Idiopathic pulmonary fibrosis (IPF) is a severely debilitating disease associated with a dismal prognosis. There are currently no effective therapies for IPF, thus the identification of novel therapeutic targets is greatly needed. The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptors whose activation has been linked to various pathologies. In healthy adult animals, RAGE is expressed at the highest levels in the lung compared to other tissues. To investigate the hypothesis that RAGE is involved in IPF pathogenesis, we have examined its expression in two mouse models of pulmonary fibrosis and in human tissue from IPF patients. In each instance we observed a depletion of membrane RAGE and its soluble (decoy) isoform, sRAGE, in fibrotic lungs. In contrast to other diseases in which RAGE signaling promotes pathology, immunohistochemical and hydroxyproline quantification studies on aged RAGEnull mice indicate that these mice spontaneously develop pulmonary fibrosis-like alterations. Furthermore, when subjected to a model of pulmonary fibrosis, RAGE-null mice developed more severe fibrosis, as measured by hydroxyproline assay and histological scoring, than wild-type controls. Combined with data from other studies on mouse models of pulmonary fibrosis and human IPF tissues indicate that loss of RAGE contributes to IPF pathogenesis. Idiopathic pulmonary fibrosis (IPF) is a debilitating disease with a dismal prognosis. Mean survival time after biopsy-confirmed diagnosis is 3 to 5 years.1,2 Traditional therapy involves the use of corticosteroids as nonspecific anti-inflammatory agents. This treatment produces an objective response in only 10 to 20% of patients and has a minimal effect on the fatal course of IPF.2-4 Thus, the need for new therapeutic modalities is evident.The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin super family of cell surface receptors. 5 In most healthy adult animal tissues, RAGE is expressed at low to undetectable levels.6,7 Activation of membrane-bound RAGE (mRAGE) by its ligands (including advanced glycation end products, HMGB1/amphoterin, S100/calgranulins, and amyloid-␤ peptide) often leads to proinflammatory signaling as well as up-regulation of RAGE itself.8 This signaling by mRAGE is believed to play an important role in disease progression for several nonpulmonary diseases, including various diabetic complications, chronic inflammation, and Alzheimer's disease, among others. 9,10 In contrast to other healthy adult tissues, RAGE mRNA and sRAGE protein are highly expressed in normal adult lungs. 6,7,11 Most recently it has been suggested that RAGE is a marker of type I alveolar epithelial cells 12 and type II alveolar epithelial cell transdifferentiation, a component of pulmonary re-epithelialization and repair.

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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.

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. Fragile X mental retardation protein (FMRP), a putative translation suppressor, is absent or significantly reduced in FXS. One prevailing hypothesis is that rates of protein synthesis are increased by the absence of this regulatory protein. In accord with this hypothesis, we have previously reported increased rates of cerebral protein synthesis (rCPS) in the Fmr1 knockout mouse model of FXS and others have reported similar effects in hippocampal slices. To address the hypothesis in human subjects, we applied the L[1- 11 C]leucine PET method to measure rCPS in adults with FXS and healthy controls. All subjects were males between the ages of 18 and 24 years and free of psychotropic medication. As most fragile X participants were not able to undergo the PET study awake, we used dexmedetomidine for sedation during the imaging studies. We found no differences between rCPS measured during dexmedetomidine-sedation and the awake state in ten healthy controls. In the comparison of rCPS in dexmedetomidine-sedated fragile X participants ( n = 9) and healthy controls ( n = 14) we found no statistically significant differences. Our results from in vivo measurements in human brain do not support the hypothesis that rCPS are elevated due to the absence of FMRP. This hypothesis is based on findings in animal models and in vitro measurements in human peripheral cells. The absence of a translation suppressor may produce a more complex response in pathways regulating translation than previously thought. We may need to revise our working hypotheses regarding FXS and our thinking about potential therapeutics.

Sleep abnormalities are prevalent in autism spectrum disorders (ASD). Moreover, the severity of ASD symptoms are correlated with the degree of disturbed sleep. We asked if disturbed sleep during brain development itself could lead to ASD-like symptoms, particularly behavioral manifestations. We reasoned that sleep is known to be important for normal brain development and plasticity, so disrupted sleep during development might result in changes that contribute to behavioral impairments associated with ASD. We sleep-restricted C57BL/6J male mice [beginning at postnatal day 5 (P5) and continuing through P52] 3 h per day by means of gentle handling and compared the data with a stress group (handled every 15 min during the 3-h period) and a control group (no additional handling). From P42–P52, we assessed the behavioral effects of sleep-restriction in this pre-recovery phase. Then, we allowed the mice to recover for 4 weeks and tested behavior once again. Compared to the control group, we found that sleep restricted-mice had long-lasting hypoactivity, and impaired social behavior; repetitive behavior was unaffected. These behavior changes were accompanied by an increase in the downstream signaling products of the mammalian target of rapamycin pathway. These data affirm the importance of undisturbed sleep during development and show that, at least in this model, sleep-restriction can play a causative role in the development of behavioral abnormalities. Assessing and treating sleep abnormalities in ASD may be important in alleviating some of the symptoms.

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.

Fragile X syndrome (FXS) is caused by silencing of the FMR1 gene and consequent absence of its protein product, fragile X mental retardation protein (FMRP). FMRP is an RNA‐binding protein that can suppress translation. The absence of FMRP leads to symptoms of FXS including intellectual disability and has been proposed to lead to abnormalities in synaptic plasticity. Synaptic plasticity, protein synthesis, and cellular growth pathways have been studied extensively in hippocampal slices from a mouse model of FXS (Fmr1 KO). Enhanced metabotropic glutamate receptor 5 (mGluR5)‐dependent long‐term depression (LTD), increased rates of protein synthesis, and effects on signaling molecules have been reported. These phenotypes were found under amino acid starvation, a condition that has widespread, powerful effects on activation and translation of proteins involved in regulating protein synthesis. We asked if this non‐physiological condition could have effects on Fmr1 KO phenotypes reported in hippocampal slices. We performed hippocampal slice experiments in the presence and absence of amino acids. We measured rates of incorporation of a radiolabeled amino acid into protein to determine protein synthesis rates. By means of western blots, we assessed relative levels of total and phosphorylated forms of proteins involved in signaling pathways regulating translation. We measured evoked field potentials in area CA1 to assess the strength of the long‐term depression response to mGluR activation. In the absence of amino acids, we replicate many of the reported findings in Fmr1 KO hippocampal slices, but in the more physiological condition of inclusion of amino acids in the medium, we did not find evidence of enhanced mGluR5‐dependent LTD. Activation of mGluR5 increased protein synthesis in both wild type and Fmr1 KO. Moreover, mGluR5 activation increased eIF2α phosphorylation and decreased phosphorylation of p70S6k in slices from Fmr1 KO. We propose that the eIF2α response is a cellular attempt to compensate for the lack of regulation of translation by FMRP. Our findings call for a re‐examination of the mGluR theory of FXS.

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