Signaling mechanisms downstream of quinolinic acid targeting the cytoskeleton of rat striatal neurons and astrocytes

AbstractThe studies of signaling mechanisms involved in the disruption of the cytoskeleton homeostasis were performed in a model of quinolinic acid (QUIN) neurotoxicity in vitro. This investigation focused on the phosphorylation level of intermediate filament (IF) subunits of astrocytes (glial fibrillary acidic protein — GFAP) and neurons (low, medium and high molecular weight neurofilament subunits — NFL, NFM and NFH, respectively). The activity of the phosphorylating system associated with the IFs was investigated in striatal slices of rat exposed to QUIN or treated simultaneously with QUIN plus glutamate receptor antagonists, calcium channel blockers or kinase inhibitors. Results showed that in astrocytes, the action of 100μM QUIN was mainly due to increased Ca2+ influx through NMDA and L-type voltage-dependent Ca2+ channels (L-VDCC). In neuronal cells QUIN acted through metabotropic glutamate receptor (mGluR) activation and influx of Ca2+ through NMDA receptors and L-VDCC, as well as Ca2+ release from intracellular stores. These mechanisms then set off a cascade of events including activation of PKA, PKCaMII and PKC, which phosphorylate head domain sites on GFAP and NFL. Also, Cdk5 was activated downstream of mGluR5, phosphorylating the KSP repeats on NFM and NFH. mGluR1 was upstream of phospholipase C (PLC) which, in turn, produced diacylglycerol (DAG) and inositol 3,4,5 triphosphate (IP3). DAG is important to activate PKC and phosphorylate NFL, while IP3 contributed to Ca2+ release from internal stores promoting hyperphosphorylation of KSP repeats on the tail domain of NFM and NFH. The present study supports the concept of glutamate and Ca2+ contribution in excitotoxic neuronal damage provoked by QUIN associated to dysfunction of the cytoskeleton homeostasis and highlights the differential signaling mechanisms elicited in striatal astrocytes and neurons

Hyperhomocysteinemia selectively alters expression and stoichiometry of intermediate filament and induces glutamate- and calcium-mediated mechanisms in rat brain during development

The aim of the present work was to investigate the actions of a chemically induced chronic hyperhomocysteinemia model on intermediate filaments (IFs) of cortical and hippocampal neural cells and explore signaling mechanisms underlying such effects. Results showed that in hyperhomocysteinemic rats the expression of neural IF subunits was affected. In cerebral cortex, glial fibrillary acidic protein (GFAP) expression was donwregulated while in hippocampus high and middle molecular weight neurofilament subunits (NF-H and NF-M, respectively) were up-regulated. Otherwise, the immunocontent of IF proteins was unaltered in cerebral cortex while in hippocampus the immunocontent of cytoskeletal-associated low molecular weight neurofilament (NF-L) and NF-H subunits suggested a stoichiometric ratio consistent with a decreased amount of core filaments enriched in lateral projections. These effects were not accompanied by an alteration in IF phosphorylation. In vitro results showed that 500 μM Hcy-induced protein phosphatases 1-, 2A- and 2B-mediated hypophosphorylation of NF subunits and GFAP in hippocampal slices of 17-day-old rats without affecting the cerebral cortex, showing a window of vulnerability of cytoskeleton in developing hippocampus. Ionotropic and metabotropic glutamate receptors were involved in this action, as well as Ca2+ release from intracellular stores through ryanodine receptors. We propose that the mechanisms observed in the hippocampus of 17-day-old rats could support the neural damage observed in these animals