Effects Of Calpain Activation On Transmembrane AMPA Receptor Regulatory Protein And Its Mechanism Of Action

Part OneThe Effects Of Situ Calpain Activation on Stargazin/TARP?-8 In Vitro In Young RatsChanges in AMPA receptors have been proposed to underlie changes in synaptic efficacy in hippocampus and other brain regions. Calpain activation has also been discussed as a potential mechanism to produce lasting modifications of synaptic structure and function. Stargazin is a member of the family of transmembrane AMPA receptor associated proteins (TARPs), which participate in trafficking of AMPA receptors and regulate their kinetic properties. In the study we used the thin frozen rat brain sections with or without calcium and calpian inhibitor?to detect the levels of stargazin and TARP-?8 in various brain regions by immunohistochemistry. To further confirm the results from immunohischemistry, we also did the western blot of the frozen sections with or without calcium and calpain inhibitor?, and the western blot of the brain membrane with or without calcium and calpain. We found that the levels of stragazin decreased in cerebellum, hippocampus and cortex with the treatment of calcium. The effect can completely blocked by the treatment with calpain inhibitor?. Interestingly, the same treatment did not modify the immunoreactivity for another TARP member, TARP-?8, although it increased immunoreactivity in cell bodies in hippocampus. The effect was not blocked by calpain inhibition?. The western blot results of the frozen sections confirmed that the calcium treatment decreased stargazin levels, which was completely blocked by calpian inhibitor. The results of the brain member ance by western blot also showed that levels of stargazin decrease following the treatment with calcium and calpian. On the contrast the levels of TARP-?8 did not change following the same treatment. The results indicate that in situ calpain activation produces a decreased immunoreactivity for stargazin in the neuropil throughout the brain. The results also strongly suggest that the involvement of calpain in the regulation of AMPA receptor targeting and function through truncation of stargazin. Part TwoThe role of calpain activation mediated C-terminal truncation on stargazin in kainic acid induced seizures.Glutamate is a primary excitatory neurotransmitter in the mammalian central nervous system, and alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors play a major role in the pathophysiology progress of the excitotoxicity. Kainic acid induced seizure is the classical model to study epilepsy and excitotoxicity in the central nervous system. Kainic acid is also a compound to activate the calpain in vivo. In this research we used kainic acid to induce the seizure in young rats to investigate the levels of stargazin and TARP-?8 in various brain regions by immunofluorescence, and the level of the co-expression with GluRl. To further confirm the results fromImmunofluorescence, we also did the western blot of the different brain regions from the kainic acid injected rats and the controls. We found that stargzain co-expressed with the GluRl in various brain regions, the levels of stragazin decreased in purkinje cells of cerebellum, hippocampus and cortex with the injection of kainic acid. As well as the stargzain, the expression of GluRl also decreased in the brain. There was not any different between the groups with 2 hours seizure and 4 hours seizure. The immunoreactivity for another TARP member, TARP-?8, decreased in the hippocampus but not in the cortex, although it did not change in vitro. The western blot results confirmed that the stargazin levels decreased after the seizure induced with kainic acid. The results suggest that the calpian activation mediated the truncation of stargazin in vivo. Calpain-mediated stargazin degradation might be a protective mechanism to decrease neuronal excitability, and may provide a new way to control the seizure of epilepsy. TARP-?8 was not the substrate of calpain, but the distribution of TARP-?8 depended on the calcium. There may be an unknown compound that involved the degradation of TARP-?8,...