocytes (GFAP, CD44). Quantification of RFX4⁺ ISH signals per nuclei in GM and WM of control samples validates RFX4 as a canonical astrocyte marker (ctrl, n= 5); quantification of GPC5⁺ and CD44⁺ ISH signals per RFX4⁺ astrocytes confirms validates GPC5 as protoplasmic GM and CD44 as fibrous WM marker. Two-tailed Mann-Whitney tests were performed. Data presented as mean ± SEM. (b) Upregulation of astroglial CRYAB, MT3 (black arrowheads) and endothelin type B receptor transcript EDNRB (white arrowhead) in reactive astrocytes in subcortical lesions. (c) tSNE plots showing OL-specific expression of myelin genes MBP, CNP and transcription factor ST18; note co-expression of ST18 with PLP in control WM by ISH. (d) Visualization of enriched GO terms in myelinating OLs based on differential gene expression analysis. Binomial test with FDR correction was utilized to calculate FDR-corrected p values using 151 genes differentially expressed in OLs. (e) Co-expression spatial transcriptomic studies confirming upregulation of heat shock protein 90 transcript HSP90AA1 in both progenitor (PDGFRA-expressing) and myelinating (PLP1-expressing) OLs at lesion rims (PPWM, black arrowheads). For tSNE and violin plots, data shown from 9 control and 12 MS samples. For astrocyte violin plots, 1,571 control and 3,810 MS nuclei are shown. Box plots inside violin plots represent median and standard deviation of gene expression. For ISH and IHC experiments, representative images from from 3 control and 4 MS individual tissue sections are shown.