Functional Consequences of Necdin Nucleocytoplasmic Localization

Background: Necdin, a MAGE family protein expressed primarily in the nervous system, has been shown to interact with both nuclear and cytoplasmic proteins, but the mechanism of its nucleocytoplasmic transport are unknown. Methodology/Principal Findings: We carried out a large-scale interaction screen using necdin as a bait in the yeast RRS system, and found a wide range of potential interactors with different subcellular localizations, including over 60 new candidates for direct binding to necdin. Integration of these interactions into a comprehensive network revealed a number of coherent interaction modules, including a cytoplasmic module connecting to necdin through huntingtin-associated protein 1 (Hap1), dynactin and hip-1 protein interactor (Hippi); a nuclear P53 and Creb-binding-protein (Crebbp) module, connecting through Crebbp and WW domain-containing transcription regulator protein 1 (Wwtr1); and a nucleocytoplasmic transport module, connecting through transportins 1 and 2. We validated the necdin-transportin1 interaction and characterized a sequence motif in necdin that modulates karyopherin interaction. Surprisingly, a D234P necdin mutant showed enhanced binding to both transportin1 and importin b1. Finally, exclusion of necdin from the nucleus triggered extensive cell death. Conclusions/Significance: These data suggest that necdin has multiple roles within protein complexes in different subcellular compartments, and indicate that it can utilize multiple karyopherin-dependent pathways to modulate its localization

Breathing deficits of the Prader-Willi syndrome

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Module decomposition of the necdin network.

<p>Modules were detected using the betweenness-centrality clustering algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033786#pone.0033786-Newman1" target="_blank">[21]</a>. Only modules containing more than 8 nodes are displayed. Blue edges denote published interactions, red are interactions detected in the present screen. Note that necdin-Eid1 and necdin-Nucleobindin1 are connected with 2 color edges. Modules - <b>A</b>. necdin; <b>B</b>. p75; <b>C</b>. Grin-Ywhab; <b>D</b>. p53-Crebbp; <b>E</b>. Transportin; <b>F</b>. Huntingtin; <b>G</b>. MAGE-D1; <b>H</b>. APP; <b>I</b>. Clock. Necdin (Ndn) is shown for each module, together with interactions with the module members.</p

Yeast RRS screen with necdin as bait.

<p>Representative pictures of yeast <i>cdc25-2</i> colonies after transformation with different candidates at the permissive temperature (24°C), and at the restrictive temperature (36°C). All colonies grow on galactose medium at 24°C, while only colonies expressing candidates that interact with the bait can grow on galactose at 36°C, but not on YPD (prey candidates are under a galactose promoter).</p

The necdin network comprising first and second degree necdin neighbors.

<p>The necdin (Ndn) network was assembled based on the mouse IntAct database, complemented with published necdin interactors and our RRS results. The network contains 205 proteins and 346 interactions. Blue edges denote published interactions, red edges are interactions detected in the present screen. Nodes with more than 5 interactions are marked in red.</p

Exclusion of necdin from the nucleus causes cell death.

<p><b>A</b>. Schematic of necdin constructs, fused at the C-terminus to either three repeats encoding DPKKKRKV (NLS) or YLVQIFQELTL (NES). <b>B</b>. Representative confocal images of PC12 cells 48 hr after transfection, fixed and stained as indicated. Scale bar 10 µm. <b>C</b>. Quantification of nuclear localization of the indicated constructs in PC12 cells, * denotes p<0.05, ** denotes p<0.01, n = 16. <b>D</b>. Live/dead staining of PC12 transfected as indicated and plated on Poly-L lysine coated cover slips. 30 hours after transfection, cells were washed and incubated with 1 µM Calcein AM and 1 µM EthD-1 in D-PBS for 25 minutes, followed by fluorescence imaging. Live cells are green, dead cells are red. Scale bar 100 µm. <b>E</b>. Cell death in necdin-NES transfected PC12 and HEK293 cells as observed by XTT assay. <b>F</b>. Viability of PC12 cells transfected with the indicated constructs, 48 hours after transfection. * indicates p<0.02, n = 3.</p

Control of Social Withdrawal of Mice Deficient for the Autism Gene Magel2 by Restoration of Vasopressin-Oxytocin Dialogue in Septum

Intellectual and social disabilities are common comorbidities in adolescents and adults with Magel2 gene deficiency characterizing the Prader-Willi and Schaaf-Yang neurodevelopmental syndromes. The cellular and molecular mechanisms underlying the risk for autism in these syndromes are unexplored. Here we used Magel2 knockout mice combined with optogenetic/pharmacological tools to characterize disease modifications in the social brain network. We find that the degree of social novelty moderates a dialogue between vasopressin and oxytocin in the lateral septum, a region organizing sequential content of sensory experiences. Social withdrawal of mice lacking Magel2 is alleviated by restoration of dialogue-lead by vasopressin. This preclinical study identifies the collective actions of vasopressin and oxytocin in the lateral septum as a key factor in the pathophysiology