Dysregulation of Protein Synthesis in Fragile X Syndrome and Other Developmental Disorders

本项目涉及的协议为06-M-0214，NCT00362843。 在2016年资助期内，我们致力于以下内容：1)用L-1-C-11亮氨酸PET方法测定FXS患者和服用右旋美托咪胺的健康志愿者的RCP，2)二甲双胍治疗对Fmr1 KO小鼠RCP和行为的影响，3)磷酸二酯酶-4D(PDE-4D)治疗对Fmr1 KO小鼠行为和RCP的影响，4)TSC2+/-小鼠的RCPS，5)Fmr1 KO和TSC2+/-小鼠的睡眠异常，6)睡眠限制对WT小鼠成年期行为表型的影响。 1)脆性X综合征。我们继续对FXS患者的RCPS进行研究。我们用L-1-C-11亮氨酸正电子发射计算机体层摄影方法测定了患者和健康志愿者在右美托咪定镇静状态下的RCP。右美托咪定是一种α-2-肾上腺素能受体激动剂。通过这一策略，我们希望避免异丙酚对患者RCP的选择性影响，因为右旋美托咪定通过完全不同的机制起作用。如果可能，我们也在研究使用修改后的方案唤醒的患者。我们的假设是，与年龄匹配的对照组相比，FXS患者的RCPS发生了改变。这些研究正在进行中。 2)二甲双胍对Fmr1KO小鼠的治疗作用。T.Jonens(宾夕法尼亚大学)最近报道，在果蝇模型FXS(Dfmr1)中，胰岛素信号的升高与行为表型的表达有关。我们正在通过测试二甲双胍对RCP和Fmr1 KO小鼠的行为的疗效来跟进这一发现。二甲双胍是一种用于治疗2型糖尿病的双胍类药物。小鼠被长期当作成人对待，并进行记忆测试和RCP测量。 3)用PDE-4D抑制剂治疗Fmr1KO小鼠。Berry-Kravis(拉什医学院)的研究表明，FXS患者外周细胞中的cAMP减少，神经母细胞瘤细胞中cAMP的产生受FMRP调节。此外，T·容金斯实验室的工作表明，抑制PDE-4可以改善dfmr1苍蝇的记忆缺陷，并将Fmr1 KO小鼠的mGluR依赖LTD恢复到WT水平。与利乐发现合作伙伴合作，我们正在测试他们开发的PDE-4D的负变构调节剂(BPN-14770)的有效性。我们正在用PDE-4D NAM慢性治疗Fmr1KO小鼠，并测量其对行为和RCP的影响。 4)SNPM正在研究的另一种自闭症症状形式是结节性硬化症(TSC)。TSC是一种常染色体显性遗传性神经遗传疾病，表现为癫痫、智能障碍和自闭症的高发生率。TSC是由TSC1或TSC2的突变引起的，TSC1或TSC2编码的蛋白质形成复合体，并与小的GTP结合蛋白Rheb相互作用，抑制mTORC1。MTORC1是核糖体生物发生和翻译起始的中心调节因子，而TSC1/2功能的丧失导致mTORC1活性增加。我们假设小鼠的TSC2(TSC2+/-)单倍体不足会导致RCP增加。到目前为止，我们在自由活动的成年TSC2+/-小鼠体内对RCPS的测量表明，RCPS在选择性脑区域减少。目前的实验正在解决TSC2突变对组织中蛋白质降解产生的亮氨酸循环的影响；这是RCPS方程式中的一个关键因素。 5)睡眠和神经发育障碍。睡眠异常是被诊断为神经发育综合征的患者中最常见的并发障碍之一。在这些患者中，行为异常的严重程度与睡眠异常的严重程度是相关的。鉴于睡眠在发育可塑性中的重要性，我们试图在两种单基因神经发育障碍动物模型FXS和TSC中检查睡眠行为。我们使用家庭笼子监测来调查光明和黑暗阶段的总睡眠时间。我们发现，一个月大的Fmr1KO小鼠在光明和黑暗两个阶段的睡眠时间都少于WT小鼠。在两个月龄的TSC+/-小鼠中，我们发现总睡眠时间低于WT，尤其是在黑暗阶段。未来的研究将解决药物的能力，这些药物可以促进对照动物的睡眠，逆转减少的睡眠时间，并改善行为表型。 6)我们还试图确定发育过程中慢性睡眠限制对WT小鼠随后成年行为的影响。我们通过温和的处理方法，对发育中的P5-P42的WT小鼠每天进行3个小时的睡眠限制，并将行为输出与每天处理10分钟的对照组进行比较。在睡眠限制后和恢复四周后，我们立即检测了患者的野外活动、社会行为、重复行为和焦虑。在六周大的时候，在长期睡眠限制之后，老鼠在开阔的场地上就不那么活跃了。男性和女性的社交能力都有所提高，但重复行为没有变化。经过4周的随意睡眠恢复期后，一些行为异常持续存在，有些变得明显。睡眠受限的小鼠在开场测试开始时活动量减少。雌性老鼠继续增加了社交能力，此外，还增加了对社会新鲜感的偏好。我们发现雄鼠的社交能力有下降的趋势。在睡眠受限的雌性小鼠中，重复行为减少，而在雄性小鼠中，重复行为增加。在睡眠受限的小鼠中，焦虑的测量没有受到影响。这些结果表明，发育过程中的长期睡眠限制可能会导致受性别影响的长期行为变化。我们的研究可能对儿童时期的睡眠障碍在神经发育障碍的发展中所起的作用有所启示。

The protocol involved in this project is 06-M-0214, NCT00362843. During the 2016 funding period, we addressed the following: 1) rCPS measured with the L-1-C-11leucine PET method in patients with FXS and healthy volunteers sedated with dexmedetomidine, 2) The effects of treatment with metformin on rCPS and behavior in Fmr1 KO mice, 3) The effects of treatment with an inhibitor of phosphodiesterase-4D (PDE-4D) on behavior and rCPS in Fmr1 KO mice, 4) rCPS in Tsc2+/- mice, 5) Sleep abnormalities in Fmr1 KO and Tsc2+/- mice, 6) The effects of sleep restriction in WT mice on behavioral phenotype in adulthood. 1) Fragile X syndrome. We continue our studies of rCPS in patients with FXS. We are measuring rCPS with the L-1-C-11leucine PET method in patients and healthy volunteers while sedated with dexmedetomidine. Dexmedetomidine is an alpha-2-adrenergic receptor agonist. With this strategy we expect to avoid the selective effects that propofol had on rCPS in patients because dexmedetomidine acts via a completely different mechanism. When possible we are also studying patients awake with a modified protocol. Our hypothesis is that rCPS is altered in patients with FXS compared with age-matched controls. These studies are ongoing. 2) Metformin treatment of Fmr1 KO mice. T. Jongens (University of Pennsylvania) reported recently that elevated insulin signaling is involved in the expression of behavioral phenotypes in the Drosophila model of FXS (dfmr1). We are following up this finding by testing the efficacy of metformin on rCPS and behavior in Fmr1 KO mice. Metformin is a biguanide drug used in the treatment of type 2 diabetes. Mice are treated chronically as adults and tested on tests of memory and rCPS are measured. 3) Treatment of Fmr1 KO mice with an inhibitor of PDE-4D. Work from Berry-Kravis (Rush College of Medicine) indicates that cAMP is reduced in peripheral cells from patients with FXS and that in neuroblastoma cells cAMP production is regulated by FMRP. Moreover, work from the laboratory of T. Jongens shows that inhibition of PDE-4 ameliorates memory deficits in the dfmr1 fly and restores mGluR-dependent LTD to WT levels in the Fmr1 KO mouse. In collaboration with Tetra Discovery Partners, we are testing the efficacy of a negative allosteric modulator (NAM) of PDE-4D that they have developed (BPN-14770). We are chronically treating Fmr1 KO mice with the PDE-4D NAM and measuring its effects on behavior and rCPS. 4) Another syndromic form of autism under study in the SNPM is tuberous sclerosis complex (TSC). TSC is an autosomal dominant neurogenetic disorder manifested by a high incidence of seizures, intellectual disability, and autism. TSC is caused by mutations in either TSC1 or TSC2, which encode for proteins that form a complex and interact with a small GTP-binding protein, RHEB, to inhibit mTORC1. mTORC1 is a central regulator of ribosomal biogenesis and translation initiation, and loss of TSC1/2 function results in increased activity of mTORC1. We hypothesized that haploinsufficiency of TSC2 (Tsc2+/-) in mice would lead to increased rCPS. To date, our in vivo measurements of rCPS in freely-moving awake, adult, male Tsc2+/- mice indicate that rCPS is decreased in selective brain regions. Current experiments are addressing the effects of the mutation in TSC2 on recycling of leucine derived from protein degradation in the tissue; this is a critical factor in the equation for rCPS. 5) Sleep and neurodevelopmental disorders. Sleep abnormalities are one of the most prevalent concurrent disorders in patients diagnosed with neurodevelopmental syndromes. In these patients, the severity of behavioral abnormalities and the severity of sleep abnormalities are correlated. Given the importance of sleep in developmental plasticity, we sought to examine sleep behavior in two animal models of single gene neurodevelopmental disorders, FXS and TSC. We used home cage monitoring to investigate total sleep times during the light and dark phases. We found that Fmr1 KO mice at one month of age spend less time sleeping than WT mice in both the light and dark phases. In Tsc+/- mice at two months of age we found that total sleep time was lower than WT particularly in the dark phase. Future studies will address the ability of drugs that can facilitate sleep in control animals to effect reversal of the reduced sleep duration and to improve behavioral phenotypes. 6) We also sought to determine the effects of chronic sleep-restriction during development on subsequent adult behavior in WT mice. We sleep-restricted developing WT mice from P5-P42 for three hours per day by means of gentle handling and compared behavioral outputs to controls who were handled ten minutes daily. We assayed activity in the open field, social behavior, repetitive behavior, and anxiety immediately following sleep restriction and after four weeks recovery. At six weeks of age, immediately following chronic sleep-restriction, mice were less active in an open field arena. Sociability was increased, but repetitive behaviors were unchanged in both males and females. After a 4-week period of recovery with sleep ad libitum, some behavioral abnormalities persisted and some became apparent. Sleep-restricted mice had decreased activity in the beginning of an open field test. Female mice continued to have increased sociability and, in addition, increased preference for social novelty. We saw a trend toward decreased sociability in male mice. Repetitive behavior was decreased in sleep-restricted female mice and increased in males. Measures of anxiety were not affected in the sleep-restricted mice. These results indicate that chronic sleep restriction during development can lead to long-lasting behavioral changes that are modulated by sex. Our study may have implications for a role of sleep disorders in childhood on the unfolding of neurodevelopmental disorders.