Studies On Protein Synthesis And Long-Term Adaptive Resp

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 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. To localize such long-term changes we developed the quantitative autoradiographic L-[1-14C]leucine method for 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. [unreadable] In the current year work progressed in the following two areas:[unreadable] [unreadable] 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 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 confounding effects of recycling of tissue amino acids derived from protein breakdown into the precursor pool for protein synthesis. Without correction for recycling one cannot distinguish true changes in rCPS from apparent changes resulting from alterations in recycling of tissue amino acids. In animal studies we evaluate the effects of recycling in parallel terminal experiments. For PET studies we developed a kinetic modeling approach to correct for effects of recycling of tissue amino acids and we have validated this approach by showing that quantitatively accurate and reproducible measurement of rCPS is possible with L-[1-11C]leucine and PET (3). [unreadable] [unreadable] We are currently studying anesthetized rhesus monkeys to test the reproducibility and sensitivity of the L-[1-11C]leucine PET method. Each animal is studied twice under baseline conditions and once following an infusion of phenylalanine. Kinetic rate constants and the correction factor for recycling are estimated and rCPS are determined in multiple brain regions. Results will improve our understanding of this new PET tracer. [unreadable] [unreadable] We have initiated L-[1-11C]leucine PET studies in human subjects (06-M-0214). Studies are designed to establish the method in human subjects. We will determine: [unreadable] a) Normal values of rCPS and reproducibility in conscious healthy volunteers. [unreadable] b) Effects of sedation on rCPS in healthy volunteers. This is an important question for future studies as some patient groups may have to be sedated for PET studies. Results will also address the sensitivity of the method to detect activity-dependent protein synthesis.[unreadable] c) Effects of fragile X syndrome (FrX) on rCPS (see below).[unreadable] [unreadable] 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. [unreadable] [unreadable] In FrX, an X-linked inherited form of mental retardation, 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. The most striking neuropathological feature of FrX is the long, thin, and tortuous appearance of cortical dendritic spines. FMRP has been postulated to function as a suppressor of translation. Our in vivo studies of rCPS in fmr1 KO mice suggest that this indeed may be the case, at least in selective brain regions. In fmr1 KO mice rCPS were higher than WT primarily in hippocampus, hypothalamus, and thalamus (1). [unreadable] [unreadable] In an effort to understand the regional selectivity of effects on rCPS we asked if the effects on rCPS occur in areas known to have pathological changes in dendritic spines. We have quantified 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 good correspondence. In contrast to the findings in neocortex where spine pathology occurs only in immature fmr1 KO mice, in subcortical regions we found spine pathology even in mature brain. A manuscript reporting these results is in preparation. [unreadable] [unreadable] Our initial studies of behavior and functional activity as assessed by regional cerebral metabolic rates for glucose (rCMRglc) in fmr1 KO 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 rCMRglc in females. To ascertain if the sex difference could be understood in terms of differences in behavioral phenotype we subjected male and female fmr1 KO mice to a battery of behavioral tests. Female fmr1 KO mice were, like males, hyperactive, deficient on a learning and memory test, and susceptible to audiogenic seizures. The only male-female difference was in the acoustic startle response in which male fmr1 KO mice had a diminished response compared with WT, but females of all three genotypes had similar responses. Whether estrogen affords females some protection from the effects of the mutation remains to be determined (2).[unreadable] [unreadable] Our finding of increased rCPS and abnormal spine morphology in hypothalamic nuclei in the fmr1 KO mouse together with clinical reports that children with FrX are more easily stressed and have elevated salivary cortisol suggests that there may be a dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis in FrX. We have examined normal HPA function and HPA response to and recovery from stress in the fmr1 KO mouse. We determined the normal circadian fluctuations in plasma concentrations of ACTH and corticosterone and studied the response of the HPA system to two stressors introduced at the nadir of the circadian fluctuations in corticosterone. The stressors were i) immobilization and ii) spatial novelty. Control and fmr1 KO mice had increased serum ACTH and corticosterone in response to both stressors, and the level of response and the time course of recovery were similar for both genotypes. These data indicate that in acute stress, regulation of HPA hormones is normal.[unreadable] [unreadable] We are studying another genetic mouse model 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). We have studied the adult PKU 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 rCMRglc. Results show that the PKU mouse has regionally selective decreases in rCMRglc. Effects are noteworthy in regions of cortex involved in executive functions.