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Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions
Published on: October 21, 2017
Citrullinated isomer of myelin basic protein can induce inflammatory responses in astrocytes
Marika Chikviladze1, Nino Mamulashvili1, Maia Sepashvili1,2
1Institute of Chemical Biology, Ilia State University, Tbilisi, Georgia.
Purpose:
During the course of demyelinating inflammatory diseases, myelin-derived proteins, including myelin basic protein(MBP), are secreted into extracellular space. MBP shows extensive post-translational modifications, including deimination/citrullination. Deiminated MBP is structurally less ordered, susceptible to proteolytic attack, and more immunogenic than unmodified MBP. This study investigated the effect of the deiminated/citrullinated isomer of MBP(C8) and the unmodified isomer of MBP(C1) on cultured primary astrocytes.
Methods:
MBP charge isomers were isolated/purified from bovine brain. Primary astrocyte cultures were prepared from the 2-day-old Wistar rats. For evaluation of glutamate release/uptake a Fluorimetric glutamate assay was used. Expression of peroxisome proliferator-activated receptor-gamma(PPAR-γ), excitatory amino acid transporter 2(EAAT2), the inhibitor of the nuclear factor kappa-B(ikB) and high mobility group-B1(HMGB1) protein were assayed by Western blot analysis. IL-17A expression was determined in cell medium by ELISA.
Results:
We found that MBP(C8) and MBP(C1) acted differently on the uptake/release of glutamate in astrocytes: C1 increased glutamate uptake and did not change its release, whereas C8 decreased glutamate release but did not change its uptake. Both isomers increased the expression of PPAR-γ and EAAT2 to the same degree. Western blots of cell lysates revealed decreased expression of ikB and increased expression of HMGB1 proteins after treatment of astrocytes by C8. Moreover, C8-treated cells released more nitric oxide and proinflammatory IL-17A than C1-treated cells.
Conclusions:
These data suggest that the most immunogenic deiminated isomer C8, in parallel to the decreases in glutamate release, elicits an inflammatory response and enhances the secretion of proinflammatory molecules via activation of nuclear factor kappa B(NF-kB).
Summary Statement:
The most modified-citrullinated myelin basic protein charge isomer decreases glutamate release, elicits an inflammatory response and enhances the secretion of proinflammatory molecules via activation of nuclear factor kappa B in astrocytes.
Insights
The citrullinated myelin basic protein (MBP) isomer C8, unlike the unmodified C1, decreases glutamate release and triggers inflammation in astrocytes by activating nuclear factor kappa B (NF-kB). This highlights the immunogenic nature of modified MBP in demyelinating diseases.
Area of Science:
- Neuroimmunology
- Cellular Neuroscience
- Protein Chemistry
Background:
- Myelin basic protein (MBP) undergoes post-translational modifications like deimination/citrullination during inflammatory demyelinating diseases.
- Deiminated MBP is more immunogenic and structurally altered compared to unmodified MBP.
- Astrocytes play a crucial role in neuroinflammation and maintaining the brain's microenvironment.
Purpose of the Study:
- To investigate the differential effects of citrullinated MBP (C8) and unmodified MBP (C1) on primary astrocyte functions.
- To elucidate the molecular mechanisms underlying the inflammatory response induced by modified MBP.
Main Methods:
- Isolation and purification of MBP charge isomers (C1 and C8) from bovine brain.
- Primary astrocyte cultures from Wistar rats.
- Assessment of glutamate uptake/release using a fluorimetric assay.
- Western blot analysis for PPAR-γ, EAAT2, ikB, and HMGB1 protein expression.
- ELISA for IL-17A quantification in cell medium.
Main Results:
- MBP C8 decreased glutamate release, while C1 increased glutamate uptake.
- Both MBP isomers upregulated PPAR-γ and EAAT2 expression.
- MBP C8 treatment led to decreased ikB and increased HMGB1 expression in astrocytes.
- C8-treated astrocytes exhibited increased nitric oxide and IL-17A secretion compared to C1-treated cells.
Conclusions:
- The immunogenic deiminated MBP isomer C8 elicits an inflammatory response in astrocytes.
- MBP C8 enhances the secretion of proinflammatory molecules, including IL-17A.
- Activation of nuclear factor kappa B (NF-kB) is implicated in the inflammatory effects of MBP C8.
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