Regulation of cell adhesion and migration in cortical neurons

Abstract

Accumulating evidence indicate that Rho family small GTPases, including RhoA, Rac1 and Cdc42, control cytoskeletal organization and cell adhesion, and thereby cell migration in vitro and in vivo. Recently, the involvement of other small GTPases, such as Rab and Arf family proteins in cell migration has also been evaluated. Rab5, Rab11 and Rab7, which regulate endocytosis, recycling and lysosomal degradation pathways, respectively, are shown to have essential roles in the migration of immature neurons during the development of cerebral cortex in vivo. These Rab proteins control distinct steps of neuronal migration through the regulation of N-cadherin-mediated cell adhesion. In this extra view paper, I will discuss the functions of Rho and Rab family small GTP ases in cell migration with particular focus on the migrating neurons in the developing cerebral cortex.

Small GTPases regulate various cellular events, such as cell adhesion, migration, proliferation and signal transduction. Among five subfamilies (Ras/Rap/Ral, Rho, Rab, Arf/Sar and Ran), Rho family small GTPases are mainly involved in cytoskeletal regulation, whereas Rab family proteins regulate membrane trafficking. It is widely accepted that three major Rho family members, RhoA, Rac1 and Cdc42, play crucial roles in cell migration and morphological changes mostly through cytoskeletal reorganization.¹ While Cdc42 and Rac1 promote the formation of actin-based membrane protrusions, filopodia and lamellipodia, respectively, RhoA regulates the assembly of focal adhesions, integrin-mediated cell-to-extracellular matrix (ECM) adhesions and actin stressfiber formation. In migrating fibroblasts, these sequential and coordinated regulations of cytoskeleton and adhesion occur at the cell front to build up the forward membrane extension and new adhesion to the ECM (Fig. 1A). RhoA activity is also required for tail retraction at the cell rear via promoting myosin-dependent contractility, although RhoA may be partially inactivated at the cell rear to induce the disassembly of focal adhesions and detachment from ECM.^1–3 In addition to these fundamental observations, recent in vivo studies using particular cell types have revealed the various roles and regulatory mechanisms of Rho family small GTPases in the migration and morphological changes of cells in different tissues at embryonic stages or adulthood.^4,5

Figure 1.

Roles of Rho and Rab family small GTPases in the migration of fibroblasts. (A) Migrating fibroblasts are polarized toward the direction of movement (Right side). Orange lines, red circles and small purple dots show F-actins, focal adhesions and focal complexes, respectively. Whereas Cdc42 and Rac1 regulate filopodia and lamellipodia, respectively, RhoA enhances the formation of focal adhesions and actin stressfibers (thick orange lines). In addition, integrin heterodimers (shown in green) are internalized and transported toward the cell front by several Rab family proteins to form new adhesions. (B) Rab family small GTPases differentially regulate specific membrane trafficking pathways. Dynamin, an atypical large GTPase, enhances endocytosis via its membrane fission activity. Rab5 and its family protein, Rab21, promote endocytosis and trafficking to early endosomes. Rab4, Rab11 and Rab7 regulate direct recycling, indirect recycling via recycling endosomes and lysosomal degradation pathways, respectively.

On the other hand, research into the roles of Rab family small GTPases in cell migration and morphological changes is just beginning. Several Rab family proteins, including Rab4, 11, 21, 25, were recently reported to regulate the intracellular trafficking of integrins (Fig. 1B).⁶ Furthermore, a very recent study has uncovered the in vivo roles of Rab proteins in the migration of cortical neurons.⁷ In this extra view paper, I will focus on the functions of Rho family as well as Rab family small GTPases in cell adhesion and cytoskeletal organization of migrating neurons in the mouse developing cerebral cortex.

Cortical Development and Rac1 Functions

In the developing mammalian cerebral cortex, neural progenitors are located near the ventricle, in the ventricular zone or subventricular zone, although they possess a very long process, called a radial glial fiber (Fig. 2A). The nuclei of neural progenitors move up-and-down in the ventricular zone according to the cell cycle; during the G₂ phase, the nuclei move to the ventricular side and to the opposite (pial surface) side in G₁. This unique phenomenon is called interkinetic nuclear movement.

Figure 2.

Rho and Rab family small GTPases differentially regulate specific steps of neuronal migration. (A) The cell bodies of neural progenitors are located near the ventricle (at the bottom of the figure), but they have very long processes, called radial glial fibers. After exhibiting multipolar morphologies (light purple cells), neurons form a leading process and migrate long distances along the radial glial fibers (green cells). Cdc42 and Rac1, but not RhoA, promote neuronal migration through the recruitment of IQGAP1/CLIP-170 complex or enhancement of JNK-mediated microtubule dynamics, respectively. At the final phase of migration, the locomoting neurons switch into a terminal translocation mode and undergo dendritic maturation. (B) The migration of locomoting neurons along radial glial fibers is dependent on N-cadherin (purple rectangles). N-cadherin undergoes Rab5-dependent endocytosis and is recycled to the forward plasma membrane via Rab11-dependent pathway. On the other hand, the requirement of Rab7-dependent lysosomal degradation pathway is increased at the final phase of neuronal migration. EE: early endosome, RE: recycling endosome, RGF: radial glial fiber.

Neurons, generated near the ventricle, migrate from the ventricular side toward the pial surface with multi-step morphological changes.⁸ A majority of neurons first exhibit multipolar morphologies and subsequently transform into bipolar-shaped neurons with a leading process, a thick neurite extending toward the pial surface (Fig. 2A). The bipolar-shaped neurons, called locomoting neurons, migrate over a long distance along the radial glial fibers of their mother cells with elongating their axons backward (the locomotion mode of migration). Thus, neural progenitors not only give rise to neurons but also provide a scaffold for locomoting neurons. While the locomotion mode of migration covers most of the neuronal journey,⁹ the locomoting neurons switch into another migration mode, called a terminal translocation mode, at the final phase of migration when their dendrites mature. This complicated multi-step migration of neurons is essential for the formation of the functional six-layered cerebral cortex and closely associated with neuronal maturation. Furthermore, defects in neuronal migration are known to cause several neurological disorders, such as lissencephaly and periventricular heterotopia.

An in vivo gene transfer method, in utero electroporation, as well as genetic approaches for human diseases have accelerated the research for understanding the molecular mechanisms of cortical neuronal migration.⁸ The first reported molecules involved in neuronal migration by the use of in utero electroporation were Rac1 and its upstream and downstream proteins.¹⁰ In utero electroporation-mediated expression of a dominant negative form for Rac1 (DN-Rac1) inhibits neuronal migration and the formation of multipolar and leading processes (Fig. 2A). Consistent with this, BrdU-pulse labeling experiments using Rac1-conditional knockout mice show that neuronal migration is delayed in both the cerebrum and cerebellum, although the migration-defect in the cerebral cortices of the conditional knockout mice is milder than that observed in the dominant negative experiments probably due to compensation by Rac3 or other Rho family small GTPases.^11,12 Furthermore, in vivo suppression of STEF/Tiam1, guanine nucleotide exchange factors (GEFs) for Rac1, strongly inhibits neuronal migration, whereas functional suppression of P-Rex1, another Rac1 GEF, exhibits much milder neuronal migration defects, suggesting that several GEFs differentially regulate Rac1 activity to promote neuronal migration.^10,13

The fact that DN-Rac1 disturbs multipolar cell morphology implicates that one of the downstream pathways of Rac1 is an actin regulatory pathway because multipolar processes contain abundant F-actin. Another downstream target of Rac1 in migrating neurons is the JNK pathway. Although JNK was believed to regulate cell death and transcription in the nucleus, JNK is predominantly activated in the cytoplasm and neuronal processes of cultured cortical neurons and required for the leading process formation and neuronal migration through regulating microtubule dynamics.¹⁰ Interestingly, JNK phosphorylates several microtubule-associated proteins, MAP1B and DCX.^10,14,15 Since the DCX protein is one of the causative gene products of lissencephaly, the JNK pathway may be involved in human neurological disorders.⁸

During the migration, neurons acquire neuronal polarity and elongate their axons. Rac1 deficiency results in severe defects in axon formation. The corpus callosal and hippocampal commissure axons fail to cross the midline in Rac1 conditional knockout brains.¹¹ Rac1 also regulates neuronal polarization via the recruitment of WAVE, an effector of Rac1, to the plasma membrane in cerebellar granule neurons.¹² Furthermore, Rac1 is reported to function in neural progenitors prior to neural differentiation and migration. Rac1 deficiency causes the reduction of neural progenitors due to enhanced cell cycle exit and decreased cell survival.^16,17 Functional suppression of Rac1 also disturbs the interkinetic nuclear movement of neural progenitors in the ventricular zone.¹⁸ These findings suggest that Rac1 regulates several aspects of neural development, including neuronal migration, axon formation and neural progenitor proliferation and survival.

The Roles of Cdc42 and RhoA in Neuronal Migration

Cdc42 is known to regulate both neuronal migration and neural progenitor proliferation (Fig. 2A).^19–21 A heterozygous Lis1 mutation, which causes lissencephaly in humans, decreases the activation of Cdc42 and Rac1, and perturbs the distribution of IQGAP1, an effector protein of Cdc42 and Rac1, and its binding protein, CLIP-170.¹⁹ Together with the fact that CLIP-170 localizes at the plus ends of microtubules and forms a tripartite complex with Cdc42 and IQGAP1,²² Cdc42 promotes neuronal migration via the regulation of microtubules in cooperation with Lis1 and other microtubule binding proteins. During corticogenesis, neural progenitors in the ventricular zone give rise to secondary progenitor cells, called basal progenitors or intermediate progenitors (Fig. 2A). Conditional deletion of Cdc42 in the mouse cerebral cortex results in an increase of basal progenitor mitoses.²⁰ Cdc42 deficiency also induces disruption of adherens junctions between (apical) neural progenitor cells in the ventricular zone, suggesting that Cdc42 is required for cadherin-mediated cell-to-cell adhesion. Although Rac1 is involved in the regulation of cell adhesion in non-neuronal cells, it is unclear whether Rac1 controls cell adhesion in neurons and/or neural progenitors.

In contrast to Rac1 and Cdc42, RhoA has some inhibitory roles in cortical neuronal migration (Fig. 2A). It has been reported that expression of DN-RhoA slightly enhances neuronal migration in the developing cerebral cortex.²³ Therefore, RhoA activity should be suppressed during neuronal migration. A cyclin-dependent kinase (CDK) inhibitor protein, p27^kip1, is reported to have crucial roles in neuronal migration and be functionally associated with RhoA.²⁴ Similar to its role in other cells, p27^kip1 inhibits CDK activity by direct binding to the Cyclin-CDK complex and controls G₁ length and cell cycle exit in neural progenitors. However, p27^kip1 is phosphorylated and stabilized by CDK5, an unconventional CDK that is activated in G₀-arrested neurons, and regulates actin reorganization in the multipolar processes of migrating neurons and neuronal migration.²⁴ The Cdk5-p27^kip1 pathway promotes the activity of cofilin, an actin-binding protein, through the suppression of RhoA, but not Rac1, activity. Interestingly, the migration defect of p27^kip1-knockdown cells can be rescued by co-expression of DN-RhoA,²⁵ suggesting that p27^kip1 promotes neuronal migration by suppressing RhoA activity. Thus, Rho family small GTPases, Rac1, Cdc42 and RhoA, are central regulators for cytoskeleton and cell adhesion of migrating neurons and neural progenitors in the developing cerebral cortex.

Rab5-Mediated Regulation of N-Cadherin Distribution and Neuronal Migration

Compared with Rho family proteins, the involvement of Rab family small GTPases in cell migration is less well understood. Rab family small GTPases consist of more than 60 proteins, and each of them is thought to regulate distinct membrane trafficking pathway in cells (Fig. 1B). Rab5 is one of the more well-studied Rab proteins and involved in many types of endocytosis and early endosome formation. In utero electroporation-mediated expression of DN-Rab5 or short hairpin RNA for Rab5 strongly suppresses cortical neuronal migration (Fig. 2B).⁷ Because endocytosis can be blocked by the expression of DN-Dynamin, which results in similar migration defects, this suggested that Rab5- and Dynamin-mediated endocytosis is important for neuronal migration. Although some Rab5-knockdown locomoting neurons show irregular morphologies, including an abnormally branched leading process, the majority exhibit a normal bipolar morphology with a leading process, and attach normally to the radial glial fibers.

The fact that the locomoting neurons in developing cerebral cortex attach to and migrate along the radial glial fibers was first reported in 1972,²⁶ but the molecular mechanisms were unknown. A recent study showed that N-cadherin is expressed in both locomoting neurons and radial glial fibers and that knockdown of N-cadherin perturbs the migration of locomoting neurons along radial glial fibers.⁷ Furthermore, Rab5-knockdown cells exhibit an increase in the cell surface level of N-cadherin and disruption of its surface distribution pattern, suggesting that increased surface N-cadherin in Rab5-knockdown neurons leads to defects of proper detachment from radial glial fibers that allow movement toward the pial surface (Fig. 2B).

It is possible that Rab5 may be involved in the trafficking of other adhesion molecules in addition to N-cadherin to promote neuronal migration. In the developing cerebellum, astrotactin is expressed in cerebellar granule neurons and required for neuron-glial binding.^27,28 It is unclear whether astrotactin is also involved in the interaction between migrating neurons and radial glial fiber in the developing cerebral cortex. However, weak knockdown of N-cadherin restores the migration defects of Rab5-knockdown neurons, suggesting that N-cadherin is a major cargo molecule of Rab5 in the regulation of neuronal migration (Fig. 2B).

Rab11 and Rab7 Regulate Distinct Steps of Neuronal Migration

Rab5 is required for endocytosis and early endosome formation. As the early endosome is also called the sorting endosome, there are many trafficking pathways that originate from it (Fig. 1B).^6,7 Rab4 and Rab11 regulate fast (direct) or slow (indirect via recycling endosome) recycling pathways from the early endosome to the plasma membrane, respectively. In contrast, Rab7 is involved in the lysosomal degradation pathway, although a recent study revealed that Rab7b controls another trafficking pathway.²⁹ Among these downstream trafficking pathways of Rab5-dependent endocytosis, the Rab11-dependent recycling pathway has important roles in neuronal migration along radial glial fibers. In vivo suppression of Rab11 delays neuronal migration and perturbs subcellular localization of N-cadherin in the locomoting neurons. Taken together with the fact that N-cadherin is abnormally accumulated at transferrin receptor-positive recycling endosomes in Rab11-knockdown primary cortical neurons, this suggests that Rab11 controls N-cadherin trafficking via recycling endosomes to promote neuronal migration along radial glial fibers (Fig. 2B). Whether the defect in forward nuclear movement of migrating neurons is secondary to the abnormal adhesion or whether Rab5 and Rab11 directly regulate nuclear movement is still unclear. A very recent report showed that Rab5 and Rab11 are required for maintaining apical localization of the nuclei in Drosophila photoreceptor cells,³⁰ suggesting that Rab5- and Rab11-dependent pathways might be directly involved in nuclear movement.

On the other hand, Rab7 suppression hardly affects the locomotion mode of neuronal migration, but disturbs terminal translocation at the final phase of migration.⁷ Therefore, each of endocytic pathways has specific functions in distinct steps of neuronal migration during mammalian brain formation. Consistent with previous observations that neurons with terminal translocation migrate independent of radial glial fibers, N-cadherin degradation is enhanced at the final phase of migration. Interestingly, a constitutive active form of Fyn, a Src family tyrosine kinase, enhances Rab7-dependent N-cadherin degradation in vitro. Because Fyn activity may be increased in response to Reelin signal at the final phase of migration, Fyn might be a key regulator for changing the migration mode of neurons from the locomotion mode to the terminal translocation mode.

Conclusion

Recent reports focusing on the in vivo roles of small GTPases have shed light on the molecular mechanisms for each step of neuronal migration. (1) Neural progenitor proliferation and interkinetic nuclear movement are regulated by Rac1 and Cdc42. (2) After neuronal differentiation, neurons extend their multipolar processes, which contain abundant F-actin. Cdk5 and p27^kip1 regulate the multipolar morphology through the suppression of RhoA activity. (3) Subsequently, multipolar cells form a leading process extending toward the pial surface and switch migration mode into a locomotion mode; the leading process formation is dependent on Rac1- and JNK-mediated regulation of microtubule dynamics. (4) The leading process attaches to radial glial fibers via N-cadherin-mediated cell-to-cell adhesion, and this interaction may stabilize leading process morphology. (5) To migrate toward the pial surface, N-cadherin is partially internalized via Rab5-mediated endocytosis. Rab11 enhances the trafficking of internalized N-cadherin to the forward plasma membrane via recycling endosomes. This recycling of N-cadherin promotes piadirected neuronal migration along the radial glial fibers. (6) At the final phase of neuronal migration, the requirement of Rab7-dependent lysosomal degradation pathway is increased, and the preferential N-cadherin degradation occurs.

Thus, cortical neuronal migration requires coordinated regulation of cell adhesion, cytoskeleton and membrane trafficking, and these cellular events are controlled by not only Rho family but also Rab family small GTPases.

Extra View to: Kawauchi T, Sekine K, Shikanai M, Chihama K, Tomita K, Kubo K, et al. Rab GTPases-dependent endocytic pathways regulate neuronal migration and maturation through N-Cadherin trafficking. Neuron. 2010;67:588–602. doi: 10.1016/j.neuron.2010.07.007.