Ph.D. 1996 University of Leeds, UK
im4n@virginia.edu
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The hippocampus is a region of the brain that has been functionally associated with learning and memory. Neural plasticity is believed to underlie these processes and is manifest in the generation of new synapses and the shedding of existing synaptic connections. The sites of synaptic interaction among neurons of the central nervous system are protrusions known as spines that are found on dendritic processes. Dendritic spines are known to change in density, morphology and connectivity in response to a variety of stimuli and are prime candidates as the loci of neural plasticity. Changes in spine density or connectivity in the neurons of the hippocampus may be associated with changes in the capacity for learning and memory formation. Therefore, a full understanding of the mechanisms by which spine density is regulated at the molecular level may illuminate the underlying molecular correlates of learning and memory. The hormone estrogen is known to induce a dramatic increase in dendritic spine density on neurons in the CA1 region of the hippocampus during the estrus cycle of the female rat. Spine density begins to rise during diestrus 2 of the cycle and peaks during proestrus. At estrus, the density of spines falls to "basal" levels that are approximately 30% lower than the peak at proestrus. The temporal profile of the increase in spine density coincides with the increases in estrogen, which peaks during proestrus. Indeed, using ovariectomised (OVX) rats, administration of ß-estradiol is sufficient to cause spine density to increase by similar amounts to those observed during proestrus. Interestingly, it is known that progesterone administration reverses ß-estradiol-mediated increases in spine density, suggesting that the peak of progesterone observed on estrus could be responsible for the decay in spine density to "basal" levels. Using these paradigms we are currently engaged in the identification and functional charaterisation of factors that mediate and regulate spine formation. To identify such factors, we are employing differential display technology as well as 2D gel analysis with nanospray mass spectroscopic peptide sequencing using tissue derived from the CA1 region of the hippocampus from naturally cycling rats as well as OVX rats with ß-estradiol. Using this model, we have already identified a number of transcripts that are expressed specifically in the female hippocampus. Each of the transcripts is differentially expressed during the estrus cycle and regulated in the OVX rat by ß-estradiol. We are currently attempting to characterise these transcripts and their function in the regulation of spine density in the adult hippocampus during the estrus cycle. The ability of estrogen to stimulate spine formation has been observed in primary cultures of dissociated hippocampal pyramidal neurons. We will utilize this model to identify regulatory factors by antisense mutagenesis using cDNA and antisense oligonucleotides. In this way will abrogate expression of target genes and observe the effect on estrogen-dependent spine formation by light microscopy as well as electron microscopy. In a parallel approach, we will demonstrate that the target cDNA is involved in spine formation by expressing the cDNA under the control of an inducible promoter. In this way, we would expect to stimulate spine formation under the conditions required for the induction of the promoter thereby rendering the process estrogen-independent. Using the hippocampal pyramidal neuron culture system, we should be able to identify a number of regulatory factors that are expressed differentially in tissue that is initiating a dramatic increase in spine formation. Additionally, we will show that these factors are able to regulate spine formation in primary culture system. To further demonstrate the role of these genes in the adult animal, we will use the considerable expertise that exists in our lab to generate large numbers of transgenic mice. In this context, we will place a candidate factor under the control of a promoter that leads to expression of the gene in response to an exogenous signal. To this end, we will employ the lacI repressible system that was developed in our lab. In this system we would identify regulatory factors that are involved in spine formation through the reliance of the process on the administration of IPTG, which represses the lacI repressor leading to induction of expression of the candidate gene or antisense construct. | ||
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Selected Publications McCaffery I., Favis R. L., Warty N., Wu W. and Rutherford C. L. A nuclear trans-acting factor and C-rich sequence elements regulate cAMP-responsive transcription of the GP2 gene in Dictyostelium discoideum. Submitted to Molecular and Cellular Biology. McCaffery I., Seger, J., Donovan J.T.J. and Hames B.D. Regulation of Transcription of a Gene Expressed During Late Development of Dictyostelium discoideum. Submitted to Biochemical Journal. Favis R., McCaffery I., Ehrenkaufer G. and Rutherford, C.L. (1998). Induction of the Dictyostelium glycogen phosphorylase-2 gene is regulated by multi-protein complexes. Developmental Genetics; 23 (3) 230-246. Rutherford C. L., McCaffery I. and Favis R. L. (1997) Regulation of Glycogen Phosphorylase and Synthase Genes During Cell Differentiation of Dictyostelium. pp. 363-378. In "Dictyostelium-A Model Organism for Cell and Developmental Biology." (Y. Maeda, K. Inouye and I. Takeuchi, Eds.) University Academic Press, Tokyo. McCaffery I., Williamson B.D. and Rutherford C. L. A Novel System for the Rapid Generation of Precise DNA Deletions (1996). Nucleic Acids Research 24 (24) 5048-5050.
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