A concerted neuron-astrocyte program declines in ageing and schizophrenia

Abstract

Human brains vary across people and over time; such variation is not yet understood in cellular terms. Here we describe a relationship between people's cortical neurons and cortical astrocytes. We used single-nucleus RNA sequencing to analyse the prefrontal cortex of 191 human donors aged 22-97 years, including healthy individuals and people with schizophrenia. Latent-factor analysis of these data revealed that, in people whose cortical neurons more strongly expressed genes encoding synaptic components, cortical astrocytes more strongly expressed distinct genes with synaptic functions and genes for synthesizing cholesterol, an astrocyte-supplied component of synaptic membranes. We call this relationship the synaptic neuron and astrocyte program (SNAP). In schizophrenia and ageing-two conditions that involve declines in cognitive flexibility and plasticity^1,2-cells divested from SNAP: astrocytes, glutamatergic (excitatory) neurons and GABAergic (inhibitory) neurons all showed reduced SNAP expression to corresponding degrees. The distinct astrocytic and neuronal components of SNAP both involved genes in which genetic risk factors for schizophrenia were strongly concentrated. SNAP, which varies quantitatively even among healthy people of similar age, may underlie many aspects of normal human interindividual differences and may be an important point of convergence for multiple kinds of pathophysiology.

Fig. 1. Identification of concerted multicellular gene-expression changes common to schizophrenia and ageing.

a, Generation of snRNA-seq data, in a series of 20-donor ‘villages’. The diagram was created using images by thekua (person icon), B. Lachner (laboratory tools) and pnx (brain exterior side view) under a Creative Commons licence CC0 1.0. b, Uniform manifold approximation and projection (UMAP; coloured by donor) analysis of the RNA-expression profiles of 1,217,965 nuclei analysed from 191 donors. c, Assignments of nuclei to cell types (same projection as in b). d,e, Assignments of nuclei to glutamatergic (n = 524,186) (d) and GABAergic (n = 238,311) (e) neuron subtypes. CT, corticothalamic; ET, extratelencephalic; IT, intratelencephalic; NP, near-projecting. f, Latent factor analysis. Cell-type-resolution expression data from all donors and cell types were combined into a single analysis. Latent factor analysis identified constellations of gene-expression changes that consistently appeared together. g, The cell type specificity of the latent factors inferred from 180 donors, shown as the cell type distributions of the 1,000 most strongly loading gene–cell type combinations per factor. Factors 4–7 and 10 are strongly driven by gene-expression co-variation spanning multiple cell types. h, The association of schizophrenia (SCZ) with interindividual variation in the expression levels of the ten latent factors in g, shown as a quantile–quantile plot comparing the observed schizophrenia associations with the ten factors (−log₁₀[P]) to the distribution of association statistics expected by chance; only LF4 significantly associated with schizophrenia. See also Supplementary Fig. 6. i, The relationship between quantile-normalized LF4 donor expression levels and age (Spearman’s ρ; n = 180 donors). The shaded regions represent the 95% confidence intervals. j, Quantile-normalized LF4 donor scores (n = 93 controls, 87 cases), adjusted for age. The P value was calculated using a two-sided Wilcoxon rank-sum test. For the violin plot, the box limits show the interquartile range, the whiskers show 1.5× the interquartile interval, the centre lines show the median values and the notches show the confidence intervals around the median values. Source Data

Fig. 2. Genes recruited by SNAP in neurons and astrocytes.

a, Comparisons of SNAP gene recruitment between cell types. For each pairwise cell type comparison, the LF4 gene loadings of all genes expressed (≥1 unique molecular identifier (UMI) per 10⁵) in both cell types in the comparison (Spearman’s ρ; n = 10,346, 11,232 and 11,217 genes, respectively) are shown. b, Concentrations of synaptic gene sets (as annotated by SynGO) in LF4’s neuronal components. FDR, false-discovery rate. c, The fraction of gene expression (UMIs) devoted to synaptic-vesicle-cycle genes in subtypes of glutamatergic and GABAergic neurons, across 180 donors. P values for case–control comparisons were calculated using two-sided Wilcoxon rank-sum tests. CGE, caudal ganglionic eminence; MGE, medial ganglionic eminence. d, The distributions of astrocyte LF4 gene loadings for all expressed genes (black; n = 18,347) and genes annotated for functions in cholesterol biosynthesis (blue; n = 21; hereafter, cholesterol biosynthesis genes according to their GO annotation, although subsets contribute to cholesterol export and/or to synthesis of additional fatty acids) (left). Right, the proportion of astrocytic gene expression devoted to the annotated cholesterol biosynthesis genes shown, across 180 donors. The P value was calculated using a two-sided Wilcoxon rank-sum test. e, Concerted gene-expression variation in neurons and astrocytes. The relationships (across 180 donors) between astrocytic gene expression related to three biological activities (synapse adhesion, neurotransmitter uptake and cholesterol biosynthesis) and neuronal gene expression related to synapses (Spearman’s ρ). Quantities plotted are the fraction of all detected nuclear mRNA transcripts (UMIs) derived from these genes in each donor’s astrocytes (x axis) or neurons (y axis) relative to the median expression among control donors. The shaded regions represent the 95% confidence intervals for the estimated slopes. For the box plots nested within the violin plots in c and d, the box limits show the interquartile range, the whiskers show 1.5× the interquartile interval, the centre line shows the median value and the notches show the confidence intervals around the median values. Source Data

Fig. 3. Biological states and transcriptional programs of astrocytes and L5 IT glutamatergic neurons in schizophrenia.

a–c, UMAP analysis of RNA expression patterns from 179,764 astrocyte nuclei from 180 donors. Nuclei are coloured by astrocyte subtype (a), schizophrenia affected/unaffected status (b) and expression of the astrocyte component of SNAP (SNAP-a) (c). d, The relationship between donor quantile-normalized SNAP-a expression scores and age (Spearman’s ρ). n = 180 donors. The shaded regions represent the 95% confidence intervals. e, The distributions of SNAP-a donor scores (age adjusted and quantile normalized) for people with and without schizophrenia. n = 93 controls, 87 cases. The P value was calculated using a two-sided Wilcoxon rank-sum test. For the box plots, the box limits show the interquartile range, the whiskers show 1.5× the interquartile interval, the centre line shows the median value and the notches show the confidence intervals around the median values. f–j, Similar plots to those in a–e, respectively, but for the L5 IT glutamatergic neuron contribution to SNAP (SNAP-n). n = 75,929 nuclei. Exc, excitatory neuron subtype. k, Variation in the expression levels across 180 individual persons (columns, ordered from left to right by SNAP expression levels) of a select set of strongly SNAP-recruited genes (rows) in astrocytes (left panel) and L5 IT glutamatergic neurons (right panel) of the 180 brain donors. One set of genes (SNAP-a; top) exhibits co-regulation in astrocytes; and a distinct set of genes (SNAP-n; bottom) exhibits co-regulation in neurons. Genes indicated by asterisks and hashes are at genomic loci associated with common and rare genetic variation in schizophrenia, respectively. The grey bars indicate that regulon activity was not detected. Source Data

Fig. 4. The relationship between SNAP and schizophrenia genetics.

a, Enrichment of schizophrenia genetic association (from common variants, using MAGMA to generate a schizophrenia association z score for each gene) in the 2,000 genes most preferentially expressed in glutamatergic neurons and astrocytes (cell identity gene expression, upper bars), or the 2,000 genes of which the expression is most strongly recruited by SNAP-n and SNAP-a (cellular programs, lower bars). Values plotted are −log₁₀[P] from a joint regression analysis in which each gene set is an independent and competing predictive factor. See also the Supplementary Note. b, The relationship between donor SNAP expression (quantile normalized) and donor schizophrenia polygenic risk scores (Spearman’s ρ; n = 180 donors; PGC3 GWAS from ref. ). The shaded regions represent the 95% confidence intervals. c, NRXN1 expression (per 10⁵ detected nuclear transcripts) in each cell type in individual donors. n = 93 controls, 87 cases. P values were calculated using two-sided Wilcoxon rank-sum tests. For the box plots, the box limits show the interquartile range, the whiskers show 1.5× the interquartile interval, the centre line shows the median value and the notches show the confidence intervals around the median values d, NRXN1 expression in individual astrocytes (using the same projection as in Fig. 3a–c) (left). The values represent Pearson residuals from variance stabilizing transformation. Right, the relationship between the 180 donors’ NRXN1 expression in astrocytes and SNAP-a expression (Spearman’s ρ). e,f, Similar plots to those in c and d, but for C4. Source Data

Extended Data Fig. 1. Ages of brain tissue donors.

a, Distribution of the ages of brain donors (n = 191 donors). b, Distributions of donors’ ages by schizophrenia status, displayed as a quantile-quantile plot that compares ages of unaffected control donors (n = 97 donors) to ages of donors with schizophrenia (n = 94 donors). c–d, Distributions of donors’ ages separated by schizophrenia status (n = 97 unaffected and 94 affected), displayed as (c) histograms and (d) violin plots. e–f, Distributions of donors’ ages, separated by sex (n = 75 women and 116 men), displayed as (e) histograms and (f) violin plots. Note that while female brain donors are on average older than male donors, expression of SNAP (LF4) did not associate with sex in either a naive or age-adjusted analysis (Extended Data Fig. 4d,e), nor in a simultaneous regression on age, sex, and schizophrenia affected/unaffected status (Supplementary Table 3). Source Data

Extended Data Fig. 2. Single-donor assignment and sequencing metrics.

a, Density plot showing the fraction of all nuclei that were determined to be “singlets” (containing alleles from just one donor); n = 1,262,765 assignable singlets out of 1,271,830). b, Density plot showing donor-assignment likelihoods (as false discovery rates, on a log scale) for the 1,271,830 singlet nuclei. c, Validation of the computational assignment of nuclei to individual brain donors whose genomes have been analysed (individually) by SNP array-genotyping plus imputation. The matrix displays the concordance of single-donor assignment between whole-genome sequencing (WGS) (y-axis) and SNP array + imputation (x-axis) for a pilot set of 11 donors whose genomes were analysed by both methods. (Accuracy of donor assignment when WGS data are available has been previously shown by). Each row/column corresponds to one of the 11 donors, and each entry in the table displays the number of nuclei that were assigned to a given donor (at a false discovery rate of 0.05). d, Number of nuclei assigned to each donor in each of 11 batches or (rightmost panel) across all batches, separated by schizophrenia case-control status (n = 10 controls and 10 schizophrenia cases per batch). P-values from a two-sided Wilcoxon rank-sum test comparing the affected to the unaffected donors are reported at the top of each panel. Central lines represent medians. e, Median number of UMIs ascertained per donor in each batch or (rightmost panel) across all batches, separated by schizophrenia case-control status (n = 10 controls and 10 schizophrenia cases per batch). P-values from a two-sided Wilcoxon rank-sum test comparing the affected to the unaffected donors are reported at the top of each panel. Central lines represent medians. f, Relationship of median UMIs/nucleus (normalized to the median value of the donors in each donor’s batch) to (top) post-mortem interval (PMI) and (bottom) RIN score (Spearman’s ρ). Colours represent different batches. Shaded regions represent 95% confidence intervals. Source Data

Extended Data Fig. 3. Properties of the latent factors inferred from snRNA-seq data.

a, Total % variance in expression explained by latent factors with different numbers of requested factors k. b, Fraction of variance explained by each latent factor in an analysis with 10 requested factors. c–d, Independence of latent factors, visualized as Pearson correlation heatmaps of factors’ (c) gene loadings (n = 125,437 gene/cell-type combinations) and (d) donor scores (n = 180 donors). e, Expression level of each latent factor (panels) in each donor (points), split by batch (n = 20 donors per batch). f, Relationship of latent factors to markers of superficial and deep cortical layers from. Markers label dominant classes of glutamatergic neurons (superficial: LAMP5, LINC00507, RORB; deep: THEMIS, FEZF2) or spatially restricted subtypes (superficial: Exc L2 LAMP5 LTK, marked by CUX2 and LINC01500; deep: Exc L5-6 THEMIS C1QL3, marked by SATB2 and LINC00343). Factor 2 exhibits the most distinct segregation of these superficial and deep layer markers when genes are ranked by their loadings onto each factor. n = 18,830 genes expressed in glutamatergic neurons; coloured dots are plotted over the dots of genes not among the markers listed above (grey). Source Data

Extended Data Fig. 4. Properties of Latent Factor 4 (LF4).

a, Expression of each latent factor by case-control status (n = 93 controls and 87 cases). P-values are from a two-sided Wilcoxon rank-sum test. Box plots show interquartile ranges; whiskers, 1.5x the interquartile interval; central lines, medians; notches, confidence intervals around medians. b, Expression of LF4 by case-control status, split by sex (female: n = 31 controls and 39 cases; male: n = 62 controls and 48 cases). P-values are from a two-sided Wilcoxon rank-sum test. Box plots show interquartile ranges; whiskers, 1.5x the interquartile interval; central lines, medians; notches, confidence intervals around medians. Note that the more-modest p-value for the females-only analysis relative to the males-only analysis appears to represent the smaller sample (70 females vs. 110 males) rather than a weaker relationship to schizophrenia status; please see also Extended Data Fig. 10h. c, Similar plots as in b, here displaying LF4 expression values adjusted for donor age. d, Expression of LF4 by sex, split by case-control status (controls: n = 31 females and 62 males; cases: n = 39 females and 48 males). P-values are from a two-sided Wilcoxon rank-sum test. Box plots show interquartile ranges; whiskers, 1.5x the interquartile interval; central lines, medians; notches, confidence intervals around medians. e, Similar plots as in d, here displaying LF4 expression values adjusted for donor age. f–k, Relationship of LF4 expression measurements to other available donor and tissue characteristics: (f) time of death in zeitgeber time (ZT), with rhythmicity analyses performed as in; (g) post-mortem interval; (h) number of nuclei sampled; (i) number of UMIs sampled; (j) use of psychiatric medications (left column) across each donor’s lifespan or (right column) in the last 6 months prior to death; and (k) use of clozapine. Correlation coefficients in g–j are Spearman’s ρ. P-values in k are from a two-sided Wilcoxon rank-sum test. Box plots show interquartile ranges; whiskers, 1.5x the interquartile interval; central lines, medians; notches, confidence intervals around medians. l, See also Fig. 2a. LF4 involves broadly similar gene-expression effects in glutamatergic and GABAergic neurons, and a distinct set of gene-expression effects in astrocytes. Genes plotted are the protein-coding genes that are expressed (at levels of at least 10 UMIs per 10⁵) in both cell types (Spearman’s ρ; n = 1,538, 1,067, and 1,131 genes respectively). m, Concentrations of the strongest enriched neuronal gene-expression changes in LF4 among synaptic functions as annotated by SynGO. Plots show categories of SynGO biological processes. Source Data

Extended Data Fig. 5. Relationship of synaptic vesicle cycle gene expression in neuronal subtypes to advancing age.

a−b, See also Fig. 2c. Neuronal expression of synaptic vesicle cycle genes in the most abundant subtypes of (a) glutamatergic and (b) GABAergic neurons (across 180 donors), plotted against donor age (Spearman’s ρ). Expression values are the fraction of all UMIs in each donor (from the indicated subtype) that are derived from these genes, normalized to the median expression among control donors. Shaded regions represent 95% confidence intervals. The observed decline in schizophrenia and aging was consistent with earlier observations that expression of genes for synaptic components is reduced in schizophrenia and with advancing age. Source Data

Extended Data Fig. 6. Relationship of gene-set expression in astrocytes and neurons to advancing age and schizophrenia.

a, Expression of gene sets enriched in the astrocyte and neuronal components of LF4 (across 180 donors), plotted against donor age (Spearman’s ρ). Expression values are the fraction of all UMIs in each donor (from the indicated cell type) that are derived from these genes, normalized to the median expression among control donors. Shaded regions represent 95% confidence intervals. b, Expression (by donor, separated by schizophrenia case-control status; n = 180 donors) of gene sets enriched in the astrocyte and neuronal components of LF4. Expression values are the fraction of all UMIs in each donor (from the indicated cell type) that are derived from these genes, adjusted for donor age. P-values from a two-sided Wilcoxon rank-sum test comparing the affected to the unaffected donors are reported at the top of each panel. Box plots show interquartile ranges; whiskers, 1.5x the interquartile interval; central lines, medians; notches, confidence intervals around medians. Source Data

Extended Data Fig. 7. Expression of cholesterol-biosynthesis genes in cortical cell types.

a, See also Fig. 2d. For each cortical cell type: (Left) Distributions of LF4 gene loadings for (black) all expressed genes and (blue) specifically for genes annotated by GO as having roles in cholesterol biosynthesis (core genes contributing to the enrichment of GO:0045540 (“cholesterol biosynthesis genes”) in that cell type’s component of LF4. (Right) Each cell type’s expression of cholesterol biosynthesis genes (by donor, split by schizophrenia case-control status; n = 180 donors). Expression values are the fraction of all UMIs in each donor (from the indicated cell type) that are derived from these genes. P-values are from a two-sided Wilcoxon rank-sum test comparing the affected to the unaffected donors. Box plots show interquartile ranges; whiskers, 1.5x the interquartile interval; central lines, medians; notches, confidence intervals around medians. b, Expression in astrocytes of cholesterol biosynthesis genes by donor, separated by statin intake among donors with available medication data (n = 63 donors not taking statins and 16 donors taking statins). Expression values are the fraction of all UMIs in each donor’s astrocytes that are derived from these genes. P-value is from a two-sided Wilcoxon rank-sum test. Box plots show interquartile ranges; whiskers, 1.5x the interquartile interval; central lines, medians; notches, confidence intervals around medians. Source Data

Extended Data Fig. 8. Concerted synaptic investments by neurons and astrocytes, adjusted for age and schizophrenia case-control status.

a–c, See also Fig. 2e. Relationship of donors’ neuronal gene expression to astrocyte gene expression (Spearman’s ρ), adjusted for age and case-control status. Astrocyte gene sets plotted on the x-axes represent (left) cholesterol biosynthesis, (middle) synaptic adhesion, and (right) neurotransmitter reuptake transporters. Neuronal gene sets plotted on the y-axes represent (a) trans-synaptic signalling, (b) integral component of postsynaptic density, and (c) presynapse genes. Expression values are the fraction of all UMIs in each donor (from the indicated cell type) that are derived from these genes, adjusted for donor age and schizophrenia case-control status. Shaded regions represent 95% confidence intervals. Source Data

Extended Data Fig. 9. Astrocyte subtype classification and proportions across donors.

a, Two-dimensional projection of the RNA-expression profiles of 179,764 astrocyte nuclei from 180 donors, reproduced from Fig. 3a. Nuclei are coloured by their assignments to subtypes of astrocytes using classifications from and. The same projection is used in panels b to d. b−d, Expression levels of marker genes for subtypes of (b) protoplasmic astrocytes (SLC1A3+) and non-protoplasmic astrocytes (SLC1A3− and GFAP+) comprising the (c) fibrous (AQP1+) and (d) interlaminar (AQP1− and ID3+, SERPINI2+, and WDR49+) subtypes. Markers are from or from transcriptomically similar subtypes in. Values represent Pearson residuals from variance stabilizing transformation (VST). e, Proportions of astrocyte subtypes in BA46 by schizophrenia status (n = 93 unaffected and 87 affected). P-values from a two-sided Wilcoxon rank-sum test comparing the affected to the unaffected donors are reported at the top of each panel. Box plots show interquartile ranges; whiskers, 1.5x the interquartile interval; central lines, medians; notches, confidence intervals around medians. f, Relationship of sampled astrocyte subtype proportions to donor age (Spearman’s ρ). Source Data

Extended Data Fig. 10. Astrocyte gene-expression programs inferred by cNMF (SNAP-a) and their relationship to SNAP.

a, Visualization of the trade-off between error and stability of cNMF factors as a function of the number of factors k. 11 factors were requested based on these results. b, Clustergram of consensus matrix factorization estimates. Each colour on the x- and y-axes represents one of 11 cNMF factors. c-d, Relationship of SNAP-a to SNAP by (c) gene loadings (n = 33,611 genes) and (d) donors’ expression levels of each factor (n = 180 donors) (Spearman’s ρ). Shaded regions represent 95% confidence intervals. e, UMAP of RNA-expression patterns from 179,764 astrocyte nuclei from 180 donors, using the same projection from Fig. 3a–c. Nuclei are coloured by (left) each donor’s expression of SNAP or (right) each cell’s expression of the astrocyte component of SNAP (cNMF2, also referred to as SNAP-a). SNAP-a is reproduced from Fig. 3c for comparison with SNAP. f, Distributions of SNAP-a expression levels among astrocytes in each donor, split by experimental batch. Box plots show interquartile ranges; whiskers, 1.5x the interquartile interval; central lines, medians. g, Density plots showing distributions of SNAP-a expression levels among astrocytes in each donor for one representative batch (batch 4) out of 11 batches. Labels in top-right corners indicate anonymized research IDs at the Harvard Brain Tissue Resource Center. Colours represent case-control status (green: controls; purple: schizophrenia cases). At the single-astrocyte level, SNAP-a expression exhibited continuous, quantitative variation rather than discrete state shifts by a subpopulation of astrocytes, supporting the idea that astrocyte biological variation extends beyond polarized states^,,, particularly in genes strongly loading onto SNAP-a^–. h, Distributions of SNAP-a expression levels by case-control status, split by sex. P-values from a two-sided Wilcoxon rank-sum test comparing the affected to the unaffected donors are reported at the top of each panel. Box plots show interquartile ranges; whiskers, 1.5x the interquartile interval; central lines, medians; notches, confidence intervals around medians. i, Distributions of SNAP-a expression levels by case-control status, split by astrocyte subtype. P-values from a two-sided Wilcoxon rank-sum test comparing the affected to the unaffected donors are reported at the top of each panel. Box plots show interquartile ranges; whiskers, 1.5x the interquartile interval; central lines, medians; notches, confidence intervals around medians. Source Data

Extended Data Fig. 11. Relationship of reactive astrocyte marker expression to SNAP-a expression.

Relationship of donors’ expression levels of reactive astrocyte marker genes to SNAP-a expression (Spearman’s ρ). Markers are from and represent (a) pan-reactive (PAN), (b) A1, and (c) A2 reactive astrocytes. Source Data

Extended Data Fig. 12. Biological states and transcriptional programs of L5 IT glutamatergic neurons in schizophrenia.

a–b, Relationship of SNAP-a to SNAP-n (Spearman’s ρ). Values plotted are (a) quantile-normalized and (b) donor age-adjusted, quantile-normalized donor scores for each factor. Shaded regions represent 95% confidence intervals. c, UMAP of regulon activity scores (as inferred by pySCENIC) from L5 IT glutamatergic neuron nuclei from 180 donors, using the same projection from Fig. 3f–h. Regulons plotted are the most strongly enriched in L5 IT glutamatergic neurons with high versus low SNAP-n expression. (+) indicates that the targets of the indicated regulon were found to be upregulated in expression. Source Data

Extended Data Fig. 13. Astrocyte gene-expression programs underlying SNAP-a.

a, See also Fig. 3k. Concerted expression in (left) astrocytes and (right) GABAergic neurons of genes strongly recruited by SNAP-a. These were enriched in genes encoding synaptic-adhesion proteins, intrinsic components of synaptic membranes such as transporters and receptors, as well as genes strongly implicated in human genetic studies of schizophrenia. Genes in the “Schizophrenia genetics” heatmap are from among the prioritized genes from (FDR < 0.05) or. Genes annotated by ^ are from among all genes at loci implicated by common variants in, regardless of prioritization status. b, UMAP of regulon activity scores (as inferred by pySCENIC) from 179,764 astrocyte nuclei from 180 donors, using the same projection from Fig. 3a–c. Regulons plotted are the most strongly enriched in astrocytes with high versus low SNAP-a expression. (+) indicates that the targets of the indicated regulon are predicted to be upregulated in expression. c–d, Transcriptional investments (by donor, separated by schizophrenia case-control status) in (c) genes encoding synaptic receptors and transporters and (d) cholesterol biosynthesis genes, in subtypes of astrocytes. Quantities plotted are the fraction of all UMIs in each subtype that are derived from these genes. P-values from a two-sided Wilcoxon rank-sum test comparing the affected to the unaffected donors are reported at the top of each panel. Box plots show interquartile ranges; whiskers, 1.5x the interquartile interval; central lines, medians; notches, confidence intervals around medians. e, Relationship of SNAP-a expression to association with super-enhancers. Genes expressed in astrocytes were grouped based on whether their promoters were predicted to contact super-enhancers in astrocytes (using bulk H3K27ac HiChIP and scATAC-seq data from), and SNAP-a loadings were compared between the two groups. (Left) Distributions of SNAP-a gene loadings for (blue) 1,286 genes whose promoters are predicted to contact super-enhancers in astrocytes and (black) the set of 32,325 remaining expressed background genes. (Right) Binomial smooth results of scaled SNAP-a gene loadings versus log₁₀-scaled mean expression values in astrocytes, shown separately for the two groups. Shaded regions represent 95% confidence intervals. Source Data

Extended Data Fig. 14. Expression of well-characterized transcriptional programs in SNAP-a and SNAP-n.

a, Concerted expression in (left) astrocytes and (right) L5 IT glutamatergic neurons of target genes of known transcriptional programs specifically active in SNAP-a or SNAP-n. Genes are listed in decreasing order by their importance for each regulon as scored by pySCENIC. b, Relationship of donors’ expression levels of known SREBP1 target genes (involved in fatty acid biosynthesis)^,, to SNAP-a expression (Spearman’s ρ). Target-gene expression levels in astrocytes are shown. c, Relationship of donors’ expression levels of known JUNB target genes (that are late-response genes)^,, to SNAP-n expression (Spearman’s ρ). Target-gene expression levels in L5 IT glutamatergic neurons are shown. Source Data

Extended Data Fig. 15. Relationship of astrocytic NRXN1 and C4 expression to advancing age.

a, Relationship of NRXN1 expression to age in astrocytes (Spearman’s ρ). Shaded region represents 95% confidence interval. b, Expression of NRXN1 in astrocytes in control donors, split by donor age (n = 56 donors younger than 70 years old and 37 donors 70 years old or older). P-value is from a two-sided Wilcoxon rank-sum test. Box plots show interquartile ranges; whiskers, 1.5x the interquartile interval; central lines, medians; notches, confidence intervals around medians. c, Validation of a metagene computational approach for identifying RNA transcripts (UMIs) from the C4 genes. Standard analysis approaches tend to discard sequence reads from C4A or C4B because these genes are almost identical in sequence, differing only at a few key positions (far from the 3’ end), such that most reads are discarded due to low mapping quality. To measure expression of these genes, UMIs were either aligned to a custom reference genome that contained only one C4 gene (x-axis) or were processed through a custom pipeline that identified UMIs associated with sets of gene families with high sequence homology, including C4A/C4B (y-axis). The two approaches (custom reference approach and joint expression of C4A/C4B via the metagene approach) arrived at concordant C4 UMI counts in 15,664 of 15,669 cells tested. Note that these measurements do not distinguish between C4A and C4B. d, Relationship of C4 expression to age in astrocytes (Spearman’s ρ). Shaded region represents 95% confidence interval. e, Expression of C4 in astrocytes in control donors, split by donor age (n = 56 donors younger than 70 years old and 37 donors 70 years old or older). P-value is from a two-sided Wilcoxon rank-sum test. Box plots show interquartile ranges; whiskers, 1.5x the interquartile interval; central lines, medians; notches, confidence intervals around medians. Source Data