In this study, we aimed to define ageing-related changes in microglial autofluorescence in the hippocampus and ARC, and whether these were associated with spectral and lifetime signatures that can be identified with plasma lipid profiles. First, we established that our mice had a typical ageing brain phenotype. As shown in previous literature, this is marked by reduced numbers of immature neurons, greater microglia numbers, and greater tissue autofluorescence in the brain (Culig et al. 2022 ; Wong 2013), all of which we recapitulate here. Similar to previous studies in rodents and humans, we also see ageing-related changes to central expression of genes indicative of disease-associated states, or at least inflammaging, including genes relating to inflammation (Itgam, Tlr4, Mhc-II, Aif1), Alzheimer’s disease (Sorl1, Mapt, Abca7), and lipid pathways (Hexb, Ch25h, Lpl) (Boche and Gordon 2022 ; Kim 2018 ; Long et al. 2022).
In association with this typical and well-established ageing-brain profile, we revealed that ageing brains have a propensity to accumulate larger-sized aggregates in all hippocampal regions assessed (DG, CA1, and CA3) and the ARC. When assessing the emission spectra of autofluorescence aggregates of varying sizes in young and aged brains, we identified consistent eight spectral peaks common to both age groups. Notably, the red-shifted intensity ratio we observed consistently distinguished aged from young brains, suggesting the potential utility of an overall emission spectral shift as a future spectral biomarker of ageing. To our knowledge, this is the first study to systematically compare spectral autofluorescence signatures between young and aged brains. We also found that aged tissue exhibited consistently shorter intensity-weighted average lifetimes across all three detection windows compared with young tissue, indicating altered autofluorescence decay dynamics with age. Supporting this interpretation, a previous study examining autofluorescent inclusions in female APPswe: PS1dE9 transgenic mice reported that both plaque-surrounding and plaque-free cells displayed shorter autofluorescence lifetimes than their wild-type counterparts (Gómez et al. 2018). This suggests that changes in autofluorescence lifetime reflect systemic neurodegenerative physiological alterations and are not solely attributable to the presence of disease-specific markers such as amyloid-beta plaques.
One working hypothesis is that these differences likely reflect fundamental physiological changes that accompany ageing. With advancing age, increasing iron accumulation promotes reactive oxygen species formation, which in turn drives lipid peroxidation and the buildup of cross-linked, undegradable biomaterials within lysosomal and autophagosomal compartments of post-mitotic cells (as reviewed in (Yin 1996)). Defective lysosomal acidification–a hallmark of both ageing and neurodegenerative diseases such as Alzheimer’s (Lo and Zeng 2023)–further impairs autophagic clearance, exacerbating the accumulation of autofluorescent material (Escrevente et al. 2021). Collectively, these processes may illustrate how ageing-related alterations in cellular maintenance mechanisms, particularly lysosomal and autophagic dysfunction, can create a self-perpetuating cycle of oxidative stress and impaired degradation that underlies the pathogenesis of ageing-related diseases.
