Phenotypic rescue via mTOR inhibition in neuron-specific Pten knockout mice reveals AKT and mTORC1-site specific changes

Phosphatase and Tensin Homolog (PTEN) is a dual-specific protein and lipid phosphatase that regulates AKT and downstream signaling of the mechanistic target of rapamycin (mTOR). PTEN functions as a tumor suppressor gene whose mutations result in PTEN Hamartoma Tumor Syndrome (PHTS) characterized by increased cancer risk and neurodevelopmental comorbidity. Here, we generated a novel neuron-specific Pten knock-out mouse model (Syn-Cre/Pten HOM) to test the ability of pharmacologic mTOR inhibition to rescue Pten mutation-associated disease phenotypes in vivo and in vitro. We found that treatment with the mTOR inhibitor, everolimus, increased the survival of Syn-Cre/Pten HOM mice while some neurologic phenotypes persisted. Transcriptomic analyses revealed that in contrast to mice harboring a neuron-specific deletion of the Tuberous Sclerosis Complex 2 gene (Syn-Cre/Tsc2 KO), genes that are under AKT regulation were significantly increased in the Syn-Cre/Pten HOM mice. In addition, genes associated with synapse, extracellular matrix, and myelination were broadly increased in Syn-Cre/Pten HOM mouse neocortex. These findings were confirmed by immunostaining of cortical sections in vivo, which revealed excessive immunoreactivity of myelin basic protein and perineuronal nets (PNN), the specialized extracellular matrix surrounding fast-spiking parvalbumin (PV) interneurons. We also detected increased expression of Synapsin I/PSD95 positive synapses and network hyperactivity phenotypes in Syn-Cre/Pten HOM mice neurons compared to wild-type (WT) neurons in vitro. Strikingly, everolimus treatment rescued the number of synapses and network hyperactivity in the Syn-Cre/Pten HOM mice cortical neuron cultures. Taken together, our results revealed in vivo and in vitro molecular and neuronal network mechanisms underlying neurological phenotypes of PHTS. Notably, pharmacologic mTOR inhibition by everolimus led to successful downstream signaling rescue, including mTOR complex 1 (mTORC1) site-specific suppression of S6 phosphorylation, correlating with phenotypic rescue found in our novel neuron-specific Syn-Cre/Pten HOM mice. Overall design: Pten homozygous and heterozygous mutant mice of the indicated genotypes were sacrificed at P45 (n=4 HET, 4 HOM), and the cortex was collected. RNA extraction was performed using TRIzol (Invitrogen). Total RNA was used as an input for cDNA library preparation using the NEBNext Ultra II RNA Library Prep Kit by Illumina, as per manufacturer's protocols. Sequencing was performed on an Illumina analyzer, and 150bp paired-end reads were obtained. Reads passing quality filters were mapped using Hisat2 (http://daehwankimlab.github.io/hisat2/; v2.0.5) onto the mouse genome. For differential expression, raw gene counts from protein coding genes from heterozygous and homozygous animals were used as input for EdgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html). Functional annotation was performed using DAVID (https://david.ncifcrf.gov/summary.jsp), and redundant or non-specific categories were manually removed. Comparison with ASD candidate genes was performed by comparing differentially expressed genes to SFARI genes (category 1-3). For RNA sequencing of Tsc2 model (previously described by Yuan and colleagues29), mice from the indicated genotypes were sacrificed at P30 and frontal cortex was dissected. Total RNA was isolated using a Qiagen RNAeasy kit, and cDNA sequencing libraries were constructed with the Kapa Hyperprep library kit using polyA selection (Roche). Sequencing was performed on an Illumina analyzer, and 75bp single-end reads were obtained. Reads passing quality filters were mapped onto the mouse genome using Hisat2. For WGCNA, the WGCNA package from CRAN was used, and gene counts from protein coding genes from both Pten mutant and Tsc2 mutant mice were normalized using log2 counts per million separately. Co-expression networks were created from genes with a coefficient of variation > 0.005 using b=6, and clusters were isolated using the cutreeDynamic package. Module overlap was determined by calculating the Pearson correlation of the kME between two modules.

张力蛋白同源磷酸酶（Phosphatase and Tensin Homolog, PTEN）是一种双特异性蛋白脂质磷酸酶，可调控AKT及雷帕霉素靶蛋白（mechanistic target of rapamycin, mTOR）的下游信号通路。PTEN是一种抑癌基因，其突变可引发PTEN错构瘤肿瘤综合征（PTEN Hamartoma Tumor Syndrome, PHTS），该疾病以癌症风险升高及神经发育共病为特征。本研究构建了一种新型神经元特异性Pten敲除小鼠模型（Syn-Cre/Pten HOM），旨在探究雷帕霉素靶蛋白（mTOR）的药物抑制策略，能否在体内及体外挽救Pten突变相关的疾病表型。
研究发现，使用mTOR抑制剂依维莫司（everolimus）处理可提升Syn-Cre/Pten HOM小鼠的存活率，但部分神经表型仍持续存在。转录组分析显示，与神经元特异性敲除结节性硬化症2基因（Tuberous Sclerosis Complex 2, Tsc2）的小鼠（Syn-Cre/Tsc2 KO）相比，Syn-Cre/Pten HOM小鼠体内受AKT调控的基因表达水平显著升高。此外，Syn-Cre/Pten HOM小鼠新皮层中，与突触、细胞外基质及髓鞘形成相关的基因表达普遍上调。
上述发现经体内皮层切片免疫染色得到验证：髓鞘碱性蛋白（myelin basic protein）及神经元周围网状结构（perineuronal nets, PNN）——包绕快速放电小白蛋白（parvalbumin, PV）中间神经元的特异性细胞外基质——的免疫反应性异常升高。体外实验中，与野生型（wild-type, WT）神经元相比，Syn-Cre/Pten HOM小鼠神经元中突触素I（Synapsin I）/突触后致密蛋白95（PSD95）阳性突触的表达量及神经网络过度兴奋表型均有所升高。值得注意的是，依维莫司处理可挽救Syn-Cre/Pten HOM小鼠皮层神经元培养物中的突触数量及神经网络过度兴奋表型。
综上，本研究揭示了PHTS神经表型背后的体内外分子及神经网络机制。值得关注的是，依维莫司介导的mTOR药物抑制可成功挽救下游信号通路，包括特异性抑制雷帕霉素靶蛋白复合物1（mTOR complex 1, mTORC1）的S6磷酸化位点，这与我们构建的新型神经元特异性Syn-Cre/Pten HOM小鼠的表型挽救结果相一致。

实验设计：将指定基因型的Pten纯合及杂合突变小鼠于出生后第45天（P45）处死（杂合组n=4，纯合组n=4），采集其皮层组织。采用TRIzol试剂（Invitrogen）提取总RNA，按照制造商方案，使用Illumina NEBNext Ultra II RNA文库制备试剂盒构建cDNA文库。随后在Illumina测序仪上完成测序，获得150bp双端读段。通过质量过滤的读段使用Hisat2（http://daehwankimlab.github.io/hisat2/; v2.0.5）比对至小鼠基因组。差异表达分析方面，以杂合及纯合突变小鼠的蛋白编码基因原始计数作为输入，通过EdgeR工具（https://bioconductor.org/packages/release/bioc/html/edgeR.html）完成分析。功能注释采用DAVID数据库（https://david.ncifcrf.gov/summary.jsp），并手动去除冗余或非特异性的分类条目。将差异表达基因与SFARI基因（1-3类）进行比对，以分析其与自闭症谱系障碍（ASD）候选基因的重叠情况。
针对Tsc2模型的RNA测序（此前由Yuan及其团队29报道）：将指定基因型的小鼠于出生后第30天（P30）处死，分离其额叶皮层。采用Qiagen RNAeasy试剂盒提取总RNA，使用Kapa Hyperprep文库试剂盒并通过polyA富集（Roche）构建cDNA测序文库。随后在Illumina测序仪上完成测序，获得75bp单端读段。通过质量过滤的读段使用Hisat2比对至小鼠基因组。加权基因共表达网络分析（Weighted Gene Co-expression Network Analysis, WGCNA）采用CRAN的WGCNA软件包完成，将Pten突变及Tsc2突变小鼠的蛋白编码基因计数分别以每百万计数的log2值进行标准化。选取变异系数>0.005的基因，以参数b=6构建共表达网络，并使用cutreeDynamic软件包分离基因簇。通过计算两个模块间kME值的皮尔逊相关系数，确定模块间的重叠程度。