Decreased rates of cerebral protein synthesis in conscious young adults with fragile X syndrome demonstrated by L-[1-11C]leucine PET.

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. Fragile X mental retardation protein, a putative translation suppressor, is significantly reduced in FXS. The prevailing hypothesis is that rates of cerebral protein synthesis (rCPS) are increased by the absence of this regulatory protein. We have previously reported increased rCPS in the Fmr1 knockout mouse model of FXS. To address the hypothesis in human subjects, we measured rCPS in young men with FXS with L-[1-11C]leucine PET. In previous studies we had used sedation during imaging, and we did not find increases in rCPS as had been seen in the mouse model. Since mouse measurements were conducted in awake animals, we considered the possibility that sedation may have confounded our results. In the present study we used a modified and validated PET protocol that made it easier for participants with FXS to undergo the study awake. We compared rCPS in 10 fragile X participants and 16 healthy controls all studied while awake. Contrary to the prevailing hypothesis and findings in Fmr1 knockout mice, results indicate that rCPS in awake participants with FXS are decreased in whole brain and most brain regions by 13-21% compared to healthy controls.

Altered cerebral protein synthesis in fragile X syndrome: studies in human subjects and knockout mice.

Dysregulated protein synthesis is thought to be a core phenotype of fragile X syndrome (FXS). In a mouse model (Fmr1 knockout (KO)) of FXS, rates of cerebral protein synthesis (rCPS) are increased in selective brain regions. We hypothesized that rCPS are also increased in FXS subjects. We measured rCPS with the L-[1-(11)C]leucine positron emission tomography (PET) method in whole brain and 10 regions in 15 FXS subjects who, because of their impairments, were studied under deep sedation with propofol. We compared results with those of 12 age-matched controls studied both awake and sedated. In controls, we found no differences in rCPS between awake and propofol sedation. Contrary to our hypothesis, FXS subjects under propofol sedation had reduced rCPS in whole brain, cerebellum, and cortex compared with sedated controls. To investigate whether propofol could have a disparate effect in FXS subjects masking usually elevated rCPS, we measured rCPS in C57Bl/6 wild-type (WT) and KO mice awake or under propofol sedation. Propofol decreased rCPS substantially in most regions examined in KO mice, but in WT mice caused few discrete changes. Propofol acts by decreasing neuronal activity either directly or by increasing inhibitory synaptic activity. Our results suggest that changes in synaptic signaling can correct increased rCPS in FXS.

Propofol anesthesia does not alter regional rates of cerebral protein synthesis measured with L-[1-(11)C]leucine and PET in healthy male subjects.

We report regional rates of cerebral protein synthesis (rCPS) in 10 healthy young males, each studied under two conditions: awake and anesthetized with propofol. We used the quantitative L-[1-(11)C]leucine positron emission tomography (PET) method to measure rCPS. The method accounts for the fraction (lambda) of unlabeled leucine in the precursor pool for protein synthesis that is derived from arterial plasma; the remainder comes from proteolysis of tissue proteins. Across 18 regions and whole brain, mean differences in rCPS between studies ranged from -5% to 5% and were within the variability of rCPS in awake studies (coefficient of variation range: 7% to 14%). Similarly, differences in lambda (range: 1% to 4%) were typically within the variability of lambda (coefficient of variation range: 3% to 6%). Intersubject variances and patterns of regional variation were also similar under both conditions. In propofol-anesthetized subjects, rCPS varied regionally from 0.98+/-0.12 to 2.39+/-0.23 nmol g(-1) min(-1) in the corona radiata and in the cerebellum, respectively. Our data indicate that the values, variances, and patterns of regional variation in rCPS and lambda measured by the L-[1-(11)C]leucine PET method are not significantly altered by anesthesia with propofol.

Use of acute hyperphenylalaninemia in rhesus monkeys to examine sensitivity and stability of the L-[1-11C]leucine method for measurement of regional rates of cerebral protein synthesis with PET.

We have previously shown by direct comparison with autoradiographic and biochemical measurements that the L-[1-(11)C]leucine positron emission tomography method provides accurate determinations of regional rates of cerebral protein synthesis (rCPS) and the fraction (lambda) of unlabeled leucine in the precursor pool for protein synthesis derived from arterial plasma. In this study, we examine sensitivity of the method to detect changes in lambda and stability of the method to measure rCPS in the face of these changes. We studied four isoflurane-anesthetized monkeys dynamically scanned with the high resolution research tomograph under control and mild hyperphenylalaninemic conditions. Hyperphenylalaninemia was produced by an infusion of phenylalanine that increased plasma phenylalanine concentrations three- to five-fold. In phenylalanine-infused monkeys, plasma leucine concentrations remained relatively constant, but values of lambda were statistically significantly decreased by 11% to 15%; rCPS was unaffected. Effects on lambda are consistent with competitive inhibition of leucine transport by increased plasma phenylalanine. The effect on lambda shows that competition for the transporter results in a reduction in the fraction of leucine in the precursor pool for protein synthesis coming from plasma. Even under these hyperphenylalaninemic conditions, rCPS remains unchanged due to the compensating increased contribution of leucine from protein degradation to the precursor pool.

Postadolescent changes in regional cerebral protein synthesis: an in vivo study in the FMR1 null mouse.

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), and consequently fragile X syndrome (FrX), an X-linked inherited form of mental retardation. Absence of FMRP in Fmr1 null mice imparts some characteristics of the FrX phenotype, but the precise role of FMRP in neuronal function remains unknown. FMRP is an RNA-binding protein that has been shown to suppress translation of certain mRNAs in vitro. We applied the quantitative autoradiographic L-[1-14C]leucine method to the in vivo determination of regional rates of cerebral protein synthesis (rCPS) in adult wild-type (WT) and Fmr1 null mice at 4 and 6 months of age. Our results show a substantial decrease in rCPS in all brain regions examined between the ages of 4 and 6 months in both WT and Fmr1 null mice. Superimposed on the age-dependent decline in rCPS, we demonstrate a regionally selective elevation in rCPS in Fmr1 null mice. Our results suggest that the process of synaptic pruning during young adulthood may be reflected in decreased rCPS. Our findings support the hypothesis that FMRP is a suppressor of translation in brain in vivo.

Measurement of regional rates of cerebral protein synthesis with L-[1-11C]leucine and PET with correction for recycling of tissue amino acids: I. Kinetic modeling approach.

Measurements of regional rates of cerebral protein synthesis (rCPS) require correction for the effect of recycling of tissue amino acids back into the precursor pool for protein synthesis. The fraction of the precursor pool derived from arterial plasma, lambda, can be evaluated as the steady-state ratio of the specific activity of leucine in the tissue tRNA-bound fraction to that in arterial plasma. While lambda can be directly measured in terminal experiments in animals, an alternative method is required for use with PET. We report a method to estimate lambda based on a kinetic model of labeled and unlabeled leucine and labeled CO2 in the tissue. The kinetic model is also used to estimate the amount of labeled protein and rCPS. We measured time courses of [14C]leucine, [14C]protein, and 14CO2 in the blood and brain of anesthetized rats and estimated parameters of the kinetic model from these data. Simulation studies based on the kinetic parameters were then performed to examine the feasibility of this approach for use with L-[1-11C]leucine and PET. Lambda and rCPS were estimated with low bias, which suggests that PET can be used for quantitative measurement of rCPS with L-[1-11C]leucine and a kinetic modeling approach for correction for recycling of tissue amino acids.

Measurement of regional rates of cerebral protein synthesis with L-[1-11C]leucine and PET with correction for recycling of tissue amino acids: II. Validation in rhesus monkeys.

The confounding effect of recycling of amino acids derived from tissue protein breakdown into the precursor pool for protein synthesis has been an obstacle to adapting in vivo methods for determination of regional rates of cerebral protein synthesis (rCPS) to positron emission tomography (PET). We used a kinetic modeling approach to estimate lambda, the fraction of the precursor pool for protein synthesis derived from arterial plasma, and to measure rCPS in three anesthetized adult monkeys dynamically scanned after a bolus injection of L-[1-11C]leucine. In the same animals, lambda was directly measured in a steady-state terminal experiment, and values showed excellent agreement with those estimated in the PET studies. In three additional monkeys rCPS was determined with the quantitative autoradiographic L-[1-14C]leucine method. In whole brain and cerebellum, rates of protein synthesis determined with the autoradiographic method were in excellent agreement with those determined with PET, and regional values were in good agreement when differences in spatial resolution of the two methods were taken into account. Low intrasubject variability was found on repeated PET studies. Our results in anesthetized monkey indicate that, by using a kinetic modeling approach to correct for recycling of tissue amino acids, quantitatively accurate and reproducible measurement of rCPS is possible with L-[1-11C]leucine and PET.