The molecular mystery of neuronal migration: FAK and Cdk5

doi:10.1016/j.tcb.2003.10.010

Trends in Cell Biology

Volume 14, Issue 1, January 2004, Pages 1-5

https://doi.org/10.1016/j.tcb.2003.10.010 Get rights and content

Abstract

The basic building blocks of a cell are its cytoskeletal proteins, the orderly but dynamic organization of which is essential. How signalling molecules regulate the cytoskeleton in the developing nervous system is still largely unknown. A recent breakthrough sheds light on a pathway involving Cdk5 (cyclin-dependent kinase 5) and FAK (focal adhesion kinase), demonstrating their role in regulating microtubule structure and thus nuclear positioning in radially migrating cortical neurones.

Section snippets

Cdk5 phosphorylates FAK in migrating neurones

Cdk5 is a major regulator of cytoskeletal components known to affect neuronal MTs, microfilaments and intermediate filaments during development and neurodegeneration (Box 2) [8]. Animal models with compromised Cdk5 activity (genetically modified to lack Cdk5 or its activating protein, p35) have disrupted radial migration of neurones from the ventricular zone to the cortical plate (Box 1) [3]. Thus, how does Cdk5 control neuronal migration? The recent findings of Tsai and colleagues [6] have

Regulation of MTs by FAK

The kinase activity of FAK is believed to be secondary to its chaperoning role in assembling complexes containing other kinases (Src, Fyn and PI3K) and their substrates (Box 2) [11]. Accordingly, the phosphorylation of FAK at S732 does not affect its catalytic activity and yet the MT fork is malformed when FAK phosphorylation is decreased. Why? A logical assumption is that the phosphorylation of FAK by Cdk5 positively or negatively affects its ability to assemble a particular protein complex.

Neuronal and non-neuronal FAK

In the adult brain, FAK is regulated by neurotransmitters and controls synaptic plasticity (Box 2). The higher levels of FAK seen in embryonic cortex when neuronal migration and neurite outgrowth predominate suggest a nonsynaptic function for this kinase 6, 12. In differentiating neurones, FAK is detected in growth cones and can regulate growth-factor- and integrin-induced neurite outgrowth 9, 13, 14. Xie et al. [6] have revealed that FAK also controls neuronal migration by affecting their MTs.

New tricks to study neuronal migration

A large part of what is known about the mechanism of neuronal migration comes from the studies of human diseases and animal models, both of which take time to obtain and characterize. To understand the processes that underlie neuronal development, it is essential to utilize a manipulative system where modifications in signalling pathways can be made and the consequences can be analyzed at a cellular level. For many years, researchers have utilized slice and explant cultures to examine the

Concluding remarks

In the developing cortex, neurones undergo many complex processes involving major changes in shape, mobility and function, which are all regulated by a multitude of proteins that interact and affect the function of one other. To understand simple aspects of the signalling pathways that determine neuronal fate and function, it is essential to be able to analyze the consequences of lost or altered protein activity in an appropriate experimental system. The Tsai laboratory [6] have demonstrated

Acknowledgements

I thank all members of my laboratory for their helpful discussions and Mikio Hoshino for allowing me to use an adapted version of one of their figure (Figure 1). Our work is funded by the Wellcome Trust, BBSRC and GSK.

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Focal adhesion dynamics are altered in schizophrenia
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These observations suggest that the differences in motility of Patient and Control cells are related to the dynamics of focal adhesion assembly/disassembly. FAK is central to neuronal migration (45,46) and is a hub in the focal adhesion complex that forms and disassembles to mediate cell binding to the extracellular matrix (47). Y397-pFAK, the autophosphorylated form examined here, is essential for the subsequent FAK phosphorylations that initiate integrin-mediated cell adhesion and is present in newly forming and mature focal adhesions (48).
Evidence from genetic association studies implicate genes involved in neural migration associated with schizophrenia risk. Neural stem/progenitor cell cultures (neurosphere-derived cells) from olfactory mucosa of schizophrenia patients have significantly dysregulated expression of genes in focal adhesion kinase (FAK) signaling, a key pathway regulating cell adhesion and migration. The aim of this study was to investigate whether olfactory neurosphere-derived cells from schizophrenia patients have altered cell adhesion, cell motility, and focal adhesion dynamics.
Olfactory neurosphere-derived cells from nine male schizophrenia patients and nine male healthy control subjects were used. Cells were assayed for cell adhesion and cell motility and analyzed for integrins and FAK proteins. Focal adhesions were counted and measured in fixed cells, and time-lapse imaging was used to assess cell motility and focal adhesion dynamics.
Patient-derived cells were less adhesive and more motile than cells derived from healthy control subjects, and their motility was reduced to control cell levels by integrin-blocking antibodies and by inhibition of FAK. Vinculin-stained focal adhesion complexes were significantly smaller and fewer in patient cells. Time-lapse imaging of cells expressing FAK tagged with green fluorescent protein revealed that the disassembly of focal adhesions was significantly faster in patient cells.
The evidence for altered motility and focal adhesion dynamics in patient-derived cells is consistent with dysregulated gene expression in the FAK signaling pathway in these cells. Alterations in cell adhesion dynamics and cell motility could bias the trajectory of brain development in schizophrenia.
Dissecting the factors involved in the locomotion mode of neuronal migration in the developing cerebral cortex
2010, Journal of Biological Chemistry
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However, in contrast to the early phase of migration, it remains difficult to directly analyze the molecular mechanisms for the locomotion. A major hurdle is that no promoter whose activation marks the start of the locomotion mode has yet been identified; thus obstructing in vivo experiments using conditional knockout mice or in utero electroporation-mediated functional suppression technique (21, 22). Thus analysis of the in vivo mechanisms underlying the locomotion mode of migration is usually complicated by various secondary effects of defects arising at the early phase of migration, such as multipolar migration, leading process formation and the acquirement of neuronal polarity and axon.
Neuronal migration is essential for proper cortical layer formation and brain function, because migration defects result in neurological disorders such as mental retardation and epilepsy. Neuronal migration is divided into several contiguous steps: early phase (multipolar mode), locomotion mode, and terminal translocation mode. The locomotion mode covers most of the migration route and thereby is the main contributor to cortical layer formation. However, analysis of the molecular mechanisms regulating this mode is difficult due to the secondary effects of defects at the early phase of migration. In this study, we established an ex vivo chemical inhibitor screening, allowing us to directly analyze the locomotion mode of migration. Roscovitine and PP2, inhibitors for Cdk5 and Src family kinases, respectively, suppressed the locomotion mode of migration. In line with this, a small percentage of Cdk5- or Src family kinase (Fyn)-knockdown cells exhibited locomoting morphology but retarded migration, although the majority of cells were stalled at the early phase of migration. We also showed that rottlerin, widely used as a specific inhibitor for protein kinase Cδ (PKCδ), suppressed the locomotion mode. Unexpectedly, however, the dominant-negative form as well as RNA interference for PKCδ hardly affected the locomotion, whereas they may disturb terminal translocation. In addition, we found JNK to be a potential downstream target of rottlerin. Taken together, our novel chemical inhibitor screening provides evidence that Cdk5 and Src family kinases regulate the locomotion mode of neuronal migration. It also uncovered roles for Fyn and PKCδ in the early and final phases of migration, respectively.
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Increasing evidence implicates cyclin-dependent kinase 5 (Cdk5) in neuronal synaptic function. We searched for Cdk5 substrates in synaptosomal fractions prepared from mouse brains. Mass spectrometric analysis after two-dimensional SDS-PAGE identified several synaptic proteins phosphorylated by Cdk5-p35; one protein identified was Sept5 (CDCrel-1). Although septins were isolated originally as cell division-related proteins in yeast, Sept5 is expressed predominantly in neurons and is implicated in exocytosis. We confirmed that Sept5 is phosphorylated by Cdk5-p35 in vitro and identified Ser¹⁷ of adult type Sept5 (Sept5_v1) as a major phosphorylation site. We found that Ser¹⁷ of Sept5_v1 is phosphorylated in mouse brains. Coimmunoprecipitation from synaptosomal fractions and glutathione S-transferase-syntaxin-1A pulldown assays of Sept5_v1 expressed in COS-7 cells showed that phosphorylation of Sept5_v1 by Cdk5-p35 decreases the binding to syntaxin-1. These results indicate that the interaction of Sept5 with syntaxin-1 is regulated by the phosphorylation of Sept5_v1 at Ser¹⁷ by Cdk5-p35.
Association of cyclin-dependent kinase 5 and neuronal activators p35 and p39 complex in early-onset Alzheimer's disease
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Malfunctioning of cyclin-dependent kinase 5 (CDK5) through aberrant proteolytic cleavage of its neuronal activators p35 and p39 is involved in neurodegeneration in Alzheimer's disease (AD) and other neurodegenerative brain diseases. By extensive genetic analysis of the genes encoding CDK5 (CDK5), p35 (CDK5R1) and p39 (CDK5R2), we excluded causal mutations in 70 familial early-onset AD patients. We performed an association study with five informative SNPs in CDK5 in two independent samples of early-onset AD patients and matched control individuals from The Netherlands and northern Sweden. Association was observed with g.149800G>C in intron 5 of CDK5, and a two times increased risk was observed in both patient samples for carriers of the C-allele. Our data are indicative for a role of the CDK5 molecular complex in the genetic etiology of early-onset AD, and suggest that a yet unknown functional variant in CDK5 or in a nearby gene might lead to increased susceptibility for early-onset AD.
MAP1B phosphorylation is differentially regulated by Cdk5/p35, Cdk5/p25, and JNK
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Citation Excerpt :
Embryos were electroporated with EGFP expression vectors at E14 and coronal slices were taken at E16 and cultured for 20 h with or without 100 μM roscovitine to observe the migration of GFP-positive cells. While GFP-positive cells migrated toward the pial surface in the control slices, roscovitine treatment inhibited cell migration, mimicking the cerebral cortex phenotype of Cdk5-deficient mice as well as that of dominant negative Cdk5-electroporated neurons (Fig. 1C) [9,24,25]. Consistent with this result, treatment with 100 μM roscovitine to slice cultures resulted in suppression of FAK phosphorylation at Ser732 (Fig. 1D), suggesting that 100 μM roscovitine inhibits Cdk5 kinase activity and Cdk5-dependent neuronal migration.
Mode I phosphorylated MAP1B is observed in developing and pathogenic brains. Although Cdk5 has been believed to phosphorylate MAP1B in the developing cerebral cortex, we show that a Cdk5 inhibitor does not suppress mode I phosphorylation of MAP1B in primary and slice cultures, while a JNK inhibitor does. Coincidently, an increase in phosphorylated MAP1B was not observed in COS7 cells when Cdk5 was cotransfected with p35, but this did occur with p25 which is specifically produced in pathogenic brains. Our primary culture studies showed an involvement of Cdk5 in regulating microtubule dynamics without affecting MAP1B phosphorylation status. The importance of regulating microtubule dynamics in neuronal migration was also demonstrated by in utero electroporation experiments. These findings suggest that mode I phosphorylation of MAP1B is facilitated by JNK but not Cdk5/p35 in the developing cerebral cortex and by Cdk5/p25 in pathogenic brains, contributing to various biological events.

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Trends in Cell Biology

Research FocusThe molecular mystery of neuronal migration: FAK and Cdk5

Abstract

Section snippets

Cdk5 phosphorylates FAK in migrating neurones

Regulation of MTs by FAK

Neuronal and non-neuronal FAK

New tricks to study neuronal migration

Concluding remarks

Acknowledgements

Neuron

Cell

Int. J. Dev. Neurosci.

Trends Neurosci.

Mol. Cell. Neurosci.

J. Biol. Chem.

Neuroscience

Dev. Biol.

Mol. Cell. Neurosci.

Central nervous system neuronal migration

Annu. Rev. Neurosci.

Human brain malformations and their lessons for neuronal migration

Annu. Rev. Neurosci.

Life is a journey: a genetic look at neocortical development

Nat. Rev. Genet.

Research Focus
The molecular mystery of neuronal migration: FAK and Cdk5