Microglia are the immune system’s primary enforcers in the brain. They are cells that patrol the brain and eliminate anything hazardous they come across, from invading bacteria to cellular waste. They also eliminate plaques and prune damaged connections between neurons.
Microglia remove their target by consuming them: they encase the material in bubble-like organelles known as phagosomes. A phagosome can then merge with other organelles to degrade its contents.
Microglial phagosomes have critical roles in brain development, brain function, and a wide range of brain disorders, including neurodegeneration and cancer. As a result, understanding microglial phagosome biology could aid in the development of new treatments for currently incurable brain illnesses.
However, because existing stem cell and animal models do not adequately mimic microglia in the human brain, studying microglia and their organelles has proven challenging. Additionally, because microglia are watchful immune patrollers that react to even minute stimuli, experimental conditions can cause changes in the cells that obscure analyses.
Immunoprecipitation in Phagosome Biology
Rudolf Jaenisch, the founder of the Whitehead Institute and a biology professor at the Massachusetts Institute of Technology, Marco Prinz, a professor of neuropathology at the University of Freiburg and Emile Wogram, a neuropathologist at the University of Freiburg who started this project as a postdoctoral researcher in Jaenisch’s lab, have developed a method to quickly and gently analyze microglia phagosomes in order to overcome those challenges.
In a new research paper, they describe how they can isolate and profile phagosomes from stem cell-derived microglia and fresh human brain tissue. They also share new insights into phagosome biology in the human brain, regarding synaptic pruning and generation of NAD+, a broadly used molecule in the brain, by microglia.
The method that the researchers developed to isolate phagosomes from cells uses immunoprecipitation, in which antibodies latch on to a specific target protein on an organelle’s surface. When antibodies are collected, they drag the organelles with them.
This method avoids various chemical perturbations that could affect the microglial profile. Researchers may genetically construct antibody targets, but in order to isolate phagosomes from human brain tissue, Wogram needed to locate a naturally expressed target. Finally, he and his colleagues discovered one: the protein CD68.
Sensitive Signaling Molecules
The researchers initially extracted phagosomes from stem cell-derived microglia. They co-cultured the microglia with other brain cell types to generate a more brain-like environment, resulting in a better match of brain and stem cell-derived microglia gene expression.
They triggered some of the microglia to enter an inflammatory or disease-like state to see how that affected the phagosomes. Additionally, Wogram collaborated with the neurosurgery department at the University of Freiburg to get access to brain tissues immediately after their removal during surgery. He isolated phagosomes from brain tissue within a half hour of its removal, allowing him to profile the organelles before their contents could change much.
The researchers created profiles that included the proteins and metabolites found in the phagosomes, as well as the whole-cell gene expression profile. The profiles varied greatly between sets of phagosomes, but the researchers discovered a core of constant proteins, including many known and new phagosome proteins.
The results showed that phagosomes contain sensitive signaling molecules that allow them to react quickly to even subtle environmental stimuli.
Quinolinic Acid Pathway
Furthermore, the protein levels of the co-cultured microglia gave strong evidence that when microglia prune synapses, they preferentially prune the side that delivers the signal rather than the side that receives it. This finding could help us understand how microglia interact with synapses in health and illness.
The researchers also gained insights into a key metabolic pathway that occurs inside of microglia. In excess, the molecule quinolinic acid can be toxic to neurons; it is implicated as involved in many neurodegenerative diseases. However, cells can use quinolinic acid to make NAD+, a molecule broadly used to carry out essential cellular functions.
Microglia are the only brain cells that generate NAD+. Wogram and colleagues found that key steps in this process occur in phagosomes. Phagosomes are therefore necessary both for removing excess quinolinic acid to prevent toxicity and for helping to generate NAD+ in the brain.
Tumor and Healthy Tissue
Wogram used brain tissues to compare phagosomes from within a tumor to those in the surrounding healthy tissue. The phagosomes in the tumor contained excess quinolinic acid.
Although follow-up studies would be needed to confirm the results, these findings are consistent with research that suggests cancer cells use quinolinic acid to fuel their growth.
Collectively, these discoveries shed light on phagosome biology and the potential functions of phagosomes in normal brain growth and maintenance, as well as cancer and neurodegeneration. The researchers also believe that this method could be beneficial for characterizing other organelles, particularly when they need to be quickly separated from human tissue.
Reference:
- Emile Wogram et al. Rapid phagosome isolation enables unbiased multiomic analysis of human microglial phagosomes. Immunity (2024). DOI: 10.1016/j.immuni.2024.07.019
Image: Volume-rendered confocal image depicting a microglia (blue) and its phagosome (red) containing phagocytosed synaptic material (magenta). Credit: Emile Wogram