Alzheimer disease (AD) is associated with age-related synaptic degeneration, neuroinflammation, and impaired autophagy. The present study aimed to investigate the age-dependent neuroprotective effects of relaxin-2 (RLN2) in amyloid-beta 1-42 (Aβ1 - 42)-induced AD-like pathology and hypothesised that RLN2 would provide greater therapeutic benefit in young than aged mice. Young (3-4 months) and aged (12-14 months) mice were intracerebrally (i.c.) injected with amyloid-β (Aβ1 - 42) to develop an AD-like phenotype and treated with RLN2 or a control. Western blot analysis of hippocampal tissues was performed to assess the expression levels of ionised calcium-binding adaptor molecule 1 (Iba1) (microglial activation marker), NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome components, and mechanistic target of rapamycin (mTOR) pathway activity. Neuroinflammation was determined by interleukin-1 beta (IL-1β) and tumour necrosis factor-alpha (TNF-α), while synaptic integrity was assessed by synaptophysin and PSD-95. The Morris Water Maze (MWM) and Novel Object Recognition (NOR) tests were used to measure cognitive and memory performance. Neuroinflammation markers IL-1β and TNF-α were significantly elevated in Aβ1 - 42-injected mice, with aged mice showing a greater increase than young mice. RLN2 treatment markedly reduced IL-1β and TNF-α levels in young mice, whereas the reduction was limited in aged mice. RLN2 treatment had a significant inhibitory effect on hippocampal NLRP3 signalling, reduced mTOR signalling, modulated autophagy-related protein expression, preserved synaptic proteins, and improved cognitive and memory performance (decrease in the MWM escape latency and increase in the NOR discrimination index). RLN2 was not effective in the aged mice, and there was persistent inflammatory activation, impaired autophagy, minimal improvement in synaptic recovery, and a moderate improvement in cognition. These findings suggest that RLN2 could be a potential therapeutic candidate for early-stage AD by targeting neuroinflammation, mTOR-mediated autophagy-related dysregulation, and synaptic loss, while also potentially providing a basis for age-based stratification of treatment responsiveness; however, further studies are required to determine its utility as a biomarker of treatment response or disease progression. Overall, RLN2 exerted neuro-protective effects in i.c. Aβ1 - 42-induced AD-like pathology in an age-dependent manner.
