Isolation and functional characterization of novel neuropeptides regulating hippocampal neurogenesis

Neuropeptides (NPs) are small peptides, produced and secreted by neurons, serving as neuromodulators and chemical messengers that influence neuronal growth, proliferation, and differentiation. Despite their diverse and important roles, the discovery and functional characterization of many NPs remain incomplete. This study aimed to extract and identify novel NPs from rat brain tissue. For this purpose, NPs were purified using gel filtration chromatography and sequenced by mass spectrometry (MS). After characterization, one candidate peptide (NP1) was selected for functional evaluation in primary rat hippocampal neurons. NP1-induced changes were assessed through morphological analysis and protein expression measurements using Western blot and ELISA. Our results revealed that NP treatment markedly enhanced neurite outgrowth and increased neuronal differentiation rate in primary hippocampal cultures compared to the untreated control group. Immunofluorescence analyses showed that NP1 localizes to both the cytoplasm and nucleus of neurons, suggesting its involvement in multiple cellular processes, including signaling, gene regulation, and structural functions. SPR analysis further demonstrated that NP exhibits strong DNA-binding affinity, supporting its potential involvement in transcriptional regulation during neuronal development. Expression analysis confirmed that NP upregulated Cyclin D1, a key regulator of neuronal differentiation, without affecting BAF53A expression. Moreover, NP treatment elevated BDNF and Tau expression levels while reducing SHH expression, suggesting a BDNF-Tau-dependent mechanism. Our results suggest that the characterized NP and its homologs may play roles in neural growth-related disorders such as Rett syndrome, Fragile X syndrome, Down syndrome, and autism spectrum disorder that need further investigation.