Fig 2: Dysregulated genes and biological processes in human temporal lobe epilepsy astrocyte, OPC and neuronal populations. (a) Differential expression analysis (TLE vs. TL) for each sorted cell type is represented by a volcano plot, depicting significantly upregulated (red) and downregulated (blue) genes in the epilepsy astrocyte, OPC, and neuronal populations. Functional enrichment analyses, performed using HOMER and DAVID (D) tools, depict top-enriched biological processes dysregulated in epilepsy, for each cell type (log2 fold change < -1 for downregulated and > 1 for upregulated, Benjamini–Hochberg adjusted p-value < 0.1). See Additional file 4: Table S3, Additional file 5: Table S4 for complete list. (b) MA plot shows the relative expression of genes differentially up- and down-regulated in PAX6 + epilepsy astrocytes, marked by red and blue dots, respectively

Fig 3: Simultaneous isolation of astrocyte, neuronal, and OPC-enriched nuclei from human temporal neocortex (a-b) Fluorescence-activated nuclei sorting (FANS) using anti-NEUN, anti-PAX6, and anti-OLIG2 antibodies simultaneously isolates three distinct nuclei populations from human postmortem control (TL) and epilepsy (TLE) temporal lobe neocortex: NEUN + , PAX6 + ( NEUN–), and OLIG2 + ( NEUN–), excluding the OLIG2LOW fraction. TN = triple negative (NEUN–PAX6–OLIG2–) population. See also Additional file 1: Fig. S1a. (c) Gene expression analysis by RT-qPCR confirms high expression of the genes used as markers for isolation and shows significantly enriched expression of the astrocytic markers GFAP and ALDH1L1 in PAX6 + ( NEUN–) nuclei and of the OPC markers CSPG4 (NG2) and PDGFRA in OLIG2 + ( NEUN–) nuclei (n = 4 TL control brains). Bars represent mean ± SEM. P-values calculated from one-tailed t-test compared to NEUN + population. PAX6 + vs. NEUN + : PAX6 p = 0.055; ALDH1L1 p = 0.067; GFAP p = 0.019. OLIG2 + vs. NEUN + : OLIG2 p = 0.0014; CSPG4 p = 0.012; PDGFRA p = 0.0008. (d) Quantification of expression of the myelinating oligodendrocyte marker PLP1 by RT-qPCR, showing its significantly higher expression in the OLIG2LOW gated nuclei population compared to all others. Bars represent mean ± SEM, (n = 3 TLE brains). *p < 0.05 one-tailed t-test. (e) Representative immunofluorescence images of PAX6, OLIG2, and NEUN expression in developing germinal matrix (left), adult postmortem TL neocortex (center) and adult TLE neocortex (right). In adult TL and TLE neocortex, PAX6 expression is seen in GFAP + astrocytes (arrows) and NEUN + neurons but is absent in OLIG2 + oligodendroglial cells (arrowheads). Scale bar = 50 µM. See also Additional file 1: Fig. S1e