TrkB-dependent regulation of molecular signaling across septal cell types

Abstract

The lateral septum (LS), a GABAergic structure located in the basal forebrain, is implicated in social behavior, learning and memory. We previously demonstrated that expression of tropomyosin kinase receptor B (TrkB) in LS neurons is required for social novelty recognition. To better understand molecular mechanisms by which TrkB signaling controls behavior, we locally knocked down TrkB in LS and used bulk RNA-sequencing to identify changes in gene expression downstream of TrkB. TrkB knockdown induces upregulation of genes associated with inflammation and immune responses, and downregulation of genes associated with synaptic signaling and plasticity. Next, we generated one of the first atlases of molecular profiles for LS cell types using single nucleus RNA-sequencing (snRNA-seq). We identified markers for the septum broadly, and the LS specifically, as well as for all neuronal cell types. We then investigated whether the differentially expressed genes (DEGs) induced by TrkB knockdown map to specific LS cell types. Enrichment testing identified that downregulated DEGs are broadly expressed across neuronal clusters. Enrichment analyses of these DEGs demonstrated that downregulated genes are uniquely expressed in the LS, and associated with either synaptic plasticity or neurodevelopmental disorders. Upregulated genes are enriched in LS microglia, associated with immune response and inflammation, and linked to both neurodegenerative disease and neuropsychiatric disorders. In addition, many of these genes are implicated in regulating social behaviors. In summary, the findings implicate TrkB signaling in the LS as a critical regulator of gene networks associated with psychiatric disorders that display social deficits, including schizophrenia and autism, and with neurodegenerative diseases, including Alzheimer's.

Figure 1:. Impact of TrkB knockdown in mouse LS neurons on local gene expression.

(A) Schematic of viral strategy using cre-mediated recombination to locally knockdown TrkB expression in the LS. (B) Volcano plot of differentially expressed genes in bulk RNA-seq of LS tissue samples comparing control versus local TrkB knockdown. Upregulated and downregulated genes were highlighted based on later results (Fig. 5). Summaries of gene ontology analysis for (C) biological processes, (D) molecular functions, and (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) annotated pathways.

Figure 2:. Identification of transcriptionally-defined cell types in mouse LS.

(A) Schematic of experimental design for snRNA-seq of mouse LS tissue. Tissues from 2 mice of the same sex were pooled together for each individual sample for a total of N=4 samples (generated from n=4 male,4 female mice). (B) Uniform manifold approximation and projection (UMAP) of identified cell types, with nuclei counts per clusters. Feature plots for expression of (C) Snap25, (D) Gad2, (E) Slc1a2, and (F) Mbp across the dataset. (G) Heatmap of cell type markers used to characterize the dataset with normalized expression values (logcounts) centered and scaled.

Figure 3:. Characterization of neuronal clusters within mouse LS.

(A) Heatmap of marker genes for various brain regions used to characterize neuronal clusters. Violin plots showing (B) Trpc4, (C) Dgkg, (D) Homer2, and (E) Ptpn3 expression, which were identified as specific markers for LS neurons. (F) Heatmap of identified cell type-specific markers for each of the LS neuronal clusters. Heatmaps show normalized expression values (logcounts) centered and scaled.

Figure 4:. Pseudo-bulking the LS cluster data reveals three groups of neurons.

(A) Principal Component Analysis of LS clusters pseudo-bulked expression profiles across all samples. (B) Heatmap of genes enriched in group 1 (LS_In.C, LS_In.L, and LS_In.N), group 2 (LS_In.D, LS_In.M, LS_In.P, LS_In. Q, and LS_In.R) and previously calculated markers for LS_In.O, Sept_In.G, and Sept_In.I across our neuronal clusters. Normalized expression values (logcounts) centered and scaled. Violin plots of these genes expressed across LS populations for group 1 (C-F) and group 2 (G-J) highlight the specificity of these markers.

Figure 5:. Enrichment of differential expression signals from local TrkB knockdown across snRNA-seq LS dataset.

(A) Enrichment analysis of all, positive, and negative DEGs from the TrkB knockdown dataset across broad cellular clusters. (B) Enrichment analysis of all, positive, and negative DE genes from the TrkB knockdown dataset across all neuronal clusters from the septum. (C) Heatmap of DE genes from the TrkB knockdown dataset enriched in lateral septal and microglial broad clusters across broad clusters. DE genes from the TrkB knockdown dataset are divided into genes unique to the LS, plasticity genes, and neurodevelopmental genes. (D) Heatmap of DE genes from the TrkB knockdown dataset that are enriched in the lateral septal broad cluster across the neuronal clusters. Heatmaps show normalized expression values (logcounts) centered and scaled. Violin plots of (E) Ywhaz, (F) Sgip1, (G) Cnr1, (H) G3bp2, (I) Cpne7, (J) Atcay, (K) Zmat4, and (L) Nxph1 expression, genes with unique LS expression identified from downregulated DE genes from the TrkB knockdown dataset enriched in the LS broad cluster, in addition to Cnr1 expression.