Nuclear import of the DSCAM-cytoplasmic domain drives signaling capable of inhibiting synapse formation. Nuclear import of the DSCAM-cytoplasmic domain drives signaling capable of inhibiting synapse formation

DSCAM and DSCAML1 are immunoglobulin and cell adhesion-type receptors serving important neurodevelopmental functions including control of axon growth, branching, neurite self-avoidance and neuronal cell death. The signal transduction mechanisms or effectors of DSCAM receptors however, remain poorly characterized. We used a human ORFeome library to perform a high-throughput screen in mammalian cells and identified novel cytoplasmic signaling effector candidates including the Down syndrome kinase Dyrk1a, STAT3, USP21, and SH2D2A. Unexpectedly, we further found that the intracellular domains (ICDs) of DSCAM and DSCAML1 specifically and directly interact with IPO5, a nuclear import protein of the beta-importin family, via a conserved nuclear localization signal. The DSCAM ICD is released by gamma-secretase dependent cleavage and both the DSCAM and DSCAML1 ICDs efficiently translocate to the nucleus. Furthermore, RNA-sequencing confirms that expression of the DSCAM as well as the DSCAML1 ICDs alone can profoundly alter the expression of genes associated with neuronal differentiation and apoptosis, as well as synapse formation and function. Gain-of-function experiments using primary cortical neurons show that increasing the levels of either the DSCAM or the DSCAML1 ICD leads to an impairment of neurite growth. Strikingly, increased expression of either full-length DSCAM or the DSCAM ICD, but not the DSCAML1 ICD, significantly decreases synapse numbers in primary hippocampal neurons. Taken together, we identified a novel membrane-to-nucleus signaling mechanism by which DSCAM receptors can alter the expression of regulators of neuronal differentiation, synapse formation and function. Considering that chromosomal duplications lead to increased DSCAM expression in trisomy 21 our findings may help to uncover novel mechanisms contributing to intellectual disability in Down syndrome. Overall design: We generated four isogenic HEK293 t-Rex-Flp-In cell lines. The HEK293 t-Rex-Flp-In expression system offers stable, Tetracycline inducible and homogeneous expression of transgenes from a defined genomic locus. The four cell lines either (1) stably express the YFP-HA-tagged DSCAM ICD, (2) stably express the YFP-HA-tagged DSCAML1 ICD, (3) serve as a cytoplasmic YFP-HA control or (4) serve as a nuclear YFP-HA control. For each cell line, three independent replicates of transgene expression were analyzed by RNA deep sequencing (RNA-Seq). The global transcriptome changes of cells expressing the YFP-tagged DSCAM/L1 ICDs versus cells expressing nuclear YFP-HA (YFP-nls) as control were determined. Genes that were already differentially expressed in cytoplasmic versus nuclear YFP expressing cells (p- value ≤ 0.1) were excluded from the analysis, as these genes are unlikely affected by DSCAM/L1 ICD expression.

唐氏综合征细胞黏附分子（DSCAM）与DSCAM样蛋白1（DSCAML1）属于免疫球蛋白样细胞黏附型受体，具备重要的神经发育调控功能，包括调控轴突生长、分支、神经突自我回避以及神经元细胞死亡。然而，目前对于DSCAM受体的信号转导机制及其效应分子仍知之甚少。
我们借助人类开放阅读框文库（human ORFeome library）在哺乳动物细胞中开展高通量筛选，鉴定出包括唐氏综合征激酶Dyrk1a、STAT3、USP21及SH2D2A在内的新型胞质信号效应分子候选靶点。
意外的是，我们进一步发现DSCAM与DSCAML1的胞内结构域（intracellular domains, ICDs）可通过保守的核定位信号（nuclear localization signal, NLS），特异性直接结合β-输入蛋白家族的核输入蛋白IPO5。
DSCAM的ICD经γ-分泌酶依赖性切割后被释放，且DSCAM与DSCAML1的ICD均可高效易位进入细胞核。
此外，RNA测序（RNA-sequencing, RNA-Seq）结果证实，单独表达DSCAM或DSCAML1的ICD即可显著改变与神经元分化、凋亡、突触形成及功能相关的基因表达谱。
通过原代皮层神经元开展的功能获得性实验显示，提高DSCAM或DSCAML1 ICD的表达水平会导致神经突生长受损。
值得注意的是，全长DSCAM或DSCAM ICD的过表达（而非DSCAML1 ICD）可显著降低原代海马神经元的突触数量。
综上，我们发现了一种全新的膜-核信号转导机制，DSCAM受体可通过该机制调控神经元分化、突触形成与功能相关调控因子的表达。鉴于21三体综合征患者存在DSCAM基因的染色体重复导致其表达上调，本研究结果或有助于揭示唐氏综合征智力障碍的潜在新型致病机制。

整体实验设计：
我们构建了4株同基因系HEK293 t-Rex-Flp-In细胞系。HEK293 t-Rex-Flp-In表达系统可实现四环素诱导型、稳定均一的转基因在特定位点基因组的表达。该四组细胞系分别为：(1) 稳定表达黄色荧光蛋白-血凝素标签（YFP-HA）融合的DSCAM ICD的细胞系；(2) 稳定表达YFP-HA标签融合的DSCAML1 ICD的细胞系；(3) 胞质YFP-HA对照细胞系；(4) 核定位YFP-HA对照细胞系。针对每一组细胞系，我们通过RNA深度测序（RNA-Seq）对转基因表达的3个独立生物学重复样本进行了分析。本研究比较了表达YFP标记的DSCAM/DSCAML1 ICDs的细胞与表达核定位YFP-HA（YFP-nls）对照的细胞之间的全局转录组变化。分析过程中我们排除了胞质YFP与核YFP表达细胞之间已存在差异表达的基因（P值≤0.1），因为此类基因不太可能受DSCAM/DSCAML1 ICD表达的调控。