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Lipofuscin-like autofluorescence within microglia and its impact on studying microglial engulfment
Jacob M Stillman1,2, Francisco Mendes Lopes1, Jing-Ping Lin3
1Department of Neurobiology, Brudnick Neuropsychiatric Research Institute, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA.
Abstract:
Engulfment of cellular material and proteins is a key function for microglia, a resident macrophage of the central nervous system (CNS). Among the techniques used to measure microglial engulfment, confocal light microscopy has been used the most extensively. Here, we show that autofluorescence (AF) likely due to lipofuscin (lipo-AF) and typically associated with aging, can also be detected within microglial lysosomes in the young mouse brain by light microscopy. This lipo-AF signal accumulates first within microglia and it occurs earliest in white versus gray matter. Importantly, in gray matter, lipo-AF signal can confound the interpretation of antibody-labeled synaptic material within microglia in young adult mice. We further show that there is an age-dependent accumulation of lipo-AF inside and outside of microglia, which is not affected by amyloid plaques. We finally implement a robust and cost-effective strategy to quench AF in mouse, marmoset, and human brain tissue.
Insights
Microglia engulf cellular material, but autofluorescence (AF) in young mouse brains can mimic this. This lipofuscin-associated AF (lipo-AF) interferes with studies of microglial function and aging.
Area of Science:
- Neuroscience
- Cell Biology
- Immunology
Background:
- Microglia, the central nervous system's (CNS) resident macrophages, are crucial for clearing cellular debris and proteins.
- Confocal light microscopy is a primary technique for studying microglial engulfment.
- Autofluorescence (AF), particularly lipofuscin-associated AF (lipo-AF), is typically linked to aging.
Purpose of the Study:
- To investigate the presence and impact of lipofuscin-associated autofluorescence (lipo-AF) in microglia, especially in young brains.
- To determine if lipo-AF confounds the assessment of microglial engulfment in microscopy studies.
- To develop a method for quenching AF in brain tissue.
Main Methods:
- Light microscopy was used to detect lipo-AF in microglial lysosomes in young mouse brains.
- Comparative analysis of lipo-AF distribution in gray versus white matter.
- Assessment of lipo-AF accumulation in relation to age and amyloid plaques.
- Development and implementation of an AF quenching strategy.
Main Results:
- Lipo-AF is present in microglial lysosomes of young mice, accumulating first in white matter.
- Lipo-AF can be misinterpreted as antibody-labeled synaptic material in gray matter microglia.
- Lipo-AF accumulates with age, both within and outside microglia, independent of amyloid plaques.
- A cost-effective AF quenching method was successfully applied to mouse, marmoset, and human brain tissue.
Conclusions:
- Autofluorescence, specifically lipo-AF, is a significant confounding factor in microscopy-based studies of microglial engulfment, even in young animals.
- Understanding and mitigating lipo-AF is essential for accurate research on microglial function, aging, and neurodegenerative diseases.
- The developed AF quenching strategy offers a valuable tool for improving the reliability of brain tissue analysis across species.
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