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Updated: Apr 23, 2026
Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
α1-antitrypsin modulates microglial-mediated neuroinflammation and protects microglial cells from amyloid-β-induced
Background:
One hallmark of Alzheimer disease is microglial activation. Therapeutic approaches for this neurodegenerative disease include the modulation of microglial cells. α1-antitrypsin (A1AT) has been shown to exert anti-inflammatory effects on macrophages and lung epithelial cells and an inhibition of calpain activity in neutrophil granulocytes. Nothing is known about the effect of A1AT on microglial-mediated neuroinflammation. Our aim was to investigate the effect of A1AT on amyloid-β (Aβ)- and LPS-treated microglial cells in vitro with respect to cytokine production, stress pathways, cell viability, phagocytotic abilities and the underlying mechanisms.
Methods:
Primary microglial cells were isolated from Swiss Webster mouse embryos on embryonic day 13.5. Cytokines in the supernatants of treated primary microglial cells were analyzed with ELISAs, and accumulated nitrite was detected with Griess reagents. Intracellular stress pathways were investigated in cell lysates using western blotting. Intracellular calcium levels were detected in BV-2 microglial cells loaded with the Ca2+-sensitive (fluorescent) dye Fluo-4. Calpain activity in primary microglial cells was assessed by using a calpain activity assay. Cell viability of Aβ-treated microglial cells was analyzed using MTT assay. Phagocytosis of Aβ was evaluated with western blot analysis.
Results:
Upon co-administration, A1AT reduced pro-inflammatory mediators induced by LPS or Aβ. Interestingly, we detected a reduction in calpain activity and in the concentration of intracellular calcium that might mediate the anti-inflammatory effects of A1AT. Inhibition of the classic activation pathways, such as phosphorylation of mitogen-activated protein kinases or activation of protein kinase A were excluded as a mechanism of A1AT-mediated effects. In addition, A1AT increased the viability of Aβ-treated microglial cells and reduced Aβ phagocytosis.
Conclusions:
We provide evidence on the mechanism of action of A1AT on microglial-mediated neuroinflammation in vitro. Our in vitro data indicate that A1AT treatment modulates microglial cells in inflammatory conditions and that this modulation is due to an inhibition of calpain activity and intracellular calcium levels. The underlying mechanisms of the effects observed here are promising for future therapeutic strategies and should thus be further pursued in transgenic mouse models of Alzheimer disease.
Insights
Alpha-1 antitrypsin (A1AT) reduces neuroinflammation in Alzheimer disease models by inhibiting calpain activity and intracellular calcium. This modulation of microglial cells offers promising therapeutic strategies for neurodegenerative diseases.
Area of Science:
- Neuroscience
- Immunology
- Pharmacology
Background:
- Microglial activation is a hallmark of Alzheimer disease (AD).
- Modulating microglial cells is a therapeutic strategy for AD.
- Alpha-1 antitrypsin (A1AT) has anti-inflammatory effects but its impact on neuroinflammation is unknown.
Purpose of the Study:
- Investigate A1AT's effect on amyloid-beta (Aβ) and LPS-treated microglial cells.
- Determine A1AT's impact on cytokine production, stress pathways, cell viability, and phagocytosis.
- Elucidate the mechanisms underlying A1AT's effects on neuroinflammation.
Main Methods:
- Primary microglial cells isolated from mouse embryos.
- Cytokine and nitrite levels measured via ELISA and Griess reagents.
- Western blotting used for stress pathways and Aβ phagocytosis.
- Intracellular calcium and calpain activity assays performed.
Main Results:
- A1AT reduced pro-inflammatory mediators induced by LPS or Aβ.
- A1AT decreased calpain activity and intracellular calcium levels.
- A1AT enhanced microglial cell viability and reduced Aβ phagocytosis.
Conclusions:
- A1AT modulates microglial cells in inflammatory conditions via calpain and calcium inhibition.
- A1AT's mechanism of action in neuroinflammation elucidated in vitro.
- Findings suggest A1AT as a promising therapeutic strategy for AD, warranting further investigation in vivo.
