The Effect Of Glutamate Transporter EAAT1 On Neurodegeneration Induced By Okadaic Acid In Rat Brain

The effect of glutamate transporter EAAT1 on neurodegeneration induced by okadaic acid in rat brainAbstract: Glutamate is a major excitatory neurotransmitter of the mammalian CNS and plays important roles in fast synaptic transmission, and in enduring synaptic changes such as long-term potentiation, that are believed to mediate learning and memory processes, as well as in the developing brain. Extracellular glutamate is normally kept at low levels, and excessive glutamate has been implicated as a neurotoxic agent in neurodegenerative disease and in CNS insults such as ischemia and epilepsy. Interstitial glutamate clearance depends on its binding to the specific transporters and subsequent uptake. Glutamate uptake is accomplished primarily by a family of Na+, K+-dependent high-affinity glutamate transporters. To date, five different glutamate transporters have been identified and named excitatory amino acid transporter 1 (EAAT1) or glutamate/aspartate transporter (GLAST), EAAT2 or glutamate transporter 1 (GLT-1), EAAT3 or excitatory amino acid carrier 1 (EAAC1), EAAT4, and EAAT5, and they have differing regional, cellular, and developmental distributions, and also have specific functional properties. A role for glutamate transporters has been postulated in acute conditions such as stroke, CNS ischemia and seizure, as well as in chronic neurodegenerative diseases such as Alzheimer's disease, Huntington's disease and amyotrophic lateral sclerosis. EAAT1/GLAST is considered to play a more important role in limiting the detrimental rise in glutamate level and inhibiting the expansion of the lesion in pathologic conditions because of its higher Km value. Okadaic acid (OA), the protein phosphatase inhibitor, induces the microtubule-associated protein tau hyperphosphorylation in vitro neurons, which associated with pathogenesis of neurodegeneration. In the present study, injection of OA into the frontal cortex was used to explore the possible roles of glutamate transporter EAAT1 in the neurodegenerative process of rat brain. Immunostaining was used to observe the expression of glial fibrillary acidic protein (GFAP), AT8, a tau protein phosphorylated marker, and EAAT1. TUNEL staining and double immuno- fluorescent labeling combined with confocal laser scanning were used to analyze the relationship between tau protein phosphorylation and EAAT1 expression and/or DNAdamage. The results were follows:Part I. Okadaic acid induces GFAP expression in rat brainImmunohistochemical staining was used to observe the effect of OA on the proliferation of astrocytes. OA at the dose of 100 ng in a 1.0 μl volume was injected into the frontal cortex (Fr) of rat brain. The results showed that (1) the expression of GFAP caused by injection of OA appeared feeble in the ipsilateral Fr, hippocampal CA1 and basolateral amygdaloid nucleus (BL) at 4 h, then spread to the parietal cortex area 1 (Par1) and piriform cortex (Pir) at 3 d and further increased even at 14 d after the injection; (2) optical density (OD) was measured to semi-quantify the density and quantity of GFAP expression in the ipsilateral Fr and BL. It showed that the expression of GFAP in both regions statistical significantly increased at 3 d, peaked at 7 d (p<0.05) and persisted at 14 d (p<0.05) after the injection. More interestingly, the enhancement of GFAP expression in the group of OA co-injection with Aβ1-42 was significantly higher than that in the group of single-injection of OA at 7 d (p<0.05) in the ipsilateral BL region. The present results indicated that OA could activate the astrocytes reaction in the peri-injected area and ipsilateral BL region where exists the afferent/efferent nerve fibers from/to the frontal cortex. The mechanism and role of astrogliosis induced by OA need to be further illustrated.Part II. Okadaic acid induces tau protein phosphorylation and neurodegeneration in rat brain neurocytesTo study the effects of okadaic acid on tau protein hyperphosphorylation and neurodegeneration, OA in a 0.5 μl volume was injected...