Sex-specific changes in the hippocampal proteome of Negr1−/− mice: insight into the mechanisms of neuropsychiatric disorders

Neuronal Growth Regulator 1 (NEGR1) is a cell adhesion molecule involved in hippocampal circuit development and function. Human genetic studies have identified NEGR1 variants as risk factors for a broad spectrum of neuropsychiatric disorders. These disorders often display sex-specific differences in prevalence, progression, and behavioral impairment, reflecting underlying maladaptive changes in neural circuitry. Findings from preclinical studies using Negr1−/− mice show several hippocampal-based behavioral and anatomical endophenotypes relevant to neuropsychiatric disorders. The hippocampus, a key region implicated in these disorders, exhibits sex-dependent anatomical features that may shape the functional impact of Negr1. However, the mechanisms driving these sex-specific characteristics have not yet been elucidated. Here, we uncover sex-specific molecular signatures and pathways associated with Negr1, using Negr1−/− mice, a genetically relevant animal model for neuropsychiatric risk. We performed label-free quantitative proteomic analysis using eight replicates of hippocampi dissected from male and female wild-type, and Negr1−/− mice. Differentially abundant proteins were subjected to functional annotation for Gene Ontology and Protein-Protein interaction using STRING analysis. NEGR1 cellular localization was examined by immunofluorescent in rat brain and human hippocampal sections. Differential expression analysis identified 232 proteins in males and 172 in females. STRING analysis revealed sex-specific regulation of proteins. In males, proteins linked to neurofilament organization, myelin integrity, and postsynaptic structure were downregulated, with parvalbumin (Pvalb, PV) around the central node. In contrast, proteins related to mitochondrial and stress-response pathways were upregulated. Female Negr1−/− hippocampus showed downregulation of proteins involved in translation and amide biosynthetic processes. Colocalization of NEGR1 with PV interneurons in the rat brain and the human hippocampus was observed. We demonstrate, for the first time, distinct sex differences in the hippocampal proteome and identify molecular networks in Negr1−/− mice. Co-localization of NEGR1 and PV in human brain tissue provides anatomical and translational validation of a proteomic target. These findings provide new insight, offering a valuable resource for understanding NEGR1-related sex-specific mechanisms in neuropsychiatric disorders. The hippocampus is a key brain structure for learning, memory, and emotional regulation, and its dysfunction is central to several neuropsychiatric disorders, including major depression, schizophrenia, autism spectrum disorders, anxiety, and cognitive decline that are associated with Negr1 gene variants. The prevalence, expression, and course of neuropsychiatric disorders vary substantially between males and females. Neuronal Growth Regulator 1 (Negr1) is a member of the IgLON family of cell adhesion molecules. Negr1 is involved in the formation of neuronal circuits during prenatal and postnatal development. Negr1 deletion (Negr1−/−) in mice causes several abnormalities in the hippocampus, which underlie behavioral changes like those observed in a neuropsychiatric disorder. To understand the protein expression profile, protein-interaction network, and signaling pathways underlying Negr1-associated neurobehavioral pathology, we performed unbiased proteome analysis of the hippocampus from male and female Negr1−/− mice, which has not been done previously. We identified distinct sex-specific, differentially abundant proteins and biological pathways that differ between male and female tissues. We found that NEGR1 is located together with a special type of calming brain cell, known as parvalbumin neurons in rat and human brain, suggesting that they may work closely together in the brain. These would provide important data to further investigate the underlying sex-specific neurobiology of behavioral impairments related to Negr1 pathology.