Abstract
Doublecortin (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.
Highlights
Doublecortin (DCX) is a protein needed for cortical development, and DCX mutations cause cortical malformations in humans
To test whether DCX-G253D has a cellular defect in NF endocytosis, we turned to loss-of-function experiments using short hairpin plasmids targeting Dcx
Endogenous levels of DCX were diminished by 75% on average after 3 days of expression of short hairpin plasmids targeting Dcx (shDcx)#2-GFP. shDcx#2-GFP expression significantly diminished signal intensity of endocytosed endogenous NF in endosomes in day in vitro 3 (DIV3) hippocampal neurons compared with untransfected neurons or neurons expressing shRandom-GFP control plasmid (Fig. 1, A, B, and E), to our previous work (24)
Summary
Doublecortin (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. The disease-associated DCX-G253D mutant protein is known to be deficient in binding neurofascin, and we asked if disruption of neurofascin endocytosis underlies the DCX-G253D–associated pathology. We showed recently that a Dcx allele with loss of MT binding is incapable of promoting dendrite growth in culture (20). We demonstrated a novel function for DCX, namely promoting the endocytosis of NF (24) Based on these data, we proposed a model that DCX enhances AIS localization of NF by promoting its endocytosis from non-AIS regions, such as dendrites (24). Mutating either the AP-2– binding site (DCX-ALPA) or the NF-binding site (DCX-G253D) leads to decreased NF endocytosis in primary neurons and increased mislocalization of NF to dendrites. This raises the possibility that single Dcx mutant alleles have multiple cellular defects that differentially contribute to distinct pathological processes
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