CNS Protein Synthesis and Long-Term Adaptive Responses

National Institute Of Mental Health

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Abstract

Long-term adaptive changes in the nervous system are founded on lasting modifications in synaptic efficacy and require de novo protein synthesis. In neurons the biosynthesis of proteins is essential for growth and continued maintenance of the entire cell including axons, dendrites, and synaptic terminals. Regulation of the rate of protein synthesis and of the expression of specific proteins are crucial to the processes of development and synaptogenesis, maturation, neuronal plasticity, regeneration, and responses to hormones. In order to be able to localize such long-term changes we have developed the quantitative autoradiographic L-[1-14C]leucine method for the measurement of regional rates of cerebral protein synthesis (rCPS) in vivo. The objective of this project is to study long-term adaptive responses in the nervous system in both experimental animals and humans. A further objective is to elaborate the role of deficiencies in protein synthetic mechanisms in diseases in which long-term adaptive responses are impaired. In the current year work progressed in the following two areas: 1) Modification of the L-[1-14C]leucine method for use in man with L-[1-11C]leucine and positron emission tomography (PET). The ability to measure rCPS quantitatively with PET will provide us with a new tool to investigate the human brain and its regional adaptive responses. A longstanding obstacle to quantitative measurement of rCPS with PET has been the confounding effect of recycling of tissue amino acids derived from protein breakdown into the precursor pool for protein synthesis. In animal studies we evaluate the effects of recycling in parallel terminal experiments. PET studies have been limited to measurement of incorporation rates of amino acids supplied by the circulation only. Without correction for recycling one cannot distinguish true changes in rCPS from apparent changes resulting from alterations in recycling of tissue amino acids. We developed and validated a kinetic modeling approach to correct for the effect of recycling of tissue amino acids (see project MH-02569) and demonstrated that quantitatively accurate and reproducible measurement of rCPS is possible with L-[1-11C]leucine and PET (4). 2) Studies of protein metabolism and neuroadaptation in experimental animals. Currently these studies are focused on two genetic mouse models of mental retardation in an effort to try to understand underlying causes of the phenotype. In fragile X syndrome (FrX), an X-linked inherited form of mental retardation, methylation-induced transcriptional silencing of the fragile X mental retardation-1 (Fmr1) gene leads to absence of the gene product, fragile X mental retardation protein (FMRP). Absence of FMRP in Fmr1 knockout (KO) mice imparts many of the characteristics of the FrX phenotype. FMRP is an RNA-binding protein that has been shown to suppress translation of certain mRNAs in vitro. In brain FMRP is highly expressed in neuronal cytoplasm and is localized in dendrites and dendritic spines. The most striking neuropathological feature of FrX is the long, thin, and tortuous appearance of cortical dendritic spines, a similar morphology to that seen early in development. FMRP has been postulated to function as a suppressor of translation. Our in vivo studies of protein synthesis in Fmr1 KO mice suggest that this indeed may be the case, at least in selective brain regions. The regions most affected include areas of hippocampus, hypothalamus, and thalamus; changes in neocortex were smaller and reached statistical significance in only a few areas (2). Current studies centered on understanding the regional selectivity of the effects on rCPS. We asked if the effects of rCPS occur in areas known to have pathological changes in dendritic spines characteristic of FrX. We are quantifying changes in dendritic spines (spine length, area, and density) in several brain regions in which changes in rCPS were statistically significant and several regions in which rCPS were unaffected in the Fmr1 KO mice, and our results suggest that there is indeed correspondence. In contrast to the findings in neocortex where spine pathology occurs only in immature Fmr1 KO mice, in subcortical regions we found dendritic spine pathology even in the mature brain. A manuscript reporting these results is in preparation. Our initial studies of behavior and functional activity as assessed by regional cerebral metabolic rates for glucose (rCMRglc) in FrX mice were carried out in adult males; results showed that Fmr1 KO mice were hyperactive and deficient on a learning and memory test. Brain functional activity was generally higher than that found in WT particularly in hippocampus and primary sensory and posterior parietal cortex. We now have included in these studies female mice both heterozygous and homozygous for the null mutation, and we found no effect on functional activity in the females. We extended our studies to ascertain if the sex difference could be understood in terms of behavioral differences in effect of the null mutation. We found that female Fmr1 null mice are like the males hyperactive and deficient on a learning and memory test. Both male and female Fmr1 null mice exhibited increased susceptibility to audiogenic seizures. The only male-female difference was in the acoustic startle response. In males the response was diminished in Fmr1 null mice compared with WT. In contrast, in females all three genotypes had similar responses. Whether estrogen affords female Fmr1 null mice some protection from the effects of the mutation remains to be determined (3). We are studying another genetic mouse model (pahenu2) of mental retardation that has a mutation in the gene for the enzyme phenylalanine hydroxylase. In many respects the phenotype of animals with the mutation resembles human phenylketonuria (PKU). Phenylalanine hydroxylase activity is minimal in liver; concentrations of phenylalanine are 10-20 times normal in plasma; animals are hypopigmented and exhibit some subtle impairment in performance of several behavioral tests of cognitive function. We have studied the adult pahenu2 mouse and shown that brain size is reduced and rCPS is diminished throughout the brain. Whether this change in rCPS is a significant factor in the development of behavioral deficits or a consequence of the disease process remains to be determined. We have extended our studies of the PKU mouse to include analysis of behavioral abnormalities and regional functional activity as indicated by regional rates of cerebral glucose metabolism (rCMRglc). Our results show that the adult male pahenu2 mouse has regionally selective decreases in rCMRglc. Effects are noteworthy in regions of cerebral cortex involved in executive functions such as associative learning, working memory, and decision-making. In the dorsal hippocampus rCMRglc is not affected and performance of the pahenu2 mouse on a spatial memory task is normal. This is of interest because lesions of the hippocampus result in severe deficits in spatial memory.