GHK-Cu intranasal delivery rescues cognitive decline in middle-aged mice, while IP shows transient effects

Age-related cognitive decline (ARCD) is a widespread issue driven by conserved aging mechanisms, yet direct brain-targeting gerotherapeutics are lacking. Glycyl-L-histidyl-L-lysine complexed with copper (GHK-Cu) is an endogenous peptide known for regenerative and anti-inflammatory properties, with levels declining with age. Understanding how GHK-Cu's effects on cognitive aging are influenced by delivery route or exposure duration is crucial for its therapeutic potential.

Aged C57BL/6J mice (20-21 months) were treated with GHK-Cu (15 mg/kg) via two routes: short-term intraperitoneal (IP) for 5 days or longer-term intranasal (IN) for 8 weeks. Hippocampal-dependent escape learning was assessed using a spatial navigation task. Molecular effects in the hippocampus were evaluated using immunohistochemistry for markers like synaptophysin and GFAP, and bulk RNA sequencing. Differential gene expression was analyzed with DESeq2 and pathway-level changes via gene set enrichment analysis (GSEA).

Intranasal (IN) GHK-Cu significantly improved escape latency across Trials 2-4 in both sexes (P < 0.05), indicating sustained cognitive benefits. In contrast, intraperitoneal (IP) dosing produced only a transient improvement in males during Trial 2 (P < 0.05) with no sustained effects or improvement in females. Molecularly, IN treatment increased synaptophysin in females (P < 0.001) and decreased GFAP in both sexes (P < 0.01), suggesting enhanced synaptic plasticity and reduced neuroinflammation. IP treatment, however, reduced TGF-β, GFAP, and MCP-1 in males (P < 0.05) and decreased p21 in females (P < 0.0001), pointing to different anti-inflammatory and anti-senescence pathways. Transcriptomic analysis revealed highly divergent molecular programs:

IN GHK-Cu induced coordinated suppression of oxidative phosphorylation (male NES -5.44, female NES -4.20; FDR < 0.0001) and MYC target pathways (female NES -4.31, FDR < 0.0001), with additional attenuation of PI3K-AKT-mTOR signaling in females (NES -3.15, FDR = 0.062). Conversely, IP treatment activated oxidative phosphorylation (female NES 4.97, FDR < 0.001), DNA repair (NES 5.58, FDR < 0.001), and MYC targets (NES 4.34, FDR = 0.002), suggesting engagement of acute stress-response and repair mechanisms.