Reelin modulates cytoskeletal organization by regulating Rho GTPases

Introduction

Reelin, a conserved extracellular glycoprotein, controls the migration and laminar arrangement of neurons during the development of the neocortex, hippocampus, cerebellum and spinal cord. When newborn postmitotic neurons have reached their final position, Reelin promotes the maturation of dendrites and dendritic spines. In reeler mice, which have a naturally occurring mutation that makes them Reelin-deficient, cortical neurons migrate abnormally, resulting in an inversion of the cortical laminar organization, with later-born neurons remaining in the deeper layers of the cortex (reviewed in ref. ¹3).

Reelin binding to the lipoprotein receptors very low-density lipoprotein receptor (Vldlr) and the apolipoprotein E receptor 2 (Apoer2) induces the Src family kinase (SFK)-mediated tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1).⁴7 Hence, disruption of Dab1, absence of both lipoprotein receptors Apoer2 and Vldlr or simultaneous inactivation of the Src family kinases (SFKs) Src and Fyn leads to a reeler-like phenotype.⁸11 The phosphorylation of Dab1 activates, besides other signaling pathways, class I phosphatidylinositol-3-kinase (PI3K).¹²14 However, despite growing insights into the signaling cascades activated by Reelin in responsive neurons, the cellular mechanisms of its action on neuronal positioning and development are still poorly understood.¹⁵

Rho GTPases as Phosphatidylinositol-3-Kinase Effectors during Neurodevelopment

The large number of PI3K effectors creates a complex signaling web downstream of PI3K activation.¹⁶ Among the key players linking PI3K activity to specific cellular responses¹⁷ are small GTPases of the Rho family, which regulate the cytoskeletal and membrane rearrangements required for cell movement.¹⁸19 Small GTPases act as molecular switches, cycling between an active, GTP-bound and an inactive, GDP-bound form.²⁰21 The members of the Rho-family Rac, RhoA and Cdc42 are essential regulators of many cellular processes during neural development.²²23

Rho GTPases Link Reelin Signaling to the Neuronal Cytoskeleton

In order to investigate cellular effects of Reelin, we employed time-lapse microsopy of cultured primary neurons and found an increased motility of distal neurites.²⁴ Using this cellular effect as a readout we identified the Rho GTPase Cdc42 as a novel cellular effector of Reelin signaling. Our results indicate that Reelin and Rho GTPase-mediated signaling cascades interact during neuromorphogenesis to increase filopodia formation and growth cone motility (Fig. 1A). The activation of Cdc42 by Reelin requires signaling through Apoer2, Dab1 and PI3K (Fig. 1B). In addition, Reelin-induced localized Rac1 activation might participate in Reelin's effects on growth cone motility. Furthermore, Reelin increases neuronal transport vesicle formation and redirects neuronal vesicle transport into small neurites, the prospective dendrites (Fig. 1A). Cdc42, but not RhoA or Rac1, localizes in part to the Golgi apparatus, together with its targets N-WASP, IQGAP and the Golgi vesicle coat protein and coatomer (reviewed in ref. ²⁶ and ²⁷). Cdc42- and N-WASP-controlled actin polymerization regulates Golgi-to-ER transport²⁸ and thus is a central event of the multiple steps of vesicle trafficking. The differential subcellular localization and activity of Cdc42 and the Par complex are critical for axon specification.²⁹30 By activating Cdc42 via Apoer2 at the tips of all neurites in stage-II neurons, Reelin seems to interfere with axondendrite differentiation.²⁴

Figure 1

Reelin influences neuromorphogenesis by activating Rho GTPases. (A) Growth cone motility and neurite branch formation are activated (+) by Rac1 and Cdc42 and negatively regulated (−) by RhoA. Reelin participates in the regulation of growth cone motility and branching by regulating Rho GTPase activity (see B). Filopodia formation and the formation of neuronal transport vesicles, both known to be mediated by Cdc42, are triggered by Reelin.²⁴ (B) Binding of the extracellular matrix protein Reelin to its transmembrane receptors Apoer2 and Vldlr triggers Dab1 tyrosine phosphorylation by Src family kinases (SFK). This leads to the activation of several downstream signals, including phosphatidylinositol-3-kinase (PI3K), which activates Cdc42 via an unknown intermediate effector. There is evidence that Reelin also might locally activate Rac1. N-WASP and WAVE link Cdc42 and Rac1 activity to changes of the actin cytoskeleton, leading to increased growth cone motility, filopodia and vesicle formation and dendritic branching (see A). Cdc42 and Rac1 also contribute to activation of the PAK/LIMK pathway, which inhibits actin filament disassembly by phosphorylating and thereby inhibiting the actin-depolymerizing factor ADF/cofilin²⁵ leading to growth inhibition or stabilization of filopodia. The fine tuning of these context-dependent convergent or divergent pathways downstream of Rho GTPases is fundamental for correct neuronal development.²⁴

Reelin, Neuronal Polarization and Golgi Organization

Of note, a recent study demonstrated that Reelin-Dab1 signaling antagonizes the action of the protein kinase LKB1 on neuronal polarization.³¹ LKB1, whose effect on cell polarization is controlled by its interaction with the pseudokinase STRAD,³² was shown to form a complex involving the serine/threonine kinase Stk25 and the Golgi matrix protein GM130, which regulates Golgi morphology and axon specification in neurons. Interestingly, the kinase activity of Stk25 was dispensable for mediating these effects.³¹ Thus, Reelin seems to have prominent roles in the regulation of the Golgi apparatus and the direction of neuronal vesicle transport, thereby participating in the axon determination of maturing neurons. Importantly, it has been shown that the Golgi protein GM130 can also form a complex that includes Cdc42.³³ Future studies will have to address how Cdc42 activation by Reelin relates to the antagonizing effect of Reelin on LKB1-STRAD-Stk25-GM130 signaling.

Reelin and Neuropeptidergic Signaling Converge on the Level of Rho GTPases

In the adult brain, Reelin is mainly localized to GABA-containing peptidergic interneurons.³⁴ The functional relevance of this colocalization, which implies a cosecretion under physiological or pathophysiological conditions, is largely unknown. We could demonstrate that Reelin functionally interacts with the peptidergic VIP/PACAP38-receptor system to increase axonal branch formation.²⁴ As VIP-mediated Rho-kinase inhibition³⁵36 induces the elongation of dendrites and axons by stabilizing microtubules,³⁷ these results demonstrate that Reelin and a neuropeptidergic system can cooperate to promote neuronal development by inducing axonal branching. In addition, these findings pinpoint the influence of the tubulin cytoskeleton in mediating Reelin's effect on neuromorphogenesis, which is also targeted by other effectors of the Reelin-Dab1 signaling cascade, including the Tau kinase GSK3beta,¹² Lis1,³⁸ and Map1b.³⁹

Putative Functions of Cdc42 in Reelin-Dependent Spine Morphogenesis and Synapse Formation

In addition to its prominent role in controlling neuronal positioning in developing brain strucutres, Reelin is also involved in the development of dendrites⁴⁰41 and promotes spine morphogenesis and synapse formation.⁴²43 Given the known function of Rho GTPases in the growth and remodeling of dendrites and synaptogenesis (reviewed in ref. ²²44 and ⁴⁵), which require extensive remodeling of the neuronal cytoskeleton, it is tempting to speculate that Cdc42 cooperates with other Reelin effector molecules such as the adapter proteins Crk und CrkL,⁴⁶ Akt and mTor signaling¹⁴ to regulate dendrite morphogenesis. An additional level of complexity is added to this signaling network by the interaction of Reelin and Notch signaling,⁴⁷ which cooperates with Rho GTPases in the specification of dendrite morphology (reviewed in ref. ⁴⁸). Defects in dendrite and spine morphology and a reduction in synapse number are observed in many neuropsychiatric diseases,⁴⁹ and both have been connected to alterations in Reelin signaling as well as to defects in Rho GTPase function (reviewed in ref. ²² and ⁵⁰). We propose that the activation of Cdc42 by Reelin²⁴ links the observation that defects in Rho GTPase function and Reelin signaling both contribute to morphological defects leading to neurological and psychiatric disorders.

Role of Cdc42 Activation in Radial glia

A recent report demonstrated that Cdc42 is required for maintaining the polarized morphology of the cortical radial glial scaffold.⁵¹ Live imaging of radial glial cells in embryonic cortical slice cultures revealed dynamic inter-radial glial interactions involving transient filopodia-like radial glial protrusions and highly motile radial glial leading edges resembling neuronal growth cones. By in utero electroporation of a dominant-negative Cdc42-GFP construct under control of a radial glial-specific Blbp promoter it was demonstrated that polarized Cdc42 activation was necessary for maintaining radial glial morphology and dynamics. Crossing mice carrying floxed Cdc42 alleles with hGFAP-Cre transgenic mice expressing Cre recombinase under control of the human GFAP promoter resulted in the conditional ablation of Cdc42 in radial progenitor cells and led to cortical layering defects and neuronal ectopias. Defects in axonogenesis were also observed.⁵¹ This phenotype resembles but is not identical to that of mice lacking essential components of the Reelin signaling cascade, probably because Cdc42 integrates the input of additional ligand-receptor signaling systems. Moreover, it should be noted that the use of radial glial-specific promoters will not prevent neuronal Cdc42 inactivation, since the vast majority of neurons is derived from radial glial progenitors.⁵² To clearly distinguish the separate effects of radial glial vs. neuronal Cdc42 ablation and their relative contributions to the observed lamination defect, the comparison with mice lacking Cdc42 specifically in neurons, e.g., by using a Dcx-Cre transgenic mouse line, might be helpful. However, in light of the data generated by Yokota and colleagues, it seems likely that at least some of the effects of Cdc42 on radial glial morphology and dynamics are modulated by Reelin, whose receptors and intracellular effectors have been described to be expressed in radial glial cells of cortical structures.⁵³54

Perspective

At first sight it might seem surprising that so many different cellular effects of Reelin are modulated by a single Rho GTPase. However, one has to bear in mind that Rho-GTPases are embedded in different effector-domain complexes depending on the contextual situation. Selectivity can be achieved by engaging different guanine nucleotide-exchange factors (GEFs). GEFs are responsible for the activation of Rho-family GTPases in response to various extracellular stimuli, and regulate diverse downstream cellular responses in neurons such as migration, morphogenesis and axonal pathfinding.⁵⁵ Dbl-related GEFs represent the largest family of direct activators of Rho GTPases in humans. In addition, atypical Rho-GEFs that contain Dock homology regions (DHR-1 and DHR-2) are expressed in a variety of tissues, including the nervous system and achieve spatial and temporal restriction of Rho GTPase-signaling.⁵⁶ It remains to be determined which of these GEFs are activated by Reelin and lipoprotein receptor-mediated signaling and how they preferentially couple to different downstream effector pathways, thereby mediating Reelin's different effects on the actin and microtubule cytoskeleton via Rho GTPases. The fine tuning and signal integration of these partially convergent or divergent pathways downstream of the Rho GTPase effectors is fundamental for correct neuronal development.⁵⁷