Regulation of glutamate receptor trafficking and function in cortical neurons
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NMDA and AMPA receptors are the key receptors in the mammalian brain that are the most important mediators of synaptic plasticity processes like long term potentiation (LTP) and long term depression (LTD) and form the cellular basis for learning and memory. Dysfunctions of these receptors are associated with many neuropsychiatric disorders. The synaptic targeting, clustering and immobilization of glutamate receptors are crucial for efficient excitatory synaptic transmission. These receptors go through a dynamic process of recycling between the intracellular pools and synaptic cell surface. Several signal transduction pathways affect the trafficking and surface expression of these receptors at the synapses and their internalization into these pools. Abnormal functioning of these receptors in neurodegenerative diseases like Alzheimer's and Huntington's is a major area of focus for intensive neuroscience research. The goal of our research is to study the mechanisms that might potentially affect the trafficking of these receptors in these diseases. Cytoskeletal elements like actin and microtubules are known to play major roles in the trafficking of AMPA and NMDA receptors. A wealth of evidence shows that the integrity of F-actin in the post synaptic region of neurons is important for optimal expression of these cationic channels. Microtubules are cellular highways along which NMDA and AMPA receptor containing vesicles are delivered for insertion at the synapse. Alteration of second messenger pathways perturb the integrity of these cytoskeletal elements at the post-synaptic density, which in turn affect the synaptic expression of the glutamate receptors. Underexpression of the receptors eventually leads to an imbalance of the ionic homeostasis within the cells. In our first study, we have demonstrated how the membrane phospholipid phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates NMDA receptors in cortical neurons. Blocking PIP2 synthesis by inhibiting phosphoinositide-4 kinase, or stimulating PIP 2 hydrolysis via activation of phospholipase C (PLC), or blocking PIP 2 function with an antibody caused a significant reduction of NMDAR-mediated currents. On the other hand, inhibition of PLC or application of PIP 2 caused an enhancement of NMDAR currents. Immunocytochemical studies also showed changes in NMDAR surface clusters induced by agents that manipulate PIP 2 levels. The PIP 2 regulation of NMDAR currents was abolished by the dynamin inhibitory peptide, which blocks receptor internalization. Agents perturbing actin stability prevented PIP 2 regulation of NMDAR currents, suggesting the actin-dependence of this effect of PIP 2 . Cofilin, a major actin depolymerizing factor, which has a common binding sequence for actin and PIP 2 , was required for PIP 2 regulation of NMDAR currents. Interestingly, the PIP 2 regulation of NMDAR channels was impaired in a transgenic mouse model of Alzheimer's disease (AD), probably due to the Aβ disruption of PIP 2 metabolism. Taken together, our data suggest that continuous synthesis of PIP 2 at the membrane might be important for the maintenance of NMDARs at the cell surface. When PIP 2 is lost, cofilin is released from the PIP 2 complex and is rendered free to depolymerize actin. With the actin cytoskeleton no longer intact, NMDARs are internalized via a dynamin/clathrin-dependent mechanism, leading to reduced NMDAR currents. In the second study, we investigated whether AMPAR trafficking is impaired in Huntington's disease. Several htt-interacting proteins implicated in intracellular transport have been identified, one of which is huntingtin-associated protein 1 (HAP1). HAP1 interacts more tightly with polyQ-htt than wild-type htt, and may act as a key mediator of pathological alterations in membrane trafficking by mutant htt. In this study, we examined the impact of abnormal interaction between polyQ-htt/HAP1 on the trafficking of AMPA receptors, the major player mediating excitatory synaptic transmission in CNS. We found that knockdown of HAP1 or expression of polyQ-htt reduced the frequency and amplitude of AMPAR-mediated miniature excitatory postsynaptic current (mEPSC) in cultured cortical neurons. On the other hand, overexpression of wild-type htt increased mEPSC. Moreover, suppressing the kinesin microtubule motor, KIF-5, occluded the effect of polyQ-htt on mEPSC, which could be recovered by overexpressing the heavy and light chain elements of KIF-5 motor protein. Disruption of the GluR2/KIF-5/microtubule complex was also found in a transgenic mouse model of HD, N171-82Q, which expresses an 82Q-containing N-terminal fragment of htt. Together, these data suggest that AMPAR trafficking is impaired by mutant htt, presumably due to the interference of KIF-5-mediated microtubule-based transport of AMPA receptors. The diminished strength of excitatory synaptic transmission in HD conditions could contribute to the loss of the cortical output that is important for movement control and account for the cognitive defects encountered in HD. Overall, our studies have described two novel mechanisms by which second messenger systems can regulate NMDA and AMPA receptors via cytoskeleton dependent mechanisms.