Spatial transcriptomics identifies multiple regional signatures in human FCD IIb

We profiled spatial gene expression in four freshly frozen samples from three patients of FCD IIb, including three typical FCD IIb lesional specimens and one perilesional specimen comprising a few cellular abnormalities and containing a relatively complete neocortical structure (Supplementary Figs. 1, 2, 3). The clinicopathological features of the patient cohort were summarized in Supplementary Table 1. And the RNA integrity numbers (RINs) of all samples were in the range of 7.29–8.13 (Supplementary Table 2). Subsequently, the frozen tissue samples were placed on Visium slides for generation of sequencing data with a total of 12,754 spots, detecting on a mean of 7,601 unique molecular identifiers (UMIs) and a mean of 3,292 genes per spot (Fig. 1B).

Fig. 1

ST analysis of four samples from three patients with FCD IIb. (A) Workflow of FCD IIb patient sample processing for ST. Four freshly frozen samples from three FCD IIb patients were collected. First, preoperative MRI positive signs provide the first clue for FCD II. Second, macroscopic abnormal tissues (yellow) were freshly frozen for intraoperative cryosection diagnosis by two experienced neuropathologists, thus confirming that three samples contained FCD IIb lesions, and few cellular abnormalities were present in one perilesion. Then, its’ mirror plane tissues (blue) were prepared for formalin-fixed paraffin embedding (FFPE) and immunostaining. Finally, the qualified samples were applied the ST technology to explore the gene expression changes in FCD IIb. (B) Number of Visium spots (left), UMIs per spot (middle), and genes per spot captured (right) are shown per subject. (C) Heatmap showing representative marker genes for each cluster. (D) From left to right: the Spatial feature plots of Cluster 0 (top) and Cluster 3 (bottom), immunohistochemistry of SMI-32 (top) and Nestin (bottom), and ST feature plots of NEFH (DNs marker, top) and VIM (BCs marker, bottom) expression in lesion-1. (E) Thirteen regions (Clusters 0–12) were identified and visualized by using UMAP

We combined data from all subjects and used the Seurat v3.2 [43] R libraries for downstream analyses. Data were clustered to classify the spots and to determine the specific region type. Hereafter, we identified 13 different clusters (Clusters 0–12; Fig. 1E). Preliminary manual annotations of marker genes (STAR Methods) allowed us to identify cell classes. Cell type markers showed reliable prediction of cell identity (NEFL, NEFM and NEFH for DNs; VIM for BCs; SLC17A7 and SATB2 for excitatory neurons; GAD1, GAD2, SST, VIP, CALB2 and PVALB for inhibitory neurons; GFAP, AQP4, SLC1A2, SLC1A3 and IGFBP7 for astrocytes; MBP, PLP1, MOG, MOBP and CLDN11 for oligodendrocytes; CCL3, CCL4L2 and CCL4 for microglia; and CLDN5, VWF, A2M, APOLD1 and SLC2A1 for endothelial cells and pericytes; Fig. 1C). We next matched our results with histopathology to further confirm the classification of the clusters. These annotated cell regions include: Excitatory neurons region (Clusters 4 and 12), Inhibitory neurons region (Clusters 9 and 10), DNs region (Cluster 0), BCs region (Cluster 3), Astrocytes region (Clusters 2, 5 and 7), Microglia region (Cluster 11), Oligodendrocytes region (Clusters 1 and 6), Endothelial cells and Pericytes region (Cluster 8) (Fig. 1E, Supplementary Fig. 4). Particularly, among them, Cluster 0 and Cluster 3 expressed several known DNs or BCs markers, relatively consistent with the DNs or BCs morphological characteristics and distributions in histology (Fig. 1D, Supplementary Fig. 5).

Identification of the DNs region

We highlighted the differentially expressed genes in the DNs region. In addition to the classic DNs markers, NEFH, NEFL and NEFM, several genes had higher expression in the DNs domain, such as ENC1, NRGN, VSNL1, OLFM1, CCK, CHN1, UCHL1, CRYM, YWHAH, MDH1, SNAP25, TUBB2A, NSF, GABRD, GAP43, NPTX2, and BASP1 (Fig. 2A, Supplementary Fig. 6). To understand changes in gene expression, we performed differential expression analyses by comparing Cluster 0 with all other clusters. Based on the GO enrichment analysis (Fig. 2B, Supplementary Fig. 7A, and Supplementary Table 3), we identified 6 major functional modules, including Synapse, Potential, Cell morphogenesis and developmental growth, Ubiquitination, Autophagy, and Microtubule. Among them, the most enriched functional classes of the DNs region were the regulation of membrane potential (GO:0042391; i.e., YWHAH, SNAP25, NSF and GABRD), synapse organization (GO:0050808; i.e., NEFL, NEFH and GAP43), proteasome-mediated ubiquitin-dependent protein catabolic process (GO:0043161; UCHL1), regulation of cellular component size (GO:0032535; i.e., NEFL, NEFH, ENC1, OLFM1, CCK, CHN1, YWHAH, GAP43 and BASP1), macroautophagy (GO:0016236; i.e., ENC1), and regulation of microtubule-based process (GO:0032886; i.e., NEFL, NEFM, NEFH and TUBB2A). We performed Z-scored mean log expression heatmap on genes related to the regulation of membrane potential and proteasome-mediated ubiquitin-dependent protein catabolic process, further confirming that these genes are mainly expressed in the DNs region (Fig. 2E, F). In addition, we found that the DNs region was involved in Proteasome (hsa03050), Ubiquitin mediated proteolysis (hsa04120), Phosphatidylinositol signalling system (hsa04070), Neurotrophin signalling pathway (hsa04722), Protein processing in endoplasmic reticulum (hsa04141), Insulin signalling pathway (hsa04910), and mTOR signalling pathway (hsa04150) by using KEGG functional enrichment analysis (Fig. 2G). And the Gene-Concept Networks by KEGG analysis exhibited complex interactions between several pathways (Supplementary Fig. 7B).

Fig. 2

Identification of the DNs region. (A) Left, the DNs region (Cluster 0) was identified and visualized by using UMAP. Right, UMAP feature plots of expression for the DNs-specific genes NEFH, NEFL, and NEFM. (B) Enrichment map of GO-BP terms for Cluster 0. (C), (D) ST feature plots of representative genes GABRD and UCHL1 expression in four samples. (E), (F) Z-scored mean log expression heatmap of genes associated with the regulation of membrane potential, and proteasome-mediated ubiquitin-dependent protein catabolic process across the annotated cell regions in the ST data. (G) Bar plots of KEGG terms for Cluster 0. (H) GO-BP and KEGG analyses on the DNs region (Cluster 0 vs. Cluster rest) applied by GSEA enrichment

To further investigate the gene functions in the DNs region, we performed gene set enrichment analysis (GSEA). These collections of gene sets allowed us to analyse the activity of groups of biologically related genes. We applied GSEA to identify the DNs region (Cluster 0) compared with the other cellular regions (without Cluster 0). All of these sets were clearly related to Cluster 0 functions. The sets were (i) an annotated collection of genes involving the regulation of postsynaptic membrane potential (GO-BP) (Fig. 2H); (ii) a biologically annotated collection of genes involved in neuroactive ligand receptor interaction (KEGG) (Fig. 2H); (iii) an annotated collection of genes association with synaptic membrane (e.g., postsynaptic membrane, presynaptic membrane, GABA ergic synapse, and glutamatergic synapse), and ion channel complex (e.g., GABA receptor complex, potassium channel complex, voltage-gated calcium channel complex, and voltage-gated sodium channel complex) (GO-CC) (Supplementary Table 4); and (iv) an annotated collection of genes enriched in ion channel activity (e.g., voltage-gated cation channel activity, extracellular ligand-gated ion channel activity, GABA receptor activity, potassium channel activity, voltage-gated cation potassium channel activity, voltage-gated sodium channel activity, and voltage-gated calcium channel activity [GO-MF]) (Supplementary Table 4). We also applied GSEA to identify the DNs region (Cluster 0) compared with Neurons region (Clusters 4, 9, 10 and 12), Excitatory neurons region (Clusters 4 and 12), and inhibitory neurons region (Clusters 9 and 10), without an appropriate enriched functional gene set to meet the filter criteria.

Taken together, these data yielded unanimous results that the DNs region in FCD IIb is mainly associated with the mTOR signalling pathway, autophagy, and the ubiquitin-proteasome system, which might be the underlying mechanism contributing to the occurrence of abnormal cell morphology in FCD. Moreover, the results suggested that the DNs region may be involved in epileptic discharge via regulating membrane potential.

Identification of the BCs region

We analysed the top 20 genes that were significantly elevated in the BCs region (VIM, CRYAB, EFEMP1, TNC, CLU, IGFBP7, SPARC, GPNMB, PTGDS, MAOB, MGST1, APOE, ANXA1, SERPINA3, AEBP1, GSN, GJA1, C3, CSRP1, and AQP4) (Fig. 3A, Supplementary Fig. 8). Afterwards, we performed differential expression analyses by comparing Cluster 3 with all other clusters. Based on the GO enrichment analysis, we identified 6 major functional modules, including Actin filament, Cell morphogenesis and developmental growth, Neural precursor cell proliferation, Reactive oxygen metabolize, Complement activation, and Inflammatory response (Fig. 3B, Supplementary Table 5). We further demonstrated in detail that Cluster 3 is involved in actin filament organization (GO:0007015; i.e., VIM, CRYAB and GSN), glial cell differentiation (GO:0010001; i.e., VIM, CLU and GSN), reactive oxygen species metabolic process (GO:0072593; i.e., CRYAB, APOE and ANXA1), and developmental growth involved in morphogenesis (GO:0060560; i.e., CLU, IGFBP7, APOE, ANXA1, GSN, and GJA1). Moreover, we observed that the BCs region not only has strong associations with complement activation (GO:0006956; i.e., CLU and C3), regulation of complement activation (GO:0030449; i.e., CLU and C3), but also with effector mechanisms triggered by the complement cascade, such as phagocytosis (GO:0006909; i.e., C3, ANXA1 and GSN), and immune response (i.e., C3, CLU, and SERPINA3), including acute inflammatory response (GO:0002526), regulation of acute inflammatory response (GO:0002673), and regulation of humoral immune response (GO:0002920). Moreover, we next analysed the expression of genes related to complement activation, mostly focused on the BCs region (Fig. 3E). In addition, most of the differentially expressed genes in the BCs region were significantly enriched in Phagosome (hsa04145), Regulation of actin cytoskeleton (hsa04810), Focal adhesion (has04510), Antigen processing and presentation (hsa04612) and Allograft rejection (hsa05330) (Fig. 3C, D). Collectively, these data indicated that the inflammatory response and complement activation may play a role in the pathogenesis of BCs region.

Fig. 3

Identification of the BCs region. (A) Left, the BCs region (Cluster 3) was identified and visualized by using UMAP. Right, UMAP feature plots of expression for BCs-specific genes VIM, C3 and CLU. (B) Dot plot of GO-BP terms for Cluster 3. (C), (D) Bar plots and Gene-Term Network of KEGG terms for Cluster 3. (E) Z-scored mean log expression heatmap of genes associated with complement activation across the annotated cell regions in ST data

Identification of the lesion and perilesion in human FCD IIb

Afterwards, we investigated the differences between lesions and perilesion in human FCD IIb (Fig. 4A). The top 20 differentially expressed genes were significantly elevated in the lesions (Fig. 4B, Supplementary Fig. 9). Of note, the levels of SERPINA3, which is a marker of reactive astrocytes, were significantly elevated in lesions compared with perilesional tissue of FCD IIb. SPARC and CHI3L1 were both related to blood vessel development, and CHI3L1 expression was reported in a unique population of small cells in close proximity to BCs, most likely to be glial progenitors [26]. As a whole, genes involved in nervous system development included GFAP, PLP1, SPP1, VIM, CLDN11, CLU, S100B, SELENOP and CNP. We also identified 7 genes enriched in cell morphogenesis, such as SPARC, SPP1, IGFBP7, FAM107A, S100B, GPNMB, and CNP. Some molecules, such as SERPINA3, SPARC, CHI3L1, PLP1, SPP1, CD74, CRYAB, TAC1, CLU, C3, S100B, and GPNMB, showed reliable associations with the inflammatory response. In addition, genes such as CLU and C3 were involved in complement activation (Supplementary Table 6).

Fig. 4

Differences in the lesions and perilesion in human FCD IIb. (A) The regions of perilesion and lesions were identified and visualized by using UMAP. (B) UMAP plots of representative SERPINA3, SPARC and CHI3L1 gene expression in perilesion and lesions. (C) Dot plot of GO-BP terms for Cluster 0 (DNs region) between the lesions and perilesion. (D) Dot plot of GO-BP terms for Cluster 3 (BCs region) between the lesions and perilesion

We subsequently analysed the functional differences of the DNs or BCs region between the lesions and perilesion in more detail. Based on the GO enrichment analysis (Fig. 4C, D), the DNs and BCs regions exhibited shared functional modules, regulation of cell morphogenesis and developmental growth (i.e., GO:0008360, regulation of cell shape; GO:0032535, regulation of cellular component size; and GO:0060560, developmental growth involved in morphogenesis). Similarly, the differentially expressed genes in the DNs region were enriched in Autophagy (i.e., GO:0016236, macroautophagy; GO:0010506, regulation of autophagy; GO:0016241, regulation of macroautophagy; and GO:0061684, chaperone-mediated autophagy) and Potential (i.e., GO:0042391, regulation of membrane potential; and GO:0098656, anion transmembrane transport). Several functional modules were identified in the BCs region (Fig. 4D), including Inflammatory response (i.e., GO:0150076, neuroinflammatory response; GO:0002269, leukocyte activation involved in inflammatory response; and GO:0002282, microglial cell activation involved in immune response, etc.), Complement activation (i.e., GO:0006956, complement activation; and GO:0030449, regulation of complement activation), Reactive oxygen metabolize (i.e., GO:0072593, reactive oxygen species; and GO:2000377, regulation of reactive oxygen species metabolic process), and Neural precursor cell proliferation (i.e., GO:0061351, neural precursor cell proliferation; and GO:2000177, regulation of neural precursor cell proliferation).

Immunohistochemistry provides protein visualization of the DNs and BCs regions

To understand gene functional enrichment in the DNs and BCs regions of FCD IIb, we performed immunohistochemical staining for representative genes that encoded for proteins in the mTOR signalling pathway, autophagy, the ubiquitin-proteasome system, inflammatory response and complement activation.

Activation of the mTOR pathway has been linked to the cytopathology of FCD. As expected, genes involved in the mTOR signalling pathway, such as PIK3R3, AKT3 and RHEB, were mainly enriched in the DNs region. We also detected phosphorylated ribosomal S6 protein (pS6) expression via immunohistochemistry (IHC). DNs showed a striking cytoplasmic labelling pattern with the pS6 marker, whereas BCs also showed pS6 immunopositivity but were less intensely labelled than the DNs, thus providing evidence for mTOR pathway activation in FCD IIb.

Autophagy dysregulation is partially dependent on mTOR pathway activation. Genes associated with autophagy were mostly expressed in the neuronal regions, not specific to the DNs region in the transcriptome level. Then, we examined the expression of p62, the critical component of the autophagy pathway, in FCD. Prominent cytoplasmic accumulation of p62 protein in the DNs and a proportion of BCs indicated an abnormality of autophagy in FCD II b (Fig. 5B).

Fig. 5

Immunohistochemical staining for representative proteins in FCD IIb. (A) Left, macroscopic HE image of a case (lesion-1) with identification of the DNs region and BCs region. The white line represents the virtual border between the cortical and white matter regions. Right, microscopically, dysmorphic neurons (DNs) and balloon cells (BCs) and relevant markers, including positive pS6 immunostaining for both, positive Nestin immunoreactivity for BCs, and strong SMI-32 immunoreactivity for DNs. (B) Positive p62 immunostaining in the DNs (arrow), BCs (arrowhead) and neurons (triangle) were observed. UCHL1 staining confirming cytoplasmic labelling in the DNs. However, frequent cytoplasmic labelling of C3 and CLU proteins were observed in the BCs

The ubiquitin proteasome system (UPS), which is essential for removing abnormal proteins, cooperates with autophagy to maintain macromolecular homeostasis. Genes involved in the UPS that were mostly specific to the DNs region, such as UCHL1, HSP90AB1, FBXW7, PSMD8, UBR3, UBE2K, FBXO44, RAD23B, PSMC6, PSMD2, PSMB3, USP5, UBQLN1, and PSMD13 (Fig. 2F). UCHL1 is a multifunctional protein expressed in neurons in the brain [30]. We found that UCHL1 is mainly expressed in neurons of mMCD (Supplementary Fig. 10), but significantly aggregates in the cytoplasm of DNs (Fig. 5B), thus suggesting dysregulation of the UPS in FCD IIb, which may contribute to the occurrence of abnormal cell morphology.

Additionally, to identify whether the products of the inflammatory response and complement activation are deposited in the BCs region, we performed immunohistochemical staining for complement C3 (C3) and the complement cascade inhibitor clusterin (CLU). C3 and CLU were expressed in astrocytes of mMCD (Supplementary Fig. 10), but showing a striking cytoplasmic labelling pattern in the BCs (Fig. 5B), indicating existence of inflammatory and anti-inflammatory factors co-regulates the immune inflammatory network in the brain tissues of FCD IIb.