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

根据方案06-M-0214，NCT 00362843进行人体研究。 于二零二零年融资期间，我们处理以下事项：1）在清醒状态下研究的FXS参与者和健康志愿者中使用L-1-C-11亮氨酸PET方法测量的rCPS，2）其他神经发育障碍小鼠模型中的蛋白质合成率，3）mTORC 1活性作为某些神经发育障碍中脑蛋白质合成的中枢调节剂，4）睡眠和神经发育障碍，5）用磷酸二酯酶D抑制剂处理Fmr 1 KO小鼠对行为和rCPS的影响。 1)脆性X综合征患者rCPS的测量采用L-1-C-11亮氨酸PET法测定rCPS。我们的工作重点是在不使用镇静剂的情况下研究脆性X综合征患者。我们使用我们修改的PET协议，使一些脆弱的X参与者在清醒状态下接受研究。我们的初步结果表明，与健康对照组相比，清醒的脆性X参与者的大脑蛋白质合成率降低。我们已经完成了数据收集，并正在分析结果。 2)其他几种神经发育障碍的遗传基础表明，翻译控制缺陷可能是一个核心表型。例如，结节性硬化症（TSC）是由TSC 1或TSC 2基因中的杂合突变引起的，这两种基因编码的蛋白质都是mTOR活性的负调节因子。mTOR通路是调节蛋白质合成的节点之一。这种控制的破坏被认为是TSC中观察到的一些表型的基础。我们已经测量了TSC，TSC 2 +/-小鼠模型中的rCPS，并发现rCPS在整个大脑中降低。我们继续在其他神经发育障碍的小鼠模型（如Shank 3 KO小鼠）中研究rCPS。我们还在TSC 2的条件性敲除小鼠模型中测量rCPS。这些研究正在实验室进行。 3)据报道，在Fmr 1 KO小鼠中，mTOR通路被过度激活。我们正在研究Fmr 1 KO小鼠中mTORC 1激活增加的后果。我们用mTORC 1抑制剂雷帕霉素长期治疗Fmr 1 KO小鼠，以确定雷帕霉素治疗是否可以逆转行为表型。我们发现，pS 6在Fmr 1基因敲除小鼠中上调，并通过雷帕霉素治疗恢复正常，但是，除了致焦虑作用外，雷帕霉素并没有逆转任何行为表型。此外，雷帕霉素治疗对对照和Fmr 1 KO小鼠的睡眠和社会行为都有不良影响。在雷帕霉素处理的Fmr 1 KO小鼠中进行的rCPS研究正在进行中。我们正在体内测量rCPS并确定这些小鼠的行为表型。 4)睡眠和神经发育障碍。睡眠异常是神经发育综合征患者最常见的并发症之一。在这些患者中，行为异常的严重程度和睡眠异常的严重程度相关。鉴于睡眠在发育可塑性中的重要性，我们继续研究睡眠在神经发育障碍中的作用。我们正在收集许多神经发育障碍小鼠模型的睡眠数据，包括Shank 3 KO，催产素受体KO和Tsc 2 +/-小鼠。此外，我们正在用催眠药治疗Fmr 1 KO小鼠，以确定对睡眠和其他行为表型的影响。 5)在人类、小鼠和果蝇中的研究表明，FXS中FMRP的缺乏影响cAMP水平。 我们的研究调查了靶向cAMP水平可能对FXS具有治疗价值的可能性。我们评估了在Fmr 1 KO小鼠中长期给予磷酸二酯酶-4D负变构调节剂BPN 14770的治疗和神经化学作用。 BPN 14770给药对Fmr 1 KO小鼠的行为表型影响有限。 一些影响，如增加睡眠时间和增加社会行为发生在两种基因型。 在WT小鼠中对旷场行为的影响更显著。 WT小鼠中的BPN 14770处理倾向于在最高剂量下增加rCPS，但BPN 14770在Fmr 1 KO小鼠中的作用不太明显。 结果表明，基因型差异的翻译通过cAMP依赖性途径的调节。

The work on humans was conducted under the protocol 06-M-0214, NCT00362843. During the 2020 funding period, we addressed the following: 1) rCPS measured with the L-1-C-11leucine PET method in participants with FXS and healthy volunteers studied in the awake state, 2) protein synthesis rates in mouse models of other neurodevelopmental disorders, 3) mTORC1 activity as a central regulator of brain protein synthesis in some neurodevelopmental disorders, 4) sleep and neurodevelopmental disorders, 5) effects of treatment of Fmr1 KO mice with a phosphodiesterase D inhibitor on behavior and rCPS. 1) Measurement of rCPS in humans with fragile X syndrome. We used the L-1-C-11leucine PET method to measure rCPS. We have focused our efforts on trying to study individuals with fragile X syndrome without the use of sedatives. We used our modified PET protocol to enable some fragile X participants to undergo the study awake. Our preliminary results indicate that rates of cerebral protein synthesis in the awake fragile X participants are decreased compared to healthy controls. We have completed data collection and are analyzing results. 2) The genetic bases of several other neurodevelopmental disorders suggest that defects in translational control may be a core phenotype. For example, tuberous sclerosis complex (TSC) is caused by heterozygous mutations in either the TSC1 or TSC2 gene, both of which encode proteins that are negative regulators of mTOR activity. The mTOR pathway is one of the nodes regulating protein synthesis. Disruption of this control is thought to underlie some of the phenotypes observed in TSC. We have measured rCPS in a mouse model of TSC, TSC2+/-, and found that rCPS are decreased throughout the brain. We continue studies of rCPS in mouse models of other neurodevelopmental disorders such as Shank3 KO mice. We are also measuring rCPS in a conditional knockout mouse model of TSC2. These studies are ongoing in the laboratory. 3) It has been reported that the mTOR pathway is overactivated in Fmr1 KO mice. We are investigating the consequences of increased mTORC1 activation in Fmr1 KO mice. We treated Fmr1 KO mice chronically with an mTORC1 inhibitor, rapamycin, to determine if rapamycin treatment could reverse behavioral phenotypes. We found that pS6 was upregulated in Fmr1 KO mice and normalized by rapamycin treatment, but, except for an anxiogenic effect, rapamycin did not reverse any of the behavioral phenotypes examined. Moreover, rapamycin treatment had an adverse effect on sleep and social behavior in both control and Fmr1 KO mice. Studies of rCPS in rapamycin-treated Fmr1 KO mice are ongoing. We are measuring rCPS in vivo and determining behavioral phenotypes in these mice. 4) 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 continue studies on the role of sleep in neurodevelopmental disorders. We are collecting sleep data on numerous mouse models of neurodevelopmental disorders including Shank3 KO, Oxytocin receptor KO, and Tsc2+/- mice. Additionally, we are treating Fmr1 KO mice with hypnotics to determine the effects on sleep and other behavioral phenotypes. 5) Studies in humans, mice and Drosophila indicate that a deficit of FMRP in FXS influences cAMP levels. Our study investigated the possibility that targeting cAMP levels may have therapeutic value in FXS. We assessed the therapeutic and neurochemical effects of chronic administration of the phosphodiesterase-4D negative allosteric modulator, BPN14770, in Fmr1 KO mice. BPN14770 treatment had limited effects on the behavioral phenotype in Fmr1 KO mice. Some effects such as increased sleep duration and increased social behavior occurred in both genotypes. Effects on open field behavior were more striking in WT mice. BPN14770 treatment in WT mice tended to increase rCPS at the highest dose, but BPN14770 effects were less apparent in Fmr1 KO mice. Results suggest a genotype difference in the regulation of translation via a cAMP-dependent pathway.