Calcium homeostasis has a major role in the normal physiology of neurons, and perturbations of intracellular calcium underlie a variety of neurologic diseases including epilepsy, stroke, and chronic degenerative diseases. For example, there is strong evidence that injury of hilar neurons, which is a critical feature of temporal lobe epilepsy, is related to calcium-mediated toxicity. Recent work has suggested that one component of the neuronal calcium homeostasis system involves cytoplasmic calcium binding proteins. Calbindin-D(28K) (CaBP) is one of several of these proteins expressed in the mammalian central nervous system (CNS). Although little is known about the actual functions of CaBP, it has been suggested that CaBP acts as a calcium buffer under normal conditions, and CaBP may also play a role in the protection of neurons from the untoward effects of abnormally high concentrations of intracellular calcium. This latter tenet is based primarily on reports describing a relationship between the presence or absence of CaBP in neuronal subpopulations and selective vulnerability to certain forms of acute and chronic CNS diseases. Nonetheless, the normal calcium buffering properties of CaBP in neurons are unknown, and direct proof of the protective function of CaBP remains lacking. Similarly, although there is some indirect evidence that CaBP has other functions beyond the buffering of calcium, these have not yet been identified. The goal of this research plan is to explore the function of CaBP using both in vitro and in vivo systems. For the in vitro studies, we are creating neural cell lines that overexpress CaBP. We plan to study the calcium-buffering properties of these cells directly with fura-2 analysis, and determine whether CaBP expression confers protection from the toxic effects of abnormal increases in intracellular calcium. Next, we will create similar neural cell lines that overexpress mutated forms of CaBP, and study the effects of these mutations on calcium-buffering and calcium- mediated toxicity. Finally, the properties of CaBP will be studied in vivo by generating transgenic mice that express CaBP in specific hippocampal neurons that both l) do not normally express the protein, and 2) are exquisitely vulnerable to various forms of CNS injury such as seizures, ischemia, and trauma. Electrophysiological and anatomical techniques will then be used to study the effects of the CaBP transgene expression on dentate granule cell physiology, and to determine whether hilar neurons expressing CaBP are relatively resistant to injury induced by prolonged stimulation of the perforant path.
