Nie Duyu, Chen Zehua, Ebrahimi-Fakhari Darius, Di Nardo Alessia, Julich Kristina, Robson Victoria K, Cheng Yung-Chih, Woolf Clifford J, Heiman Myriam, Sahin Mustafa
F.M. Kirby Neurobiology Center, Department of Neurology, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts 02115.
Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts 02142, and.
J Neurosci. 2015 Jul 29;35(30):10762-72. doi: 10.1523/JNEUROSCI.4796-14.2015.
Hyperactivation of the mechanistic target of rapamycin (mTOR) kinase, as a result of loss-of-function mutations in tuberous sclerosis complex 1 (TSC1) or TSC2 genes, causes protein synthesis dysregulation, increased cell size, and aberrant neuronal connectivity. Dysregulated synthesis of synaptic proteins has been implicated in the pathophysiology of autism spectrum disorder (ASD) associated with TSC and fragile X syndrome. However, cell type-specific translational profiles in these disease models remain to be investigated. Here, we used high-fidelity and unbiased Translating Ribosome Affinity Purification (TRAP) methodology to purify ribosome-associated mRNAs and identified translational alterations in a rat neuronal culture model of TSC. We find that expression of many stress and/or activity-dependent proteins is highly induced while some synaptic proteins are repressed. Importantly, transcripts for the activating transcription factor-3 (Atf3) and mitochondrial uncoupling protein-2 (Ucp2) are highly induced in Tsc2-deficient neurons, as well as in a neuron-specific Tsc1 conditional knock-out mouse model, and show differential responses to the mTOR inhibitor rapamycin. Gelsolin, a known target of Atf3 transcriptional activity, is also upregulated. shRNA-mediated block of Atf3 induction suppresses expression of gelsolin, an actin-severing protein, and rescues spine deficits found in Tsc2-deficient neurons. Together, our data demonstrate that a cell-autonomous program consisting of a stress-induced Atf3-gelsolin cascade affects the change in dendritic spine morphology following mTOR hyperactivation. This previously unidentified molecular cascade could be a therapeutic target for treating mTORopathies.
Tuberous sclerosis complex (TSC) is a genetic disease associated with epilepsy and autism. Dysregulated protein synthesis has been implicated as a cause of this disease. However, cell type-specific translational profiles that are aberrant in this disease are unknown. Here we show that expression of many stress and/or activity-dependent proteins is highly induced while some synaptic proteins are repressed in neurons missing the Tsc2 gene expression. Identification of genes whose translation is abnormal in TSC may provide insights to previously unidentified therapeutic targets.
由于结节性硬化症复合体1（TSC1）或TSC2基因的功能丧失突变，雷帕霉素机制靶点（mTOR）激酶的过度激活会导致蛋白质合成失调、细胞大小增加和异常的神经元连接。突触蛋白的合成失调与与结节性硬化症（TSC）和脆性X综合征相关的自闭症谱系障碍（ASD）的病理生理学有关。然而，这些疾病模型中细胞类型特异性的翻译谱仍有待研究。在这里，我们使用高保真且无偏差的翻译核糖体亲和纯化（TRAP）方法来纯化与核糖体相关的mRNA，并在TSC的大鼠神经元培养模型中鉴定翻译改变。我们发现许多应激和/或活性依赖性蛋白的表达被高度诱导，而一些突触蛋白则被抑制。重要的是，激活转录因子3（Atf3）和线粒体解偶联蛋白2（Ucp2）的转录本在Tsc2缺陷神经元以及神经元特异性Tsc1条件性敲除小鼠模型中被高度诱导，并对mTOR抑制剂雷帕霉素表现出不同的反应。凝溶胶蛋白是Atf3转录活性的已知靶点，也被上调。shRNA介导的Atf3诱导阻断抑制了肌动蛋白切割蛋白凝溶胶蛋白 的表达，并挽救了Tsc2缺陷神经元中发现的树突棘缺陷。总之，我们的数据表明，由应激诱导的Atf3 - 凝溶胶蛋白级联组成的细胞自主程序会影响mTOR过度激活后树突棘形态的变化。这个以前未被识别的分子级联可能是治疗mTOR病的治疗靶点。
结节性硬化症复合体（TSC）是一种与癫痫和自闭症相关的遗传疾病。蛋白质合成失调被认为是这种疾病的一个原因。然而，这种疾病中异常的细胞类型特异性翻译谱尚不清楚。在这里我们表明，在缺失Tsc2基因表达的神经元中，许多应激和/或活性依赖性蛋白的表达被高度诱导，而一些突触蛋白则被抑制。鉴定TSC中翻译异常的基因可能为以前未被识别的治疗靶点提供见解。
