Extended Data Fig. 4: DAAs found in female 5xFAD mice and in the cortex.

a, tSNE embedding of single nuclei RNA profiles of astrocytes from hippocampus of 7-month old female (1,500 nuclei, 2 mice, left) and male (5,183 nuclei, 4 mice, right) of three WT and three 5xFAD (AD) mice. Colored by cluster assignment. The three astrocyte end states are marked on the graph (annotated by projection from Fig. 1d, Methods). b, tSNE embedding as in (a), colored by cluster 4 (DAA) and cluster 6 (Gfap-low) cluster IDs of male mice as in Fig. 1d (other cells in grey). c, Expression levels (color scale) and the percent of cells expressing (dot size) of marker genes across clusters, split by sex: for DAA (Ggta1, Gsn, Osmr, Vim, Serpina3n, Ctsb, Gfap), Gfap-low (Fabp7, Slc38a1, Myoc, Aqp4, Id1, Id3, Gfap) and Gfap-low (Mfge8, Slc7a10, Luzp2). d, The proportion of astrocytes classified as Gfap-low (clusters 1,2,5,6 in (a)), DAAs (cluster 3 in (a)) and Gfap-high (cluster 4 in (a)) in male and female mice. Bar: individual mice, colored by strain and sex. e, DAAs in 5xFAD mice cortex at age 7 and 10 months. tSNE embedding of single nuclei RNA profiles of astrocytes from WT and AD mice, from the cortex of 7 and 10 month old mice (6,062 nuclei, 4 mice) and hippocampus of 7-month old mice (5,344 nuclei, 4 mice). Colored by cluster. f, Similar astrocytes marker genes in in the hippocampus and cortex. Dot plot showing the expression level (as color scale) and the percent of cells expressing (as dot size) marker genes for DAAs, Gfap-high and Gfap-low across clusters (as in (e)), split by brain region to hippocampus and cortex. g, tSNE embedding as in (e), colored by predicted cluster ID of hippocampal astrocytes from 7-month old male mice (inferred by CCA^22,33 projections, as in Fig. 1d, Methods). h, Cortical astrocyte populations match astrocyte states identified in the hippocampus. Heat map showing the correspondence between the de novo cluster IDs (rows, from (e)) of the cross regional dataset, and the predicted cluster IDs (columns) using the hippocampal astrocytes cluster IDs as reference (as from Fig. 1d). Color scale based on the proportion of predicted IDs per de novo cross-regional cluster. i, DAAs appear in the cortex of 5xFAD (AD) mice. The proportion of astrocytes, per sample, across clusters, including clusters of Gfap-low, DAAs, and Gfap-high astrocytes. Bars: Individual mice, color annotated by region: cortex or hippocampus (Hip), age: 7 or 10 months (m), and strain: AD or WT. j, Astrocyte marker genes. Left: Average expression level (color scale) and the percent of cells expressing (dot size) marker genes for: Gfap-low (Slc7a10; Trpm3), DAA (Ctsb¹⁶; Csmd1, associated with cognitive functions; C4b, encoding complement factor 4; Vim, a marker of adult neurogenesis/NSCs¹⁸), common to DAA and Gfap-high (Cd9, expressed by neural stem cells like astrocytes), and Gfap-high (Sparcl1/Hevin, encoding astrocytes pro-synaptic protein; Aqp4, an endfeet marker ¹⁴) (cells clusters as in Fig. 1e). Right: Violin plots (n=8 animals, 10 samples, 7,345 cells), showing the expression level distributions of Csmd1 and Ctsb in WT and AD astrocytes.