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.

DSCAM与DSCAML1是免疫球蛋白（immunoglobulin）类细胞黏附型受体（cell adhesion-type receptors），在神经发育过程中发挥关键功能，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，DSCAM受体的信号转导机制及其效应分子仍未得到充分解析。我们借助人类ORFeome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（Down syndrome）激酶Dyrk1a、STAT3、USP21及SH2D2A在内的新型胞质信号效应候选分子。出乎意料的是，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），与β-输入蛋白家族的核输入蛋白IPO5发生特异性直接相互作用。DSCAM的胞内结构域经γ-分泌酶（gamma-secretase）依赖型切割后被释放，且DSCAM与DSCAML1的胞内结构域均可高效易位至细胞核内。此外，RNA测序（RNA-sequencing）证实，单独表达DSCAM或DSCAML1的胞内结构域即可显著改变与神经元分化、细胞凋亡、突触形成及功能相关的基因表达谱。利用原代皮层神经元开展的功能获得性（gain-of-function）实验显示，提高DSCAM或DSCAML1胞内结构域的表达水平均可抑制神经突生长。令人瞩目的是，过表达全长DSCAM或DSCAM胞内结构域（而非DSCAML1胞内结构域）可显著减少原代海马神经元的突触数量。综上，我们鉴定出一种全新的膜-核信号转导机制，通过该机制，DSCAM受体可调控神经元分化、突触形成及功能相关调控因子的表达。考虑到21三体综合征（trisomy 21）患者的染色体重复会导致DSCAM表达上调，我们的研究结果或有助于揭示唐氏综合征（Down syndrome）患者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HEK293 t-Rex-Flp-In细胞系。HEK293 t-Rex-Flp-In表达系统可实现从特定基因组位点稳定、四环素诱导且均一的转基因表达。这4株细胞系分别为：（1）稳定表达YFP-HA标签融合的DSCAM胞内结构域的细胞系；（2）稳定表达YFP-HA标签融合的DSCAML1胞内结构域的细胞系；（3）作为胞质YFP-HA对照的细胞系；（4）作为核YFP-HA对照的细胞系。对于每株细胞系，我们通过RNA深度测序（RNA deep sequencing，即RNA-Seq）对转基因表达的3个独立生物学重复进行分析。我们比较了表达YFP标签融合的DSCAM/L1胞内结构域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。对于在胞质YFP与核YFP表达细胞中已存在差异表达（p值=0.1）的基因，我们将其从分析中排除，因为这类基因不太可能受DSCAM/L1胞内结构域表达的影响。