Exploring Microglial Ageing: Insights from Human Genomic Data, Pre-Clinical Animal Models, and the Future of In Vitro Modelling

Neurodegenerative diseases (NDs), including Alzheimer’s disease (AD) and Parkinson’s disease (PD), have profound global impacts that are expected to increase due to population aging. Understanding how age-related changes in brain cells contribute to susceptibility to ND is critical, specifically changes within the CNS immune system. Microglia, the resident immune cells of the brain, play a central role in initiating inflammatory responses and have been linked to ND pathology. Whilst microglia have diverse roles in development and neuroinflammation, the current understanding of microglial ageing is incomplete, and methods for assessing microglia remain inconsistent. Critically, research models of ND fail to consider age, potentially contributing to the poor translational ability of results. The current thesis addresses these gaps in our understanding of aged microglia and the importance of considering age in ND research. Chapter 1 reviews existing literature on ageing models and the overall consideration of age in both in vitro and in vivo ND research and underscores the need to include age-appropriate models to recapitulate neurodegenerative environments in aged patients. Chapter 2 examines microglial ageing with specific reference to the 12 prescribed hallmarks of ageing, providing insight beyond inflammation into the cellular and molecular changes in microglia during ageing. Chapter 3 evaluates methods of microglial quantification in a human PD cohort and a rat TBI cohort. This chapter compares quantification performance of both the Halo automated microglia module and ImageJ automated analysis to manual analysis, demonstrating strong correlations between the methods in human post-mortem tissue, with Halo offering significant workflow efficiencies for microglial analysis over an automated ImageJ alternative. Chapter 4 presents a cross-species analysis of microglia across ageing in the lissencephalic rat brain compared to the gyrencephalic sheep brain. The animals utilised in this study reflect humans approximately around 45-50 years old, and thus, while not an exploration of aged microglia specifically, the findings of this study provide insight into subtle changes in microglia at a critical midlife period. Chapter 5 utilises a publicly available human gene expression database to analyse microglia-related gene expression across healthy aging, AD and traumatic brain injury (TBI). Findings suggest that TBI may exacerbate age-related changes in microglia, revealing a potential interaction between injury and ageing in microglia. Finally, Chapter 6 investigates phenotypic changes in the BV2 microglial-like cell line, exploring the potential of immortalized cells to mimic the characteristics of aged microglia. This chapter explores a novel approach in modelling microglial aging in commonly used cell lines, offering an alternative avenue to animal models and primary cell culture models, where successfully obtaining an “aged” cohort may prove challenging. Overall, this thesis expands our understanding of microglial ageing, emphasising the need for consistent methodologies and appropriate models in ND research. The insights into microglia across species has particular relevance for assessing the translation of research into humans and the exploration of potential in vitro models presents an efficient alternative for the study of molecular mechanisms in the aged brain. Ultimately, this work lays the foundation for future studies to explore the contribution of microglia in ageing and in NDs more broadly.