Effect of quinolinic acid on human astrocytes morphology and functions: implications in Alzheimer's disease

Abstract

The excitotoxin quinolinic acid (QUIN) is synthesized through the kynurenine pathway (KP) by activated monocyte lineage cells. QUIN is likely to play a role in the pathogenesis of several major neuroinflammatory diseases including Alzheimer's disease (AD). The presence of reactive astrocytes, astrogliosis, increased oxidative stress and inflammatory cytokines are important pathological hallmarks of AD. We assessed the stimulatory effects of QUIN at low physiological to high excitotoxic concentrations in comparison with the cytokines commonly associated with AD including IFN-gamma and TNF-alpha on primary human astrocytes. We found that QUIN induces IL-1beta expression, a key mediator in AD pathogenesis, in human astrocytes. We also explored the effect of QUIN on astrocyte morphology and functions. At low concentrations, QUIN treatment induced concomitantly a marked increase in glial fibrillary acid protein levels and reduction in vimentin levels compared to controls; features consistent with astrogliosis. At pathophysiological concentrations QUIN induced a switch between structural protein expressions in a dose dependent manner, increasing VIM and concomitantly decreasing GFAP expression. Glutamine synthetase (GS) activity was used as a functional metabolic test for astrocytes. We found a significant dose-dependent reduction in GS activity following QUIN treatment. All together, this study showed that QUIN is an important factor for astroglial activation, dysregulation and cell death with potential relevance to AD and other neuroinflammatory diseases.

Figure 1

(A) Ethidium bromide-stained gel showing mRNA expression of GS and GAPDH 24 hrs after cytokine or QUIN treatments. Results are expressed as a ratio of specific gene expression normalized against expression of the housekeeping gene GAPDH. Standard errors were always 5%. (B) Glutamine synthetase activity after 24 hours stimulation with QUIN. GS enzyme assay has been done in triplicate (P < 0.05 was taken as significant).

Figure 2

Expression of VIM, GFAP and IL-1β genes following 24 hours stimulation with cytokine/growth factor and QUIN. Real-time PCR results for VIM (a and b), GFAP (c and d), IL-1β (e and f). Each experiment repeated, n = 3. A statistical value of P < 0.05 was taken as significant.

Figure 3

Quantification of GFAP and vimentin using ELISA after 24 hours stimulation with IFN-γ, TNF-α, TGF-α and QUIN. GFAP and VIM indirect ELISA have been done in triplicates. All samples except IFN-γ stimulated VIM protein expression and 50 nM QUIN stimulated GFAP expression were statistically significant (p < 0.05).

Figure 4

Proliferation measurement after stimulation with QUIN for 96 hours, SF = serum free, 5% FCS = normal control and 10% FCS = positive control. Statistical values of P < 0.05 = *, P < 0.01 = ** and P < 0.001 = *** were taken as significant. Proliferation assay was optimized at 96 hours and was done in triplicates. The normal control (5% FCS) was set to 0%.