In light of the importance of the calcium ion in neuronal function, the object of this proposal is, first of all, to obtain a better understanding of the control of intraneuronal calcium by studying the functional significance of the 28,000 Mr brain calcium binding protein (calbindin). The calbindin protein will be introduced into single cells using a patch pipette and we will determine possible effects on intracellular calcium and changes in calcium channel activity. Using CA1 hippocampal cells we will also determine whether the presence of calbindin can protect against cell death which results after exposure to high levels of glutamate and subsequent excessive increase in intracellular calcium. In addition, we will transfect PC12 cells with the calbindin gene and we will determine if the presence of calbindin has an effect on neurite outgrowth which occurs in response to nerve growth factor (NGF). If changes are observed, studies using site directed mutagenesis will allow the elucidation of structure-function relationships. We also propose to study the regulation of calbindin in cultured, dissociated neurons. Preliminary findings concerning an induction of calbindin by NGF in a dispersed culture of rat cerebellar cells will be further investigated. In addition, we will examine the possibility of regulation of calbindin expression by calcium. Our second objective will be to determine whether there are alterations in other calcium related genes, such as calcium calmodulin dependent protein kinase (CaM kinase II), with seizure activity. Changes in CaM kinase II gene expression will be examined in hippocampus, cortex and subcortical structures (including amygdala, substantia nigra and striatum) of commissure and amygdaloid kindled rats. In addition, changes in CaM kinase II gene expression will also be examined in these structures after pentylenetetrazole induced seizures. Finally, in order to obtain the best understanding of the molecular basis of epileptogenesis, we will determine by differential hybridization which genes are altered at early times after kindling induced seizures (1 hour or 24 hours) or 28 days after the last kindled seizure. Changes in gene expression in hippocampus and substantia nigra will be initially investigated. DNA sequences of clones obtained by differential hybridization will be compared to known sequences. cDNAs of genes altered either at early times or 28 days after the last kindled seizure will be used to determine by Northern analysis whether changes in these genes are also observed with chemically induced seizures. We also propose to examine human brain tissue from epileptic patients for changes in these genes. These studies should enable us to identify alterations in specific genes involved in convulsive neuronal activity. It is likely from these studies that important advances can be made concerning the molecular level changes underlying epileptogenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS020270-09
Application #
2263821
Study Section
Neurology A Study Section (NEUA)
Project Start
1983-12-01
Project End
1995-02-28
Budget Start
1992-03-01
Budget End
1995-02-28
Support Year
9
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Newark
State
NJ
Country
United States
Zip Code
07107
Wang, Y Z; Christakos, S (1995) Retinoic acid regulates the expression of the calcium binding protein, calbindin-D28K. Mol Endocrinol 9:1510-21
Lee, S; Miskovsky, J; Williamson, J et al. (1994) Changes in glutamate receptor and proenkephalin gene expression after kindled seizures. Brain Res Mol Brain Res 24:34-42
Cheng, B; Christakos, S; Mattson, M P (1994) Tumor necrosis factors protect neurons against metabolic-excitotoxic insults and promote maintenance of calcium homeostasis. Neuron 12:139-53
Lee, S; Christakos, S; Small, M B (1993) Apoptosis and signal transduction: clues to a molecular mechanism. Curr Opin Cell Biol 5:286-91
Katsetos, C D; Frankfurter, A; Christakos, S et al. (1993) Differential localization of class III, beta-tubulin isotype and calbindin-D28k defines distinct neuronal types in the developing human cerebellar cortex. J Neuropathol Exp Neurol 52:655-66
Walters, M R; Fischette, C T; Fetzer, C et al. (1992) Specific 1,25-dihydroxyvitamin D3 binding sites in choroid plexus. Eur J Pharmacol 213:309-11
Reisner, P D; Christakos, S; Vanaman, T C (1992) In vitro enzyme activation with calbindin-D28k, the vitamin D-dependent 28 kDa calcium binding protein. FEBS Lett 297:127-31
Iacopino, A M; Christakos, S; Modi, P et al. (1992) Nerve growth factor increases calcium binding protein (calbindin-D28K) in rat olfactory bulb. Brain Res 578:305-10
Iacopino, A; Christakos, S; German, D et al. (1992) Calbindin-D28K-containing neurons in animal models of neurodegeneration: possible protection from excitotoxicity. Brain Res Mol Brain Res 13:251-61
Sonnenberg, J L; Frantz, G D; Lee, S et al. (1991) Calcium binding protein (calbindin-D28k) and glutamate decarboxylase gene expression after kindling induced seizures. Brain Res Mol Brain Res 9:179-90

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