Retrotransposons in Schizophrenia

 描述（由申请人提供）：精神分裂症（SZ）是一种常见的慢性精神病性脑部疾病，影响1%的美国人口，由于相关的残疾、发病率和死亡率，造成了严重的公共卫生问题。SZ的发病机制知之甚少，但它被认为是一种神经发育障碍。神经影像学证据表明，背外侧前额叶皮层（DLPFC）功能异常，在SZ，无论是在激活和休息。此外，EEG证据已经确定SZ中的γ振荡异常，表明皮质锥体神经元的小白蛋白阳性GABA能中间神经元调节功能障碍。 在过去的5年中，积累的数据证明LINE 1（L1）反转录转座子（RTPs）的激活可能发生在染色质处于松弛状态以允许转录激活的任何地方。SZ中异常的DLPFC激活和γ振荡可能驱动慢性异常转录激活，从而为L1在发育中的CNS中逆转录转座提供机会。这些体细胞新生L1可能会干扰正常的神经元活动，因为它们插入了神经元正常功能所需的基因。如果在CNS发育早期出现一个或多个功能性新生L1，则所有源自该神经元前体的子神经元也将携带L1，可能导致神经元功能障碍，从而增加SZ的风险。 该项目建立在初步结果的基础上，初步结果表明，与对照组织相比，在SZ DLPFC神经元DNA的相关基因本体（例如，突触部分和质膜部分; p = ~10-4）中，基因内新生L1的发现频率显著更高。该提案将采用来自100名SZ患者和100名匹配对照的DLPFC和前扣带回组织（尸检时获得）。使用差速离心和荧光辅助细胞分选（FACS），然后通过PCR富集DNA中的L1序列，对DNA扩增子进行测序并与参考基因组比对，以检测脑表达基因中的基因内从头（不在参考基因组中）L1。最有希望的基因内新生L1的等位基因频率将通过液滴数字PCR进行估计。选择的基因内从头L1将通过CRISPR/Cas9技术在神经元细胞系中重新产生。将确定从头L1对基因转录和翻译的功能影响。以这种方式，预计将发现增加SZ风险的基因内新生L1。

 DESCRIPTION (provided by applicant): Schizophrenia (SZ) is a common, chronic group of psychotic brain disorders, affecting 1% of the US population, creating a significant public health problem because of the associated disability, morbidity and mortality. The pathogenesis of SZ is poorly understood, but it is thought to be a neurodevelopmental disorder. Neuroimaging evidence has accumulated to indicate that the dorsolateral prefrontal cortex (DLPFC) functions abnormally in SZ, both when activated and at rest. Further, EEG evidence has identified abnormalities of γ oscillations in SZ, suggesting that parvalbumin positive GABAergic interneuron regulation of cortical pyramidal neurons is dysfunctional. In the past 5 years, data have accumulated to prove that activation of LINE1 (L1) retrotransposons (RTPs) may occur wherever chromatin assumes a relaxed state to permit transcriptional activation. The abnormal DLPFC activation and γ oscillations in SZ may drive chronic aberrant transcriptional activation, thereby providing opportunities for L1s to retrotranspose in the developing CNS. These somatic de novo L1s may interfere with normal neuronal activity because they have inserted into a gene needed by that neuron for normal function. If one or more functional de novo L1s occur early in CNS development, all the daughter neurons that derive from that neuronal precursor will also carry the L1, perhaps leading to a dysfunctional population of neurons destined to increase risk for SZ. This project builds on preliminary results indicating that intragenic de novo L1s are found significantly more often in SZ DLPFC neuronal DNA in relevant gene ontologies (eg, synapse part and plasma membrane part; p = ~10-4), compared to control tissue. This proposal will employ DLPFC and anterior cingulate tissue (obtained at autopsy) from 100 SZ patients and 100 matched controls. Using differential centrifugation and fluorescence assisted cell sorting (FACS), followed by PCR to enrich the DNA for L1 sequences, DNA amplicons will be sequenced and aligned to the reference genome to detect intragenic de novo (not in the reference genome) L1s in brain-expressed genes. Allele frequencies of the most promising intragenic de novo L1s will be estimated by droplet digital PCR. Selected intragenic de novo L1s will be re-created in neuronal cell lines via CRISPR/Cas9 technology. The functional effect of the de novo L1 on transcription and translation of the gene will be determined. In this manner, it is expected that intragenic de novo L1s, which increase risk for SZ, will be discovered.