A dominant dendrite phenotype caused by the disease-associated G253D mutation in doublecortin (DCX) is not due to its endocytosis defect

Abstract

Edited by Phyllis I. HansonDoublecortin (DCX) is a protein needed for cortical development, and DCX mutations cause cortical malformations in humans. The microtubule-binding activity of DCX is well-described and is important for its function, such as supporting neuronal migration and dendrite growth during development. Previous work showed that microtubule binding is not sufficient for DCX-mediated promotion of dendrite growth and that domains in DCX's C terminus are also required. The more C-terminal regions of DCX bind several other proteins, including the adhesion receptor neurofascin and clathrin adaptors. We recently identified a role for DCX in endocytosis of neurofascin. The disease-associated DCX-G253D mutant protein is known to be deficient in binding neurofascin, and we now asked if disruption of neurofascin endocytosis underlies the DCX-G253D-associated pathology. We first demonstrated that DCX functions in endocytosis as a complex with both the clathrin adaptor AP-2 and neurofascin: disrupting either clathrin adaptor binding (DCX-ALPA) or neurofascin binding (DCX-G253D) decreased neurofascin endocytosis in primary neurons. We then investigated a known function for DCX, namely, increasing dendrite growth in cultured neurons. Surprisingly, we found that the DCX-ALPA and DCX-G253D mutants yield distinct dendrite phenotypes. Unlike DCX-ALPA, DCX-G253D caused a dominant-negative dendrite growth phenotype. The endocytosis defect of DCX-G253D thus was separable from its detrimental effects on dendrite growth. We recently identified Dcx-R59H as a dominant allele and can now classify Dcx-G253D as a second Dcx allele that acts dominantly to cause pathology, but does so via a different mechanism. This article contains Fig. S1.

Nestin selectively facilitates the phosphorylation of the Lissencephaly-linked protein doublecortin (DCX) by cdk5/p35 to regulate growth cone morphology and Sema3a sensitivity in developing neurons

“…In order to test if DCX and nestin affected their respective cellular localizations, we transfected COS-7 cells which are large, well-spread cells which have spatially resolvable MT and vimentin IF filament systems. As shown previously by us and others, GFP-DCX decorates MTs (Ge et al, 2006;Gleeson et al, 1999;Moslehi et al, 2017;Sapir et al, 2000;Tsukada et al, 2005;Yap et al, 2018Yap et al, , 2012, but is not co-localized with vimentin ( Fig. 4A).…”

“…DCX is a multifunctional cytosolic protein expressed in developing neurons (Ge et al, 2006;Gleeson, et al 1999;Moslehi, et al, 2017;Sapir et al, 2000;Tsukada et al, 2005;Yap et al, 2018Yap et al, , 2012. Mutations in human DCX cause lissencephaly, a disease characterized by defects in neuronal migration and axon outgrowth that results in profound cortical malformations (Bahi-Buisson et al, 2013).…”

Nestin Selectively Facilitates the Phosphorylation of the Lissencephaly-Linked Protein Doublecortin (DCX) by cdk5/p35 to Regulate Growth Cone Morphology and Sema3a Sensitivity in Developing Neurons

“…In order to test whether DCX and nestin affected their respective cellular localizations, we transfected Cos7 cells, which are large, well-spread cells which have spatially resolvable MT and vimentin IF filament systems. As shown previously by us and others, GFP-DCX decorates MTs (Gleeson et al, 1999;Sapir et al, 2000;Tsukada et al, 2005;Ge et al, 2006;Moslehi et al, 2017;Yap et al, 2018Yap et al, , 2012, but it is not colocalized with vimentin ( Fig. 4A).…”

“…DCX is a cytoskeleton-associated protein expressed in developing neurons (Gleeson, et al, 1999;Sapir et al, 2000;Tsukada et al, 2005;Ge et al, 2006;Yap et al, 2012Yap et al, , 2018Moslehi et al, 2017). Mutations in human DCX cause lissencephaly, a disease characterized by defects in neuronal migration and axon outgrowth that results in profound cortical malformations (Bahi-Buisson et al, 2013).…”

Epstein-Barr Virus Early Protein BFRF1 Suppresses IFN-β Activity by Inhibiting the Activation of IRF3

“…pBFRF1-Myc, pIKKi-HA, pBZLF1-HA (Zta-HA), and pBGLF4-HA were also constructed with similar methods, using pMyc-N1 or pHA-N1 vector (regenerated from pEYFP-N1, Clontech). Besides, one pair of oligonucleotide sequences 5′-GGG TCT CTC AAC GGA TGT TGA and 5′-CTC AAC TCA CGT GTC TAG TGT C (38)(39)(40)(41)(42)(43)(44) was inserted into the good RNAi product of Oligoengine pSuper-retro-puro (Oligoengine) that can effectively remove off-target of the target gene, to construct RNA interference expression plasmids pSuper-shBFRF1-retro-puro and pSuper-shRandom-retro-puro [a good off-target control (45)(46)(47)(48)], respectively. Other gift plasmids were provided by Drs.…”