quantitative-autoradiographic-method-for-determination-regional-rates

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...

National Institutes of Health

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.

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

Several lines of clinical evidence support the idea that fragile X syndrome (FXS) may involve a dysregulation of hypothalamic-pituitary-adrenal axis function [Wisbeck et al. (2000) J Dev Behav Pediatr 21:278-282; Hessl et al. (2002) Psychoneuroendocrinology 27:855-872]. We had tested this idea in a mouse model of FXS (Fmr1 KO) and found that the hormonal response to acute stress was similar to that of wild-type (WT) mice [Qin and Smith (2008) Psychoneuroendocrinology 33:883-889]. We report here responses to chronic stress (CS) in Fmr1 KO mice. Following restraint for 120 min/d, 10 consecutive days, we assessed dendrite and spine morphology in basolateral amygdala (BLA). We also monitored behavior in an elevated plus maze (EPM) and the hormonal response to this novel spatial environment. After CS, mice of both genotypes underwent adrenal hypertrophy, but effects were greater in WT mice. Behavior in the EPM indicated that only WT mice had the expected increase in anxiety following CS. Serum corticosterone and adrenocorticotropic hormone (ACTH) levels were both increased following the spatial novelty of EPM, and there were no differences between genotypes in the hormonal responses. BLA dendritic branching increased proximal to the soma in WT, but in Fmr1 KO mice branching was unaffected close to the soma and slightly decreased at one point distal to the soma. Similarly, spine density on apical and basal dendrites increased in WT but decreased in Fmr1 KO mice. Spine length on apical and basal dendrites increased in WT but was unaffected in Fmr1 KO mice. These differences in behavioral response and effects on neuron morphology in BLA suggest a diminished adaptive response of Fmr1 KO mice.

Effects of chronic immobilization stress on anxiety-like behavior and basolateral amygdala morphology in Fmr1 knockout mice.

Individuals with fragile X syndrome (FXS), an inherited form of cognitive disability, have a wide range of symptoms including hyperactivity, autistic behavior, seizures and learning deficits. FXS is caused by silencing of FMR1 and the consequent absence of fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that associates with polyribosomes and negatively regulates translation. In a previous study of a mouse model of FXS (Fmr1 knockout (KO)) we demonstrated that in vivo rates of cerebral protein synthesis (rCPS) were elevated in selective brain regions suggesting that the absence of FMRP in FXS may result in dysregulation of cerebral protein synthesis. Lithium, a drug used clinically to treat bipolar disorder, has been used to improve mood dysregulation in individuals with FXS. We reported previously that in the Fmr1 KO mouse chronic dietary lithium treatment reversed or ameliorated both behavioral and morphological abnormalities. Herein we report that chronic dietary lithium treatment reversed the increased rCPS in Fmr1 KO mice with little effect on wild type mice. We also report our results of analyses of key signaling molecules involved in regulation of mRNA translation. Our analyses indicate that neither effects on the PI3K/Akt nor the MAPK/ERK 1/2 pathway fully account for the effects of lithium treatment on rCPS. Collectively our findings and those from other laboratories on the efficacy of lithium treatment in animal models support further studies in patients with FXS.

Lithium reverses increased rates of cerebral protein synthesis in a mouse model of fragile X syndrome.

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. 

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

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.

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

Tuberous sclerosis complex (TSC) is an autosomal dominant neurogenetic disorder affecting about 1 in 6000 people and is caused by mutations in either TSC1 or TSC2. This disorder is characterized by increased activity of mammalian target of rapamycin complex 1 (mTORC1), which is involved in regulating ribosomal biogenesis and translation initiation. We measured the effects of Tsc2 haploinsufficiency (Tsc2 ) in 3-month-old male mice on regional rates of cerebral protein synthesis (rCPS) by means of the in vivo L-[1- C]leucine method. This quantitative autoradiographic method includes an estimate of the integrated specific activity of the tracer amino acid in brain tissue. The estimate accounts for recycling of unlabeled amino acids from tissue protein breakdown by means of a factor (λ) that was determined in control and Tsc2 mice. The value of λ was higher in Tsc2 mice, indicating that a greater fraction of leucine in the tissue precursor pool for protein synthesis is derived from the plasma compared to controls, consistent with reduced rates of protein degradation. We determined rCPS in freely moving, awake male Tsc2 and control mice, and we used the determined values of λ in the calculation of rCPS. Unexpectedly, we found that rCPS were significantly decreased in 16 of the 17 brain regions analyzed in Tsc2 mice compared to controls. Our results indicate a complex role of mTORC1 in the regulation of cerebral protein synthesis that has not been previously recognized.

Decreased rates of cerebral protein synthesis measured in vivo in a mouse model of Tuberous Sclerosis Complex: unexpected consequences of reduced tuberin.

Article	Year
A mouse model of the fragile X premutation: effects on behavior, dendrite morphology, and regional rates of cerebral protein synthesis. Neurobiology of disease\| PubMed ID: 21220020	2011
Effects of chronic immobilization stress on anxiety-like behavior and basolateral amygdala morphology in Fmr1 knockout mice. Neuroscience\| PubMed ID: 21723920	2011
Lithium reverses increased rates of cerebral protein synthesis in a mouse model of fragile X syndrome. Neurobiology of disease\| PubMed ID: 22227453	2012
Cerebral protein synthesis in a knockin mouse model of the fragile X premutation. ASN neuro\| PubMed ID: 25290064	2014
R-Baclofen Reverses a Social Behavior Deficit and Elevated Protein Synthesis in a Mouse Model of Fragile X Syndrome. The international journal of neuropsychopharmacology\| PubMed ID: 25820841	2015
Decreased rates of cerebral protein synthesis measured in vivo in a mouse model of Tuberous Sclerosis Complex: unexpected consequences of reduced tuberin. Journal of neurochemistry\| PubMed ID: 29364507	2018

Tianjian Huang

Biography

JoVE Journal Publications

Other Publications