ABSTRACT: Single nucleotide polymorphisms (SNPs) within 1p13.1 region have been identified as significant genetic variations associated with depression, and our prior functional genomics pinpointed a regulatory variant rs3101339 among them. However, its precise roles in depression pathogenesis remains elusive. In this study, we employed series analytical and functional approaches, including regulatory element annotation, brain expression quantitative trait loci (eQTL), reporter gene assay, electrophoretic mobility shift assay (EMSA), and precision gene editing. Our results confirmed that rs3101339 is a causal variant within 1p13.1 with its risk allele C upregulating NEGR1 expression. To further investigate the consequences of NEGR1 upregulation, we overexpressed NEGR1 in specific region of the mouse brain (including medial prefrontal cortex (mPFC) and ventral hippocampus (vHIP)) using stereotaxic injection. Behavioral assessments revealed that elevated NEGR1 levels in the brain, particularly in the vHIP, results in working memory impairing and anxiety- and depression-like behaviors in mice. Neuronal sparse labeling assays and electron microscopy revealed that NEGR1 overexpressing in the vHIP leads to neural spine loss and synaptic ultrastructure abnormality. Immunoprecipitation-mass spectrometry (IP-MS) further identified 67 high-confidence proteins that interact with NEGR1, many of them are involved in neurotransmitter exocytosis and synaptic vesicle endocytosis. Notably, Snap25 emerged as a potential central hub within this protein-protein interaction network. Transcriptomic profiling revealed 94 differentially expressed genes in NEGR1-OE (vHIP) mice compared to control mice (P adj < 0.05). These genes were enriched in myelination-related signaling pathways (such as myelination, ensheathment of neurons, axon ensheathment in central nervous system, etc.). Moreover, several myelination-related genes, such as Itgax, Itgb4, Mag, Mbp, Sox10, Bcas1, Trf, Slc8a3, Myrf, and Plp1, were downregulated in NEGR1-OE (vHIP) mice. Together, our findings implicated that the overexpression of the NEGR1 gene in the mouse brain as a potential driver of anxiety- or depression-like phenotypes potentially through impairing synaptic function and myelination.
