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A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes
Published on: February 5, 2018
Metformin increases phagocytosis and acidifies lysosomal/endosomal compartments in AMPK-dependent manner in rat
Krzysztof Labuzek1, Sebastian Liber, Bozena Gabryel
1Department of Clinical Pharmacology, Medical University of Silesia, Medyków 18, 40-752 Katowice, Poland. labuzek@rubikon.pl
Abstract:
Recent evidence suggests that metformin shows beneficial effects in experimental models of neuroinflammatory diseases. The aim of the present study was to determine the effect of metformin on phagocytosis and acidification of lysosomal/endosomal compartments in rat primary microglia in the presence of lipopolysaccharide (LPS) and/or beta-peptides (25-35), (1-40), and (1-42). Metformin increased the phagocytosis of fluorescent microspheres in the presence or absence of all the beta-peptides. However, the drug had no effect on the phagocytosis in LPS-stimulated microglia regardless of the presence of all the beta-peptides. Metformin acidified the lysosomal/endosomal compartments in the presence or absence of the beta-peptide 1-40 in both resting and activated microglia. To elucidate the mechanism of metformin action, we used 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside as an activator of adenosine monophosphate-activated protein kinase (AMPK) and compound C as a confirmed pharmacological inhibitor of AMPK. We have shown that metformin increased AMPK activity in microglial cells and that all observed effects are AMPK-dependent because the pretreatment of microglia with compound C reversed the effects of the drug. Since degradation of proteins in lysosomal/endosomal compartments depends largely on their phagocytosis and acidification, metformin may be beneficial in proteinopathies affecting the brain.
Insights
Metformin enhances microglial phagocytosis and lysosomal acidification, crucial for clearing brain proteins. These beneficial effects on neuroinflammation are dependent on adenosine monophosphate-activated protein kinase (AMPK) activation.
Area of Science:
- Neuroscience
- Pharmacology
- Cell Biology
Background:
- Neuroinflammation plays a key role in neurodegenerative diseases.
- Metformin, a common diabetes drug, has shown potential in experimental models of neuroinflammation.
- Microglia are the primary immune cells in the brain and are involved in clearing cellular debris and pathogens.
Purpose of the Study:
- To investigate the effects of metformin on microglial phagocytosis and lysosomal/endosomal acidification.
- To determine the role of adenosine monophosphate-activated protein kinase (AMPK) in mediating metformin's effects.
- To assess metformin's impact in the presence of lipopolysaccharide (LPS) and various beta-peptides associated with neurodegenerative diseases.
Main Methods:
- Primary rat microglia were treated with metformin, LPS, and beta-peptides (25-35, 1-40, 1-42).
- Phagocytosis was measured using fluorescent microspheres.
- Lysosomal/endosomal acidification was assessed using fluorescent probes.
- AMPK activation was studied using an activator (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside) and an inhibitor (compound C).
Main Results:
- Metformin significantly increased microglial phagocytosis of microspheres, independent of beta-peptides.
- Metformin did not enhance phagocytosis in LPS-stimulated microglia.
- Metformin acidified lysosomal/endosomal compartments in both resting and activated microglia.
- Metformin increased microglial AMPK activity.
- All observed effects of metformin were reversed by the AMPK inhibitor compound C, confirming AMPK-dependency.
Conclusions:
- Metformin enhances microglial phagocytosis and lysosomal acidification through an AMPK-dependent mechanism.
- These findings suggest metformin's potential therapeutic value in proteinopathies affecting the brain by improving the clearance of aggregated proteins.
- Metformin's efficacy may be limited in conditions with significant LPS-induced microglial activation.
