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Updated: Jun 11, 2025
Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis
Published on: June 23, 2022
BV2 Microglial Cell Activation/Polarization Is Influenced by Extracellular Vesicles Released from Mutated SOD1 NSC-34
Elisabetta Carata1, Marco Muci1, Stefania Mariano1
1Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy.
Abstract:
Microglia-mediated neuroinflammation is a key player in the pathogenesis of amyotrophic lateral sclerosis (ALS) as it can contribute to the progressive degeneration of motor neurons (MNs). Here, we investigated the role of mSOD1 NSC-34 MN-like cell-derived extracellular vesicles (EVs) in inducing the activation of BV2 microglial cells. NSC-34-released EVs were isolated by culture medium differential ultracentrifugation to obtain two fractions, one containing small EVs (diameter < 200 nm) and the other containing large EVs (diameter > 200 nm). BV2 cells were incubated with the two EV fractions for 12, 24, and 48 h to evaluate 1) the state of microglial inflammation through RT-PCR of IL-1β, IL-6, IL-4, and IL-10 and 2) the expression of proteins involved in inflammasome activation (IL-β and caspase 1), cell death (caspase 3), and glial cell recruitment (CXCR1), and presence of the TGFβ cytokine receptor (TGFβ-R2). The obtained results suggest a mSOD1 type-dependent polarization of BV2 cells towards an early neurotoxic phenotype and a late neuroprotective status, with an appearance of mixed M1 and M2 microglia subpopulations. A significant role in driving microglial cell activation is played by the TGFβ/CX3CR1 axis. Therefore, targeting the dysregulated microglial response and modulating neuroinflammation could hold promise as a therapeutic strategy for ALS.
Insights
Extracellular vesicles from motor neurons in amyotrophic lateral sclerosis (ALS) can activate microglia, shifting their state. Targeting this neuroinflammation offers potential therapeutic strategies for ALS.
Area of Science:
- Neuroscience
- Cell Biology
- Immunology
Background:
- Microglia-mediated neuroinflammation is crucial in amyotrophic lateral sclerosis (ALS) pathogenesis.
- Neuroinflammation contributes to motor neuron (MN) degeneration in ALS.
Purpose of the Study:
- Investigate the role of extracellular vesicles (EVs) from mSOD1 NSC-34 MN-like cells in activating BV2 microglial cells.
- Determine the impact of small (<200 nm) and large (>200 nm) EVs on microglial inflammatory responses.
Main Methods:
- Isolated small and large EVs from NSC-34 cell culture medium via differential ultracentrifugation.
- Incubated BV2 cells with EV fractions for 12, 24, and 48 hours.
- Analyzed microglial inflammation (IL-1β, IL-6, IL-4, IL-10), inflammasome activation (IL-1β, caspase 1), cell death (caspase 3), glial recruitment (CXCR1), and TGFβ-R2 expression.
Main Results:
- mSOD1 EVs induced BV2 cell polarization towards an early neurotoxic and late neuroprotective phenotype.
- Observed mixed M1 and M2 microglia subpopulations.
- Identified the TGFβ/CX3CR1 axis as significant in driving microglial activation.
Conclusions:
- mSOD1-derived EVs modulate microglial polarization, influencing neuroinflammation in ALS.
- Targeting dysregulated microglial responses and neuroinflammation presents a promising therapeutic avenue for ALS.
