GSE122568
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.