Inseyah Bagasrawala, Nevena Radonjic and Nada Zecevic
Department of Neuroscience, Uconn Health, Farmington, CT, USA
Viral infections in an expecting mother can lead to devastating neurodevelopmental defects and part of the damage is believed to be due to pro-inflammatory cytokines secreted from reactive astrocytes and microglia. Kynurenic acid (KYNA), an antagonist at the glycine-binding site of the NMDA receptor (NMDAR), activates these glial cells. According to the KYNA hypothesis of schizophrenia (Sch), excessive production of KYNA antagonizes NR1, the obligatory subunit of the excitatory glutamate receptor NMDAR, and possibly leads to an excitation-inhibition (E/I) imbalance of cortical circuits. Animal studies point to a possible role of KYNA in generating behavioral deficits (anhedonia, prepulse inhibition), similar to those observed in Sch patients. The NMDAR-antagonizing effects of KYNA are yet to be established in the developing human cerebral cortex. We hypothesize that KYNA will downregulate NMDAR expression on human cortical progenitors, and thereby alter their proliferation and differentiation into neurons and glial cells. We studied the effects of increasing doses of KYNA (0.01 mM, 0.05 mM and 0.1 mM) on NR1 expression and on the proliferation and differentiation of radial glial cells (RGCs) isolated from human fetal cerebral cortex at 17 gestational weeks (gw). RGCs (GFAP+) are multipotent progenitors capable of generating intermediate progenitors (Tbr2+), interneuron progenitors (Nkx2.1+), glutamatergic neurons (Tbr1+), and interneurons (GABA+, Calretinin+). KYNA treatment for 48 hours resulted in a significant and dose-dependent increase in cell death (21.02%) compared to control cultures (8.1%). In addition, cell proliferation measured after 3 days in vitro (DIV) in proliferation medium (using Ki67 as a proliferation marker) was reduced in KYNA treated cultures as compared to controls (p = 0.001). More specifically, the number of GFAP+ astroglia cells from total number of cells was reduced in half (76.25% in controls to 35.06%), similar to a reduction in other progenitors (Tbr2: 72.86% to 38.16%; Nkx2.1: 67.76% to 40.80%). KYNA treatment for 7DIV influenced differentiation into neurons, reducing significantly the number of GABAergic (33.17% to 5.52%) or Calretinin (29.71% to 3.63%) cells, but not glutamatergic neurons (Tbr1: 21.18% to 21.02%). Using immunocytochemistry and Western blotting, we find that the 48 hour KYNA treatment results in a 30% reduction of NR1 expression at 3 DIV cultures in proliferation medium, and a 12% reduction after 7 DIV in differentiation medium, as compared to controls. Furthermore, KYNA treatment resulted in a decrease in NR1 expression on all studied cell types: (RGCs, intermediate progenitors, interneurons and glutamatergic neurons). These results indicate that exposure of the human fetal cerebral cortex to KYNA at midgestation can lead to an imbalance between the number of interneurons and glutamatergic neurons, and a reduction of NMDAR subunit NR1 expression, possibly laying the foundation for an imbalance in cortical circuits during development.
Funding Source: RO1 NSO41489