• The Korean Journal of Physiology and Pharmacology
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• v.1 no.3
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• pp.285-296
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• 1997

Injury to brain transforms resting astrocytes to their reactive form, the hallmark of which is an increase in glial fibrillary acidic protein (GFAP), the major intermediate filament protein of their cell type. The overall glial response after brain injury is referred to as reactive gliosis. Glial-neuronal interaction is important for neuronal migration, neurite outgrowth and axonal guidance during ontogenic development. Although much attention has been given to glial regulation of neuronal development and regeneration, evidences also suggest a neuronal influence on glial cell differentiation, maturation and function. The aim of the present study was to analyze the effects of glial-hippocampal neuronal co-culture on GFAP expression in the co-cultured astrocytes. The following antibodies were used for double immunostaining chemistry; mouse monoclonal antibodies for confirm neuronal cells, rabbit anti GFAP antibodies for confirm astrocytes. Primary cultured astrocytes showed the typical flat polygonal morphology in culture and expressed strong GFAP and vimentin. Co-cultured hippocampal neurons on astrocytes had phase bright cell body and well branched neurites. About half of co-cultured astrocytes expressed negative or weak GFAP and vimentin. After 2 hour glutamate (0.5 mM) exposure of glial-neuronal co-culture, neuronal cells lost their neurites and most of astrocytes expressed strong CFAE and vimentin. In Western blot analysis, total GFAP and vimentin contents in co-cultured astrocytes were lower than those of primary cultured astrocytes. After glutamate exposure of glial-neuronal co-culture, GFAP and vimentin contents in astrocytes were increased to the level of primary cultured astrocytes. These results suggest that neuronal cell decrease GFAP expression in co-cultured astrocytes and hippocampal neuronal-glial co-culture can be used as a reactive gliosis model in vitro for studying GFAP expression of astrocytes.

• Biomolecules & Therapeutics
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• v.31 no.1
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• pp.127-138
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• 2023

Glycogen synthase kinase-3β (GSK-3β) is an important serine/threonine kinase that implicates in multiple cellular processes and links with the neurodegenerative diseases including Alzheimer's disease (AD). In this study, structure-based virtual screening was performed to search database for compounds targeting GSK-3β from Enamine's screening collection. Of the top-ranked compounds, 7 primary hits underwent a luminescent kinase assay and a cell assay using human neuroblastoma SH-SY5Y cells expressing Tau repeat domain (Tau_RD) with pro-aggregant mutation ΔK280. In the kinase assay for these 7 compounds, residual GSK-3β activities ranged from 36.1% to 90.0% were detected at the IC₅₀ of SB-216763. In the cell assay, only compounds VB-030 and VB-037 reduced Tau aggregation in SH-SY5Y cells expressing ΔK280 Tau_RD-DsRed folding reporter. In SH-SY5Y cells expressing ΔK280 Tau_RD, neither VB-030 nor VB-037 increased expression of GSK-3α Ser21 or GSK-3β Ser9. Among extracellular signal-regulated kinase (ERK), AKT serine/threonine kinase 1 (AKT), mitogen-activated protein kinase 14 (P38) and mitogenactivated protein kinase 8 (JNK) which modulate Tau phosphorylation, VB-037 attenuated active phosphorylation of P38 Thr180/ Tyr182, whereas VB-030 had no effect on the phosphorylation status of ERK, AKT, P38 or JNK. However, both VB-030 and VB-037 reduced endogenous Tau phosphorylation at Ser202, Thr231, Ser396 and Ser404 in neuronally differentiated SH-SY5Y expressing ΔK280 Tau_RD. In addition, VB-030 and VB-037 further improved neuronal survival and/or neurite length and branch in mouse hippocampal primary culture under Tau cytotoxicity. Overall, through inhibiting GSK-3β kinase activity and/or p-P38 (Thr180/Tyr182), both compounds may serve as promising candidates to reduce Tau aggregation/cytotoxicity for AD treatment.