Figure 3. NDNF-PROX1 inhibitory neuron loss is associated with a hyperactivity signature in L2/3 excitatory neurons.

A) Logistic mixed-effect model regression of NDNF-PROX1 proportion versus cell type transcriptional signature in $A\beta +$ subjects. The dashed horizontal line represents the FDR threshold of 0.05. B) Associations (by logistic mixed-effect model) between the proportion of each inhibitory neuron type with each ExN type’s transcriptional signature in $A\beta +$ subjects. The red dots indicate the regression Z-score of the LINC00507-COL5A2 neurons with the corresponding inhibitory neuron cell type. The dashed line represents an FDR threshold of 0.05. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range. See Figure S7H for more details. C) Scatter plot comparing the logFC in the ExNs of $A\beta +$ (x-axis) and $A\beta +Tau+$ samples (y-axis). Visualization is based on the union of top 300 protein-coding DE genes (sorted by jack-knifed p-value) in either group. D) Logistic mixed-effect model regression of NDNF-PROX1 proportion versus early-specific up-regulated DE genes (green dots in C) and up-regulated DE genes shared in both $A\beta +$ and $A\beta +Tau+$ samples (blue dots in C) for each ExN cell type. The dashed lines represent an FDR threshold of 0.05. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range. E) Logistic mixed-effect model regression of NDNF-PROX1 cell fraction versus expression of neural activity signatures²³ in each ExN type in $A\beta +$ samples (one-sided). The dashed line represents a one-sided FDR threshold of 0.05. PRG, primary response genes; SRG, secondary response genes. F) Scatter plot showing normalized NDNF-PROX1 fraction (x-axis) and the percent of LINC00507-COL5A2 ExNs with high expression of the core immediate early genes FOS, JUNB, ARC, NPAS4, ERG1, and ERG2 (y-axis, Methods) in $A\beta +$ subjects. A logistic mixed-effect model was used to calculate the p-value. G) GSEA of Reactome pathways on DE results from subjects with varying $A\beta$ and tau burdens, across ExN types. Dots outlined in black denote significant terms (FDR-adjusted p-value < 0.05). $A\beta +$ individuals with $A\beta$ burden scores of 2 and 3 are grouped together. H) Concordance of DE genes between different stages of AD pathology within excitatory neuron cell types. The LINC00507+ and RORB+ were selected as upper layer excitatory neurons and FEZF2+, CTGF+, and THEMIS+ populations as lower layer. I) ExN DE genes whose products are involved in synapse vesicle cycle and trafficking (SYT1, SNAP25, and CDK5), amyloid precursor protein (APP), and receptors of oligomeric $A\beta$ (PRNP, ATP1A3, and PGRMC1) across different $A\beta$ and tau burdens. The outlined dots represent DE genes with jack-knifed FDR-adjusted p-value < 0.01. J) GSEA of human KEGG gene sets using DE genes of WIF1+ homeostatic astrocytes across increasing $A\beta$ and tau burden. Outlined dots represent significant terms (FDR-adjusted p-value < 0.1).