Brain endothelial tmTNFα overexpression in a mouse model altered cell type-specific signaling changes linked to...

Genes and coexpression networks related to immune and inflammatory responses are upregulated in blood and multiple brain regions of individuals with schizophrenia, yet their mechanistic contribution to disease pathophysiology remains unclear. We previously identified transmembrane TNFα (tmTNFα) in brain endothelial cells (ECs) as a potential regulator of immune-related gene networks in schizophrenia and showed that short-term overexpression of an uncleavable tmTNFα (uc-tmTNFα) in mouse brain ECs induces schizophrenia-relevant behaviors via TNF receptor 2 (TNFR2). To investigate the molecular mechanisms underlying the behaviors, we performed biochemical and transcriptomic analyses in mice with short-term (4 weeks) or long-term (28 weeks) tmTNFα expression, and in TNFR2 knockout mice. In the short-term model (STM), phosphorylation of TNFR2 signaling-associated proteins (AKT, IκBα, ERK, JNK) was increased in astrocytes, while cGMP levels were reduced in ECs and neurons-effects absent in TNFR2 knockouts. Long-term expression (LTM) exacerbated schizophrenia-like behaviors, further reduced cGMP levels, and increased neuronal ERK phosphorylation. Single-cell transcriptomics revealed temporally distinct, cell type-specific transcriptional responses. STM upregulated TNFR2 signaling genes in astrocytes and downregulated cGMP-PKG and synaptic genes in neurons. In contrast, LTM upregulated TNF signaling genes in ECs and downregulated autophagy and mTOR pathway genes in glial cells. Western blot analysis of autophagy markers and sGC activity assays confirmed cell type specific alterations in autophagic flux and cGMP production. Together, our data show that tmTNFα expression in brain ECs induces transcriptional and signaling changes that evolve from acute astrocyte and synaptic disruption to chronic immune activation, metabolic dysregulation, and altered post-translational regulation. These alterations may contribute to schizophrenia-relevant phenotypes.