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

Abstract

Nestin, an intermediate filament protein widely used as a marker of neural progenitors, was recently found to be expressed transiently in developing cortical neurons in culture and in developing mouse cortex. In young cortical cultures, nestin regulates axonal growth cone morphology. In addition, nestin, which is known to bind the neuronal cdk5/p35 kinase, affects responses to axon guidance cues upstream of cdk5, specifically, to Sema3a. Changes in growth cone morphology require rearrangements of cytoskeletal networks, and changes in microtubules and actin filaments are well studied. In contrast, the roles of intermediate filament proteins in this process are poorly understood, even in cultured neurons. Here, we investigate the molecular mechanism by which nestin affects growth cone morphology and Sema3a sensitivity. We find that nestin selectively facilitates the phosphorylation of the lissencephaly-linked protein doublecortin (DCX) by cdk5/p35, but the phosphorylation of other cdk5 substrates is not affected by nestin. We uncover that this substrate selectivity is based on the ability of nestin to interact with DCX, but not with other cdk5 substrates. Nestin thus creates a selective scaffold for DCX with activated cdk5/p35. Last, we use cortical cultures derived from Dcx KO mice to show that the effects of nestin on growth cone morphology and on Sema3a sensitivity are DCX-dependent, thus suggesting a functional role for the DCX-nestin complex in neurons. We propose that nestin changes growth cone behavior by regulating the intracellular kinase signaling environment in developing neurons. The sex of animal subjects is unknown.

scRNA sequencing uncovers a TCF4-dependent transcription factor network regulating commissure development in mouse

“…5 D, ). Plxna2 , a gene involved in semaphorin/plexin signaling for axon guidance ( Rohm et al, 2000 ; Mitsogiannis et al, 2017 ), and Dcx , a gene that in addition to its function in neuronal migration has been connected to neuronal morphogenesis and axon guidance ( Deuel et al, 2006 ; Pilz et al, 1998 ; Yap et al, 2016 ; Bott et al, 2020 ), were selected for further investigation. Evolutionarily conserved regions (ECRs) upstream of or at the promotor containing conserved binding sites for TCF4 and SOX11 ( …”

“…Furthermore, we present in vitro biochemical evidence for several of the predicted interactions and provide in vivo biochemical and genetic evidence for the cooperativity of TCF4 with the non-bHLH transcription factor SOX11 in the generation of the commissural system. We identified two potential common target genes of TCF4 and SOX11, Dcx and Plxna2 , which have been shown to play important roles in axonal development and axon guidance ( Rohm et al, 2000 ; Mitsogiannis et al, 2017 ; Deuel et al, 2006 ; Pilz et al, 1998 ; Yap et al, 2016 ; Bott et al, 2020 ) and are likely candidates to contribute to the observed dysgenesis of the forebrain commissures in Tcf4 KO mice. These two genes are, however, only two examples out of a larger network of factors involved in commissural formation that are dysregulated as a result of changes in GRN activities caused by the loss of TCF4B.…”

Unraveling Axon Guidance during Axotomy and Regeneration

“…They have differential expression patterns that influence the response, for instance, the atypical intermediate filament nestin, which is expressed in neuronal progenitors and newborn neurons, localizes mainly to the distal area of growing axons in embryonic, but not in adult neurons [ 67 ]. Neurons expressing nestin are more sensitive to Sema3A than the ones that do not express it [ 67 ], and this effect is dependent on Doublecortin (DCX) [ 68 ]. Nestin binds DCX and the cdk5 kinase activator p53, facilitating DCX phosphorylation by cdk5, a kinase that acts downstream of Sema3A [ 68 ].…”

“…Neurons expressing nestin are more sensitive to Sema3A than the ones that do not express it [ 67 ], and this effect is dependent on Doublecortin (DCX) [ 68 ]. Nestin binds DCX and the cdk5 kinase activator p53, facilitating DCX phosphorylation by cdk5, a kinase that acts downstream of Sema3A [ 68 ]. Interestingly, studies in Xenopus laevis , comparing the transcriptomic response after spinal cord injury (SCI) in R versus NR stages, showed a differential increase in nestin transcripts in NR-stages, while no changes in R-stages, at 6 days post-injury (dpi) [ 69 , 70 ].…”

Mitochondria in Early Forebrain Development: From Neurulation to Mid-Corticogenesis

“…More recent work has elucidated that cdk5/p35 is the mechanism through which the neuronal intermediate filament protein Nestin facilitates phosphorylation of the critical neuronal migration factor doublecortin (DCX), which is also linked to lissencephaly. This Nestin-driven phosphorylation of DCX, thought cdk5/p35, affects both growth cone morphology and sensitivity to the axonal chemorepellent Sema3a ( Bott et al, 2020 ). Thus, while mPTP is not directly implicated in these processes, the key roles of cdk5 during cortical development, particularly migration of newly differentiated neurons, suggests mPTP as an intriguing process worthy of further study as a potential mechanism further linking the two converging fields of corticogenesis and mitochondria.…”