Studies On Protein Synthesis And Long-Term Adaptive Responses in the CNS

National Institute Of Mental Health

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Abstract

In the current year work progressed in the following two areas:[unreadable] [unreadable] 1) Modification of the L-1-C-14leucine method for use in man with L-1-C-11leucine 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 was the confounding effect 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-C-11leucine and PET (Smith et al, J Cerebr Blood Flow & Metab 25: 629-640). [unreadable] [unreadable] We have completed a study of anesthetized rhesus monkeys to test the reproducibility and sensitivity of the L-1-C-11leucine PET method. Each animal was studied twice under baseline conditions and once following an infusion of phenylalanine. Kinetic rate constants and the correction factor for recycling were estimated and rCPS were determined in multiple brain regions. As predicted, the rate constant for leucine influx into brain and the correction factor for recycling were both affected in the hyperphenylalaninemic animals, but rCPS remained unaffected likely due to the compensating increased contribution to the precursor pool of leucine from protein degradation. Our results demonstrate the sensitivity and the robustness of the method to effects on the parameters. These results were presented at the meetings of the International Society for Cerebral Blood Flow & Metabolism (Smith et al, J Cereb Blood Flow & Metab 27 (Suppl 1) (XXIII-1), P02-5U, 2007) and the Society for Nuclear Medicine (Smith et al, J Nucl Med 48 (Suppl 2):238P, 2007). [unreadable] [unreadable] Studies designed to establish the L-1-C-11leucine PET method in human subjects are underway. Dynamic scans following injection of L-1-C-11leucine have been completed in six subjects. Our results indicate that in human brain 68% of the precursor pool for protein synthesis comes from arterial plasma and 32% from endogenous protein degradation. rCPS showed an expected regional pattern; rates in white matter were about 30% of grey matter rates. Coefficients of variation were 9% in whole brain and 8-18% regionally. These results were reported at the annual meeting of the Society for Nuclear Medicine (Bishu et al, J Nucl Med 48 (Suppl 2):60P, 2007).[unreadable] [unreadable] 2) Studies of protein metabolism and neuroadaptation in experimental animals. Currently these studies are focused on three genetic mouse models of developmental disorders in an effort to understand underlying causes of phenotype. [unreadable] [unreadable] In fragile X syndrome (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 (Qin et al, J Neurosci 25:5087-5095, 2005).[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 has been submitted. [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 suggested 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. A manuscript reporting these results has been submitted.[unreadable] [unreadable] The 5UTR of human FMR1 contains a CGG-repeat tract the size of which varies from 5-2000 repeats. Individuals with 55-200 repeats are carriers of FrX premutation alleles. These individuals are at risk for mild cognitive defects, behavioral problems, and/or autism spectrum disorders. In later life they are at risk for fragile X associated tremor and ataxia syndrome. K. Usdin (NIDDK) has developed a knock-in mouse model (KI) with an expanded repeat sequence that recapitulates key features seen in humans with the premutation including a direct relationship between repeat number and Fmr1 mRNA levels and an inverse relationship with FMRP levels. In addition, the KI mouse model has a regionally differential deficit in FMRP levels that might account for the full mutation symptoms seen in premutation carriers. We are studying this mouse model to ascertain the behavioral correlates of the model and to correlate differential regional effects on FMRP levels and rCPS. This study will further test the hypothesis that FMRP is a negative regulator of translation and that it is the loss of this regulation that is responsible for the clinical symptoms. In addition our results may help to establish in the mouse model the regional correlates of clinical symptoms.[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 (Qin and Smith, J Inherit Metab Dis 30:318-325, 2007).