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

<jats:title>Abstract</jats:title><jats:p>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. Lastly, we use cortical cultures derived from DCX knockout 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.</jats:p><jats:sec><jats:title>Significance Statement</jats:title><jats:p>Nestin, an intermediate filament protein highly expressed in neural progenitors, was recently identified in developing neurons where it regulates growth cone morphology and responsiveness to the guidance cue Sema3a. Changes in growth cone morphology require rearrangements of cytoskeletal networks, but the roles of intermediate filaments in this process are poorly understood. We now report that nestin selectively facilitates phosphorylation of the lissencephaly-linked doublecortin (DCX) by cdk5/p35, but the phosphorylation of other cdk5 substrates is not affected. This substrate selectivity is based on preferential scaffolding of DCX, cdk5, and p35 by nestin. Additionally, we demonstrate a functional role for the DCX-nestin complex in neurons. We propose that nestin changes growth cone behavior by regulating intracellular kinase signaling in developing neurons.</jats:p></jats:sec>

Visualising the cytoskeletal machinery in neuronal growth cones using cryo-electron tomography

“…DCX/Dcx is a modulator of neuronal growth cone morphology and the underlying MT and F-actin organisation (Bielas et al, 2007; Bott et al, 2020; Fu et al, 2013; Jean et al, 2012) and nucleates and stabilises 13-protofilament MTs in vitro (Moores et al, 2004). We therefore undertook cryo-ET of hippocampal neuron growth cones from Dcx knockout (KO) mice and analysed the detailed ultrastructural data, with a focus on the cytoskeleton (Fig.…”

Repeated administration of acrylamide for 28 days reduces late-stage progenitor cells and immature granule cells accompanying impaired neurite outgrowth in the adult hippocampal neurogenesis in young-adult rats

“…Since neural cell adhesion molecule 2 (NCAM2) and neuronal regeneration related protein (NREP, also known as P311) are known to be involved in neuronal migration and neurite outgrowth (Fujitani et al, 2004;Parcerisas et al, 2021), these results suggest that AA suppressed the neurite outgrowth of immature granule cells via the downregulation of Ncam2 and Nrep. The decrease in the transcript level of Nes might also be related to the impairment of neurite outgrowth of the DCX + cells since it has been reported that nestin forms a complex with DCX to regulate the growth cone morphology in developing cortical neurons (Bott et al, 2020). In our previous study, exposure to glycidol, a neurotoxicant causing distal axonopathy similar to that induced by AA, also resulted in gait abnormality and a decrease in the densities of type-3 NPCs and immature granule cells and suppressed the gene expressions of Ncam2, suggesting that AA share a similar target mechanism with glycidol for disruption of hippocampal neurogenesis (Akane et al, 2013(Akane et al, , 2014.…”

Microtubule Organization Is Essential for Maintaining Cellular Morphology and Function

“…Binding of DCX to tubulin increases MT homeostasis in neurons, which can be disturbed via knockout of the DCX gene sequence using an inducible transgenic mouse approach [40]. Additionally, DCX is expressed in various regions of the developing nervous system and is regarded as a gold standard biomarker for identifying neuronal precursors and migration during adult neurogenesis [41,42].…”