Increased rates of brain protein synthesis during [N1,N2] sleep: L-[1-11C]leucine PET studies in human subjects.

During sleep, reduced brain energy demands provide an opportunity for biosynthetic processes like protein synthesis. Sleep is required for some forms of memory consolidation which requires de novo protein synthesis. We measured regional cerebral protein synthesis rates (rCPS) in human subjects to ascertain how rCPS is affected during sleep. Subjects underwent three consecutive L-[1-11C]leucine PET scans with simultaneous polysomnography: 1. rested awake, 2. sleep-deprived awake, 3. sleep. Measured rCPS were similar across the three conditions. Variations in sleep stage times during sleep scans were used to estimate rCPS in sleep stages under the assumption that measured rCPS is the weighted sum of rCPS in each stage, with weights reflecting time and availability of [11C]leucine in that stage. During sleep scans, subjects spent most of the time in N2, N3, and awake and very little time in N1 and REM; rCPS in N1 and REM could not be reliably estimated. When stages N1 and N2 were combined [N1,N2], estimates of rCPS were more robust. In selective regions, estimated rCPS were statistically significantly higher (30-39%) in [N1,N2] compared with N3; estimated rCPS in N3 were similar to values measured in sleep-deprived awake scans. Results indicate increased rates of protein synthesis linked to [N1,N2] sleep.

Fragile X syndrome in a male with methylated premutation alleles and no detectable methylated full mutation alleles.

Most cases of fragile X syndrome (FXS) result from aberrant methylation of the FMR1 gene. Methylation occurs when the number of tandemly arranged cytosine guanine guanine (CGG)-repeats in the 5' end of the transcriptional unit of FMR1 exceeds a certain critical threshold, thought to be between 200 and 400 repeats. Such alleles are referred to as full mutation (FM) alleles. Premutation (PM) alleles, alleles with 55-200 repeats, are generally not aberrantly methylated and in fact may have hyperexpression of the FMR1 mRNA. We describe here a male who meets the diagnostic criteria for FXS, who is highly mosaic with a mixture of multiple PM and FM alleles and 50% methylation. However, the methylated alleles are limited to two alleles in the PM range, ~165 and ~175 repeats respectively, with the FM alleles being unmethylated. This finding has implications for FXS diagnosis as well as for efforts to delete the repeat in individuals with FXS using a CRISPR-Cas9 approach.

Substitution of venous for arterial blood sampling in the determination of regional rates of cerebral protein synthesis with L-[1-11C]leucine PET: A validation study.

We developed and validated a method to estimate input functions for determination of regional rates of cerebral protein synthesis (rCPS) with L-[1-11C]leucine PET without arterial sampling. The method is based on a population-derived input function (PDIF) approach, with venous samples for calibration. Population input functions were constructed from arterial blood data measured in 25 healthy 18-24-year-old males who underwent L-[1-11C]leucine PET scans while awake. To validate the approach, three additional groups of 18-27-year-old males underwent L-[1-11C]leucine PET scans with both arterial and venous blood sampling: 13 awake healthy volunteers, 10 sedated healthy volunteers, and 5 sedated subjects with fragile X syndrome. Rate constants of the L-[1-11C]leucine kinetic model were estimated voxel-wise with measured arterial input functions and with venous-calibrated PDIFs. Venous plasma leucine measurements were used with venous-calibrated PDIFs for rCPS computation. rCPS determined with PDIFs calibrated with 30-60 min venous samples had small errors (RMSE: 4-9%), and no statistically significant differences were found in any group when compared to rCPS determined with arterial input functions. We conclude that in young adult males, PDIFs calibrated with 30-60 min venous samples can be used in place of arterial input functions for determination of rCPS with L-[1-11C]leucine PET.

Rates of cerebral protein synthesis in primary visual cortex during sleep-dependent memory consolidation, a study in human subjects.

If protein synthesis during sleep is required for sleep-dependent memory consolidation, we might expect rates of cerebral protein synthesis (rCPS) to increase during sleep in the local brain circuits that support performance on a particular task following training on that task. To measure circuit-specific brain protein synthesis during a daytime nap opportunity, we used the L-[1-(11)C]leucine positron emission tomography (PET) method with simultaneous polysomnography. We trained subjects on the visual texture discrimination task (TDT). This was followed by a nap opportunity during the PET scan, and we retested them later in the day after the scan. The TDT is considered retinotopically specific, so we hypothesized that higher rCPS in primary visual cortex would be observed in the trained hemisphere compared to the untrained hemisphere in subjects who were randomized to a sleep condition. Our results indicate that the changes in rCPS in primary visual cortex depended on whether subjects were in the wakefulness or sleep condition but were independent of the side of the visual field trained. That is, only in the subjects randomized to sleep, rCPS in the right primary visual cortex was higher than the left regardless of side trained. Other brain regions examined were not so affected. In the subjects who slept, performance on the TDT improved similarly regardless of the side trained. Results indicate a regionally selective and sleep-dependent effect that occurs with improved performance on the TDT.

Deficient Sleep in Mouse Models of Fragile X Syndrome.

In patients with fragile X syndrome (FXS), sleep problems are commonly observed but are not well characterized. In animal models of FXS (dfmr1 and Fmr1 knockout (KO)/Fxr2 heterozygote) circadian rhythmicity is affected, but sleep per se has not been examined. We used a home-cage monitoring system to assess total sleep time in both light and dark phases in Fmr1 KO mice at different developmental stages. Fmr1 KOs at P21 do not differ from controls, but genotype × phase interactions in both adult (P70 and P180) groups are statistically significant indicating that sleep in Fmr1 KOs is reduced selectively in the light phase compared to controls. Our results show the emergence of abnormal sleep in Fmr1 KOs during the later stages of brain maturation. Treatment of adult Fmr1 KO mice with a GABAB agonist, R-baclofen, did not restore sleep duration in the light phase. In adult (P70) Fmr1 KO/Fxr2 heterozygote animals, total sleep time was further reduced, once again in the light phase. Our data highlight the importance of the fragile X genes (Fmr1 and Fxr2) in sleep physiology and confirm the utility of these mouse models in enhancing our understanding of sleep disorders in FXS.

Negative Effects of Chronic Rapamycin Treatment on Behavior in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is also highly associated with autism spectrum disorders (ASD). It is caused by expansion of a CGG repeat sequence on the X chromosome resulting in silencing of the FMR1 gene. This is modeled in the mouse by deletion of Fmr1 (Fmr1 KO). Fmr1 KO mice recapitulate many of the behavioral features of the disorder including seizure susceptibility, hyperactivity, impaired social behavior, sleep problems, and learning and memory deficits. The mammalian target of rapamycin pathway (mTORC1) is upregulated in Fmr1 KO mice and is thought to be important for the pathogenesis of this disorder. We treated Fmr1 KO mice chronically with an mTORC1 inhibitor, rapamycin, to determine if rapamycin treatment could reverse behavioral phenotypes. We performed open field, zero maze, social behavior, sleep, passive avoidance, and audiogenic seizure testing. We found that pS6 was upregulated in Fmr1 KO mice and normalized by rapamycin treatment, but, except for an anxiogenic effect, it did not reverse any of the behavioral phenotypes examined. In fact, rapamycin treatment had an adverse effect on sleep and social behavior in both control and Fmr1 KO mice. These results suggest that targeting the mTOR pathway in FXS is not a good treatment strategy and that other pathways should be considered.

A PILOT STUDY ON THE ENCODING OF A PERCEPTUAL LEARNING TASK FOLLOWING SLEEP DEPRIVATION.

Memory encoding sometimes must occur during a period of sleep deprivation. The question was whether one night of sleep deprivation inhibits encoding on a perceptual learning task (the texture discrimination task). The sample was 18 human participants (M age=22.1 yr., SEM=0.5; 8 men). The participants were randomized to a sleep deprivation or sleep control condition and, after the manipulation, were given two administrations of the texture discrimination task. All participants were given an opportunity for a 90 min. nap between the two administrations. Performance was measured by the interpolated stimulus-to-mask-onset asynchrony (i.e., the inter-stimulus interval), at which the percentage of correct responses for the stimuli in the participant's peripheral vision fell below 80%. Offline consolidation was defined as a decrease in this index between the two administrations. Participants who were sleep deprived prior to encoding exhibited similar offline consolidation (M=-5.3 msec., SEM=2.3) compared to participants who were not sleep deprived prior to encoding (M=-6.2 msec., SEM=3.9); the two-way interaction between time and condition was not significant. In light of reports in the literature, these results indicate encoding following sleep deprivation may be influenced by both the type of task encoded and the brain regions involved in memory processing.

Endocannabinoid-mediated improvement on a test of aversive memory in a mouse model of fragile X syndrome.

Silencing the gene FMR1 in fragile X syndrome (FXS) with consequent loss of its protein product, FMRP, results in intellectual disability, hyperactivity, anxiety, seizure disorders, and autism-like behavior. In a mouse model (Fmr1 knockout (KO)) of FXS, a deficit in performance on the passive avoidance test of learning and memory is a robust phenotype. We report that drugs acting on the endocannabinoid (eCB) system can improve performance on this test. We present three lines of evidence: (1) Propofol (reported to inhibit fatty acid amide hydrolase (FAAH) activity) administered 30 min after training on the passive avoidance test improved performance in Fmr1 KO mice but had no effect on wild type (WT). FAAH catalyzes the metabolism of the eCB, anandamide, so its inhibition should result in increased anandamide levels. (2) The effect of propofol was blocked by prior administration of the cannabinoid receptor 1 antagonist AM-251. (3) Treatment with the FAAH inhibitor, URB-597, administered 30 min after training on the passive avoidance test also improved performance in Fmr1 KO mice but had no effect on WT. Our results indicate that the eCB system is involved in FXS and suggest that the eCB system is a promising target for treatment of FXS.

R-Baclofen Reverses a Social Behavior Deficit and Elevated Protein Synthesis in a Mouse Model of Fragile X Syndrome.

BACKGROUND: Fragile X syndrome (FXS) is the most common known inherited form of intellectual disability and the single genomic cause of autism spectrum disorders. It is caused by the absence of a fragile X mental retardation gene (Fmr1) product, FMRP, an RNA-binding translation suppressor. Elevated rates of protein synthesis in the brain and an imbalance between synaptic signaling via glutamate and γ-aminobutyric acid (GABA) are both considered important in the pathogenesis of FXS. In a mouse model of FXS (Fmr1 knockout [KO]), treatment with R-baclofen reversed some behavioral and biochemical phenotypes. A remaining crucial question is whether R-baclofen is also able to reverse increased brain protein synthesis rates. METHODS: To answer this question, we measured regional rates of cerebral protein synthesis in vivo with the L-[1-(14)C]leucine method in vehicle- and R-baclofen-treated wildtype and Fmr1 KO mice. We further probed signaling pathways involved in the regulation of protein synthesis. RESULTS: Acute R-baclofen administration corrected elevated protein synthesis and reduced deficits on a test of social behavior in adult Fmr1 KO mice. It also suppressed activity of the mammalian target of rapamycin pathway, particularly in synaptosome-enriched fractions, but it had no effect on extracellular-regulated kinase 1/2 activity. Ninety min after R-baclofen treatment, we observed an increase in metabotropic glutamate receptor 5 expression in the frontal cortex, a finding that may shed light on the tolerance observed in human studies with this drug. CONCLUSIONS: Our results suggest that treatment via activation of the GABA (GABA receptor subtype B) system warrants further study in patients with FXS.

Cerebral protein synthesis in a knockin mouse model of the fragile X premutation.

The (CGG)n-repeat in the 5'-untranslated region of the fragile X mental retardation gene (FMR1) gene is polymorphic and may become unstable on transmission to the next generation. In fragile X syndrome, CGG repeat lengths exceed 200, resulting in silencing of FMR1 and absence of its protein product, fragile X mental retardation protein (FMRP). CGG repeat lengths between 55 and 200 occur in fragile X premutation (FXPM) carriers and have a high risk of expansion to a full mutation on maternal transmission. FXPM carriers have an increased risk for developing progressive neurodegenerative syndromes and neuropsychological symptoms. FMR1 mRNA levels are elevated in FXPM, and it is thought that clinical symptoms might be caused by a toxic gain of function due to elevated FMR1 mRNA. Paradoxically, FMRP levels decrease moderately with increasing CGG repeat length in FXPM. Lowered FMRP levels may also contribute to the appearance of clinical problems. We previously reported increases in regional rates of cerebral protein synthesis (rCPS) in the absence of FMRP in an Fmr1 knockout mouse model and in a FXPM knockin (KI) mouse model with 120 to 140 CGG repeats in which FMRP levels are profoundly reduced (80%-90%). To explore whether the concentration of FMRP contributes to the rCPS changes, we measured rCPS in another FXPM KI model with a similar CGG repeat length and a 50% reduction in FMRP. In all 24 brain regions examined, rCPS were unaffected. These results suggest that even with 50% reductions in FMRP, normal protein synthesis rates are maintained.

Identification of fragile X syndrome specific molecular markers in human fibroblasts: a useful model to test the efficacy of therapeutic drugs.

Fragile X syndrome (FXS) is the most frequent cause of inherited intellectual disability and autism. It is caused by the absence of the fragile X mental retardation 1 (FMR1) gene product, fragile X mental retardation protein (FMRP), an RNA-binding protein involved in the regulation of translation of a subset of brain mRNAs. In Fmr1 knockout mice, the absence of FMRP results in elevated protein synthesis in the brain as well as increased signaling of many translational regulators. Whether protein synthesis is also dysregulated in FXS patients is not firmly established. Here, we demonstrate that fibroblasts from FXS patients have significantly elevated rates of basal protein synthesis along with increased levels of phosphorylated mechanistic target of rapamycin (p-mTOR), phosphorylated extracellular signal regulated kinase 1/2, and phosphorylated p70 ribosomal S6 kinase 1 (p-S6K1). The treatment with small molecules that inhibit S6K1 and a known FMRP target, phosphoinositide 3-kinase (PI3K) catalytic subunit p110ß, lowered the rates of protein synthesis in both control and patient fibroblasts. Our data thus demonstrate that fibroblasts from FXS patients may be a useful in vitro model to test the efficacy and toxicity of potential therapeutics prior to clinical trials, as well as for drug screening and designing personalized treatment approaches.

Sleep, plasticity and the pathophysiology of neurodevelopmental disorders: the potential roles of protein synthesis and other cellular processes.

Sleep is important for neural plasticity, and plasticity underlies sleep-dependent memory consolidation. It is widely appreciated that protein synthesis plays an essential role in neural plasticity. Studies of sleep-dependent memory and sleep-dependent plasticity have begun to examine alterations in these functions in populations with neurological and psychiatric disorders. Such an approach acknowledges that disordered sleep may have functional consequences during wakefulness. Although neurodevelopmental disorders are not considered to be sleep disorders per se, recent data has revealed that sleep abnormalities are among the most prevalent and common symptoms and may contribute to the progression of these disorders. The main goal of this review is to highlight the role of disordered sleep in the pathology of neurodevelopmental disorders and to examine some potential mechanisms by which sleep-dependent plasticity may be altered. We will also briefly attempt to extend the same logic to the other end of the developmental spectrum and describe a potential role of disordered sleep in the pathology of neurodegenerative diseases. We conclude by discussing ongoing studies that might provide a more integrative approach to the study of sleep, plasticity, and neurodevelopmental disorders.

A mouse model of the fragile X premutation: effects on behavior, dendrite morphology, and regional rates of cerebral protein synthesis.

Carriers of FMR1 premutation alleles have 55-200 CGG repeats in the 5' untranslated region of the gene. These individuals are at risk for fragile X associated primary ovarian insufficiency (females) and, in late life, fragile X associated tremor and ataxia syndrome (males, and to a lesser extent, females). Premutation carrier status can also be associated with autism spectrum disorder, attention deficit hyperactivity disorder, and some cognitive deficits. In premutation carriers, FMR1 mRNA levels are often higher than those with normal sized alleles. In contrast, in subjects with full mutation alleles, (>200 repeats) the FMR1 gene is silenced and FMR1 mRNA and its product, FMRP, are absent. We have studied a male knock-in (KI) mouse model of the fragile X premutation (120-140 repeats) during young adulthood. In comparison to wild type, KI mice were hyperactive, exhibited less anxiety in both the open field and the elevated zero maze, were impaired on the passive avoidance test, and showed some subtle deficits on a test of social interaction. Motor learning as assessed by the rotarod test was normal. Dendritic arbors were less complex and spine densities and lengths increased in medial prefrontal cortex, basal lateral amygdala, and hippocampus compared with wild type. Regional rates of cerebral protein synthesis measured in vivo in KI mice were increased. KI mice also had elevated levels of Fmr1 mRNA and decreased levels of FMRP. Our results highlight similarities in phenotype between KI and Fmr1 knockout mice and suggest that the decreased concentration of FMRP contributes to the phenotype in young adult KI mice.

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.

Regional rates of cerebral protein synthesis measured with L-[1-11C]leucine and PET in conscious, young adult men: normal values, variability, and reproducibility.

We report regional rates of cerebral protein synthesis (rCPS) measured with the fully quantitative L-[1-(11)C]leucine positron emission tomography (PET) method. The method accounts for the fraction (lambda) of unlabeled amino acids in the precursor pool for protein synthesis derived from arterial plasma; the remainder (1-lambda) comes from tissue proteolysis. We determined rCPS and lambda in 18 regions and whole brain in 10 healthy men (21 to 24 years). Subjects underwent two 90-min dynamic PET studies with arterial blood sampling at least 2 weeks apart. Rates of cerebral protein synthesis varied regionally and ranged from 0.97+/-0.70 to 2.25+/-0.20 nmol/g per min. Values of rCPS were in good agreement between the two PET studies. Mean differences in rCPS between studies ranged from 9% in cortical regions to 15% in white matter. The lambda value was comparatively more uniform across regions, ranging from 0.63+/-0.03 to 0.79+/-0.02. Mean differences in lambda between studies were 2% to 8%. Intersubject variability in rCPS was on average 6% in cortical areas, 9% in subcortical regions, and 12% in white matter; intersubject variability in lambda was 2% to 8%. Our data indicate that in human subjects low variance and highly reproducible measures of rCPS can be made with the L-[1-(11)C]leucine PET method.

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.