Role of T-type calcium channels and calcium/calmodulin-dependent kinases in the pathogenesis of ischemic brain damage.

The present unit will explore the hypothesis that T-type voltage-gated Ca2+ channels could take part in the genesis of brain damage in stroke, one of the main form of ischemic disease in ageing. The idea that these channels interact with Ca2+/calmodulin dependent kinases during stroke will be explored as well. Background: Previous studies showed an increase in T-type channel expression in response to hypoxic conditions in cell cultured in vitro, and demonstrated that in the presence of T-type channel blockers the combined oxygen and glucose deprivation (OGD) followed by reoxygenation (Rx) in hyppocampal slices elicits a much less severe damage than in control conditions. This supports the idea that T-type channels take part in the genesis of ischemic brain damage. CaM°K II and IV are highly expressed in brain, where they exert a neuroprotective activity in stroke. CaM°K II binds and activates the CaV3.2 T-type channel isoform while, given its tight association with T-type channels, CaM°K II is probably specifically activated by Ca2+ entering the cells through these channels. Aim of the study: We intend to establish whether: 1. T-type channel expression and/or activity is modified in response to brain ischemia 2. the pharmacological blockade of T-type Ca2+ channels can reduce the extension of ischemic brain damage 3. the three different T-type channel isoforms have a different role in the progression of ischemic brain damage 4. T-type channel blockade affects neuronal CaM°K II and CaM°K IV activity or expression in normoxic and anoxic conditions 5. a CaM°K II/IV pathway takes part in the control of the expression of the three different T-type channel isoforms 6. ageing affects T-type or CaM°K brain expression. Methods: To address these questions we will perform studies: 1. in vitro on cultured NG108-15 cells and on cortical neurons exposed to the combined deprivation of oxygen and glucose (OGD) followed by reoxygenation (Rx) to replicate the metabolic effects of stroke and 2. in vivo on mice undergoing experimental stroke obtained by the transient occlusion of middle cerebral artery (tMCAO). All animal experimentations will be performed in accordance with the national and international rules and after approval by the local ethical committee; we will make any effort to keep pain and discomfort as low as possible. Western blot experiments with specific commercial antibodies and real time PCR experiments with custom designed primers will be performed to quantify T-type or CaM°K protein expression and mRNA transcription. Immunoprecipitation studies will be performed to evaluate the effect of the different treatments on CaM°K binding to CaV3.2 channels. Experimental design The involvement of T-type channels in the genesis of ischemic brain damage will be determined by evaluating the effect of T-type channel blockers on the severity of stroke in mice undergoing tMCAO. In order to understand whether the three different isoforms of these channels have a different role in the process, the severity of tMCAO-induced brain damage will be determined in ko mice for each of these isoforms. In addition, we will perform in vitro studies to explore the consequences on the overexpression of each channel isoform on the sensitivity of neuronal cells to OGD-Rx. The hypothesis that brain ischemia causes a change in T-type channel expression will be tested by comparing protein and mRNA levels for these channels in ischemic and non ischemic brain areas in mice undergoing tMCAO. The effect of ischemia on T-type channel activity will be examined by patch clamp on cultured neuronal cells undergoing OGD-Rx. To determine whether T-type channel activity affects CaM°K activity in brain ischemia we will examine the effect of T-type channel blockers on CaM°K II and IV activity in cultured neuronal cells undergoing OGD-Rx and in the ischemic brain of mice undergoing tMCAO. We will also examine whether in response to experimental stroke there is any change in the amount of CaM°K II bound to T-type channels. Parallel with these studies, experiments aiming to establish whether CaM°Ks control T-type channel expression in ischemia will be performed with the help of specific CaM°K blockers. To establish whether changes in T-type channel/CaM kinase occur in ageing we will compare the expression and activity of these channels and enzymes in old and young mice. We will also establish whether tMCAO induces the same changes in T-type channels and CaM kinases in young and old animals. Expected results: The results of the planned experiments will determine the role of T-type channels in the genesis of ischemic neuronal death in vivo, and whether such a role could be regulated by their ability to interact with CaM°Ks.