As a result of the last granting period, the applicants have made significant progress in understanding various molecular aspects of aging in rat hippocampal neurons. The partial """"""""molecular fingerprints"""""""" which they have generated require them to expand their analysis to determine how these coordinate changes in the expression of particular genes result in changes in neuronal biology during aging. Add-back experiments will be performed to determine the direct consequence of altered levels of functional protein on cellular expression profiles and physiology. They have already made significant progress in characterizing the molecular fingerprints that are generated by direct addition of c-fos/c-jun to neurons. Only a subset of mRNAs (8mRNAs out of 54 examined) in hippocampal pyramidal cells change in abundance in response to aging so it is necessary to expand the expression profile to include novel sequences so that a better representation of age correlated changes are analyzed. This will be accomplished by subtractive hybridization and/or differential display using a modified protocol and analysis system developed in the applicants' laboratory. Included in the mRNA changes which are documented in the grant are various subunits of the glutamate and GABA receptor system suggesting that changes in the balance of inhibitory/excitatory responsiveness is altered. The glutamate receptor subunit mRNAs will be further analyzed to determine whether alternative splicing or RNA-editing is altered during aging. The physiological responsiveness of individual neurons to GABA and glutamate will be correlated with the observed molecular biological changes. The abundance of CamKII mRNA, which has a somatodendritic localization in neurons, is altered in aging. Hence they will determine whether the subcellular distribution of mRNAs is altered over the same time course as that which results in a change in CamKII mRNA abundance. This will be accomplished by cloning of these mRNAs using a combination of techniques developed in the applicants' laboratory followed by determining whether their distribution is altered by aging or chronic glucocorticoid treatment. In the process of performing these experiments they have developed a model system which will enable them to prove whether the mRNAs localized in neuronal processes can in fact be translated. Critically, since protein levels do not necessarily parallel mRNA levels it is necessary to determine whether changes occur in the profile of cognate proteins encoded by the mRNAs which have been shown to change upon expression profiling. To do this they have modified the immuno-PCR technique for use in the single cell and have preliminary data showing that they can detect individual protein species from single cells. They will continue to develop this procedure so that they can determine the protein profile of multiple proteins at the same time as expression profiling of multiple mRNAs is done. These data will provide a refined mRNA and protein fingerprint of individual neurons which have been anatomically and electrophysiologically characterized from aged rats which have been behaviorally tested for age related learning deficits. It is hypothesized that this fingerprint will provide novel targets for manipulation of the physiological consequences of aging.
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