The Role of Microglia in Neuroinflammation: Metabolic and Gender Differences in Aging

Research Background

Aging is a major risk factor for neurodegenerative diseases such as Alzheimer’s Disease (AD) and Parkinson’s Disease. Even healthy aging is accompanied by a decline in cognitive function. Previous research has indicated that microglia undergo sex-specific changes during aging. Therefore, understanding the functional changes in microglia during aging and the differences between genders is particularly important. Microglia, as resident macrophages in the brain, play a critical role in maintaining brain homeostasis, clearing debris, and repairing damage. During brain aging, they may exhibit functional abnormalities and are considered a key factor in the development of neurodegenerative diseases.

Paper Information

This study, conducted by Seokjo Kang et al., was published in 2024 in the Journal of Neuroinflammation, titled “Microglia undergo sex-dimorphic transcriptional and metabolic rewiring during aging.” The research was a collaborative effort among institutions including Cedars-Sinai Medical Center, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, and University of Southern California, with Helen S. Goodridge serving as the corresponding author.

Research Objective

The study aimed to explore the transcriptomic and metabolomic changes in microglia during aging and to analyze the differences between genders in these changes. By revealing the gender-specific responses of microglia in the context of aging and neurodegenerative diseases, the study seeks to provide a theoretical basis for the development of new clinical interventions.

Research Methods

This study employed various experimental methods and techniques to dissect the transcriptional and metabolic changes in microglia during aging. The main research methods included RNA sequencing, single-cell RNA sequencing analysis, metabolomic analysis, Western blotting, and real-time metabolic rate measurement.

Animal Model

The study used C57BL/6 mice obtained from The Jackson Laboratory and the National Institute on Aging (NIA). Experiments were conducted on male and female mice aged 3 to 24 months. All experimental procedures were carried out at the animal facilities of Cedars-Sinai Medical Center and were approved by the Institutional Animal Care and Use Committee.

Microglia Isolation

Mice were perfused with Calcium-free and Magnesium-free Hank’s Balanced Salt Solution (HBSS) to avoid exogenous activation, and antibiotics and protease inhibitors were used during brain tissue dissociation. After enzymatic digestion of brain tissue using a neural tissue dissociation kit, CD11b+ microglia were isolated using immunomagnetic beads for further experimental processing.

RNA Sequencing and Analysis

Total RNA from microglia was extracted, and reverse transcription and double-stranded cDNA synthesis were performed using the Smart-Seq v4 Ultra Low Input RNA Kit. Library preparation was done with the Nextera XT Library Prep Kit. RNA samples were sequenced on the NovaSeq 6000 system. Sequence reads were aligned using STAR software, and differential expression and pathway enrichment analyses were conducted using DESeq2 and pathway analysis tools.

Research Results

Transcriptomic Changes

RNA sequencing analysis revealed that aging significantly impacts gene expression in microglia, particularly in female mice. Principal component analysis and multidimensional scaling showed significant separation between young and aged microglia, especially in females. Further analysis indicated that there are 41 gender-related genes when age is considered as a covariate, and 4290 age-related genes when gender is considered as a covariate.

Metabolic Pathway Changes

IPA analysis showed significant activation of the PI3K/AKT/mTOR signaling pathway in aged microglia, especially in female mice. The activation of the mTOR pathway was associated with increased glycolysis. These metabolic pathway changes were further validated by Western blotting and Seahorse XF analysis. Results indicated that glycolysis significantly increases in microglia with aging, a metabolic reprogramming that is more pronounced in female mice.

Infection and Immune Response

We observed a significant enhancement of complement pathway activity in aged female mice. Specifically, the expression of C3 and its receptor C3aR increased significantly in aged microglia, suggesting that this change may promote the activation of the mTOR/HIF-1α signaling pathway through an autocrine mechanism, further enhancing glycolysis and phagocytic activity.

Single-Cell RNA Sequencing Analysis

Single-cell RNA sequencing data indicated that disease-associated microglia (DAM) are more abundant in females. These DAMs exhibited higher glycolytic activity, suggesting they may play an important role in debris clearance and neuroprotection.

Conclusion

This study reveals transcriptional and metabolic reprogramming of microglia during aging, with notable gender differences. Particularly, the complement system and the mTOR/HIF-1α signaling pathway play important roles in this process. These findings not only expand our understanding of the role of microglia in aging and neurodegenerative diseases but also provide potential directions for the development of new therapeutic strategies. Targeting the autocrine C3a-C3aR signaling