Cdk5 regulates multiple cellular events in neural development, function and disease

Authors

  • Takeshi Kawauchi

    Corresponding author
    1. Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Saitama, Japan
    2. Department of Physiology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan
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Abstract

Cyclin-dependent kinases (CDKs) generally regulate cell proliferation in dividing cells, including neural progenitors. In contrast, an unconventional CDK, Cdk5, is predominantly activated in post-mitotic cells, and involved in various cellular events, such as microtubule and actin cytoskeletal organization, cell–cell and cell–extracellular matrix adhesions, and membrane trafficking. Interestingly, recent studies have indicated that Cdk5 is associated with several cell cycle-related proteins, Cyclin-E and p27kip1. Taking advantage of multiple functionality, Cdk5 plays important roles in neuronal migration, layer formation, axon elongation and dendrite arborization in many regions of the developing brain, including cerebral cortex and cerebellum. Cdk5 is also required for neurogenesis at least in the cerebral cortex. Furthermore, Cdk5 is reported to control neurotransmitter release at presynaptic sites, endocytosis of the NMDA receptor at postsynaptic sites and dendritic spine remodeling, and thereby regulate synaptic plasticity and memory formation and extinction. In addition to these physiological roles in brain development and function, Cdk5 is associated with many neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. In this review, I will introduce the physiological and pathological roles of Cdk5 in mammalian brains from the viewpoint of not only in vivo phenotypes but also its molecular and cellular functions.

Introduction

Cyclin-dependent kinase-5 (Cdk5) is a proline-directed serine/threonine kinase related to Cdc2/Cdk1 that was discovered in 1992 (Hellmich et al. 1992; Lew et al. 1992b; Meyerson et al. 1992; Xiong et al. 1992). While other CDK family kinases control cell cycle progression, Cdk5 is abundantly expressed in neural tissues and appears to be less involved in the proliferation of neural progenitors or other cycling cells (Tsai et al. 1993). Furthermore, Cdk5 is activated upon association with its specific activators, p35 or p39, but not most cyclins, such as Cyclin-A, -D and -E (Ishiguro et al. 1994; Lew et al. 1994; Tsai et al. 1994; Uchida et al. 1994; Tang et al. 1995; Lee et al. 1996), although recent reports indicate that Cyclin-I can activate Cdk5 in post-mitotic cells (Brinkkoetter et al. 2009). Cdk5 was identified as a kinase for neurofilaments and tau protein (Hisanaga et al. 1991, 1993; Ishiguro et al. 1992; Lew et al. 1992a; Baumann et al. 1993), and to date, numerous substrates have been reported. Functional analyses of Cdk5 and its substrate molecules have revealed that Cdk5 controls various cellular events, including cytoskeletal organization, cell adhesion, membrane trafficking, cell cycle exit and neuronal differentiation, and thereby regulates multiple aspects of brain development and function. In this review, I will introduce the physiological and pathological roles of Cdk5 in neural development, function and disease with particular focus on the in vivo phenotypes of Cdk5 suppression in mice, and then discuss the related molecular and cellular functions of Cdk5.

Cdk5 in brain development

Cortical neuronal migration

The mammalian cerebral cortex exhibits a six-layered structure (Fig. 1A–C). During the development of the cerebral cortex, the first cohort of newborn neurons forms the preplate, which contains several types of neurons, such as Reelin-positive Cajal-Retzius cells and GABAergic interneurons. Subsequent cohorts of neurons, generated in the ventricular zone (VZ), migrate and split the preplate into the outer marginal zone (MZ) (future Layer I) and inner subplate by forming the cortical plate (CP). The CP neurons are arranged in accordance with their birth date. While the early-born neurons are located at the deep layer, the late-born neurons pass through the existing cortical layers to reach the superficial layer of the CP. As a result, the CP neurons are aligned in a birth date-dependent inside-out sequence in Layers II–VI of the cortex (Kawauchi & Hoshino 2008; Govek et al. 2011) (Fig. 1A).

Figure 1.

Cerebral cortical development and Cdk5. (A) Newly generated cortical excitatory neurons migrate toward the pial surface and split the preplate (PP). Subsequent cohorts of neurons are aligned in a birth date-dependent inside-out manner. (B) In Cdk5- or p35-deficient brains, the first cohort of cortical excitatory neurons (Layer VI neurons) migrates and splits the preplate (PP) normally. However, subsequently generated Layer II-V neurons exhibit severe migration defects, resulting in the formation of roughly inverted cortical layers under the subplate (SP). (C) Neural progenitors proliferate in the ventricular zone (VZ) or subventricular zone (SVZ). After differentiation, post-mitotic neurons first display multipolar morphology (purple cells), and subsequently form a leading process and an axon while retracting other processes. The bipolar-shaped locomoting neurons (red cells) migrate along radial fibers that are derived from the radial glial progenitors (light blue cells) in the VZ. At the final phase of migration, neurons change their migration mode from locomotion into the terminal translocation mode (orange cell) and start dendrite maturation (yellow cell). PP, preplate; MZ, marginal zone; CP, cortical plate; SP, subplate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone.

The migration of immature excitatory neurons consists of several contiguous modes in the developing cerebral cortex (Fig. 1C). After the final cell division, post-mitotic neurons first display multipolar morphology. Subsequently, multipolar neurons form a pia-directed thick process, called a leading process, and an axon, with retraction of the other processes, transforming into bipolar-shaped, so-called “locomoting” neurons. These locomoting neurons migrate along the neural progenitor-derived radial fibers. This migration mode (the locomotion mode) covers most of the neuronal migration path. At the final phase of neuronal migration, neurons change from the locomotion mode into a radial fiber-independent terminal translocation mode and begin dendrite maturation (Kawauchi & Hoshino 2008; Marin et al. 2010; Hatanaka et al. 2012) (Fig. 1C).

Cyclin-dependent kinase-5 knockout mice display perinatal lethality and lack the cerebral cortical laminar structure and cerebellar foliation (Ohshima et al. 1996; Gilmore et al. 1998) mainly due to neuronal migration defects (Kawauchi et al. 2003) (Fig. 1B). In the developing cerebral cortex, Cdk5 controls multiple steps of cortical neuronal migration, including the formation of multipolar processes (Kawauchi et al. 2006), acquisition of neuronal polarity (Ohshima et al. 2007), formation of a leading process (Kawauchi et al. 2006) and the locomotion mode of migration (Nishimura et al. 2010) (Fig. 1C). In contrast, suppression of the Reelin signaling pathway in late-born neurons only disturbs the terminal translocation (Olson et al. 2006; Sekine et al. 2011), suggesting that the Reelin pathway and Cdk5 regulate divergent downstream events although Cdk5 is known to phosphorylate Dab1, a downstream adapter protein of Reelin (Keshvara et al. 2002).

Unlike Cdk5-deficient mice, mice lacking p35, an activator for Cdk5, survive to adulthood and suffer from sporadic adult lethality and seizures (Chae et al. 1997). The milder p35 phenotype results from functional compensation by another Cdk5 activator, p39, as p35/p39 double knockout mice exhibit perinatal lethality and disruption of cortical lamination and other brain structures, resembling Cdk5-deficient mice (Ko et al. 2001). While Cdk5- or p35-deficient early-born neurons can migrate and split the preplate to form layer VI, subsequently generated neurons (layer II–V neurons) are stalled under the subplate, and exhibit a roughly inverted cortical layer lamination (Gilmore et al. 1998; Kwon & Tsai 1998) (Fig. 1B). This phenotype is different from Reelin-deficient reeler mice, in which the preplate splitting does not occur (Tissir & Goffinet 2003).

Axon and dendrite formation

It has been reported that Cdk5 is required for neurite outgrowth in rat primary cortical neurons (Nikolic et al. 1996). Cdk5 promotes axon elongation through phosphorylation of many substrates, including Axin and LMTK1 (Fang et al. 2011; Takano et al. 2012). In Cdk5 knockout brains, the trajectories of thalamocortical axons are abnormal (Gilmore et al. 1998). In addition, cerebral cortex-specific Cdk5 knockout mice, generated by use of Emx1-Cre, exhibit abnormal axon and dendrite morphologies (Ohshima et al. 2007). While cortical layer V pyramidal neurons extend a single apical dendrite with secondary branches and a ventricle-directed axon in control mice, Cdk5-deficient layer V neurons form multiple dendrites extended directly from soma, and their axons run obliquely within the cortex (Ohshima et al. 2007). Interestingly, S-nitrosylation of Cdk5 on Cys83 is reported to regulate proper number and length of dendrites in cultured hippocampal neurons (Zhang et al. 2010b).

Cerebellar development

In the developing cerebellum, Purkinje cells are born in the ventricular zone of the fourth ventricle and migrate radially (Kapfhammer 2004; Roussel & Hatten 2011; Hoshino 2012; Seto et al. 2014), although a recent report has indicated that Purkinje cells that are generated at E10.5 in the posterior periventricular region of the lateral cerebellum migrate tangentially towards the anterior direction (Miyata et al. 2010) (Fig. 2A). While Purkinje cells exhibit stellate morphologies with disoriented dendrites after birth, they show rapid growth and expansion of the dendritic trees from P7 (Kapfhammer 2004) (Fig. 2C). In contrast, granule cells, derived from the rhombic lip, first migrate tangentially in the external granule cell layer (EGL) and subsequently migrate radially along Bergmann glial fibers toward the internal granule cell layer (IGL) (Roussel & Hatten 2011; Hatanaka et al. 2012) (Fig. 2B).

Figure 2.

Cerebellar development and Cdk5. (A–B) Illustration of developing cerebellum in embryonic (A) and postnatal (B) stages. Purkinje cells (light red cells), generated from the ventricular zone (VZ) of the fourth ventricle, migrate radially or tangentially (red arrows). Cerebellar granule cells (blue cells) first migrate tangentially in the external granule layer (EGL), and subsequently change direction towards the ventricular side. They exhibit inward radial migration along the radial fibers of Bergmann glial cells (light green cells), a process controlled by Cdk5. (C) After birth, Purkinje cells exhibit stellate morphologies with disoriented dendrites, and subsequently show rapid growth and expansion of the dendritic trees (Kapfhammer 2004). Proper dendritic branching relies on Cdk5.

Cyclin-dependent kinase-5 is involved in the radial migration of Purkinje cells in a cell-autonomous manner (Ohshima et al. 1999) (Fig. 2A). While suppression of Cdk5 results in weak tangential migration-defects of cerebellar granule cells in the EGL, it severely perturbs the radial migration toward the IGL (Umeshima & Kengaku 2013) (Fig. 2B). Midbrain-hindbrain-specific Cdk5 conditional knockout mice, generated by use of Wnt1-Cre, have Purkinje cells with abnormal dendrite morphology at P8 and P12 and defects in radial migration, in addition to granule cells with defects in inward migration (Kumazawa et al. 2013) (Fig. 2C). Consistently, the dendritic density and branching of the Purkinje cells are decreased in p35-deficient cerebellum, compared with control. These deficiencies of the Purkinje cell dendrites are at least partly derived from non-cell autonomous defects (Kumazawa et al. 2013).

Cdk5 in brain functions and disease

Synaptic plasticity and high-ordered brain functions

Upon completion of the pia-directed migration, excitatory neurons form dendritic spines and synapses to create neural networks. The neural networks are not fixed, but exhibit plasticity in an activity-dependent manner. Synaptic plasticity, such as long-term potentiation (LTP) and depression (LTD), are selective strengthening and weakening of synapses, respectively, is regulated by various cellular mechanisms, including alteration of neurotransmitter release at the presynaptic site, modulation of cell surface levels or molecular properties of neurotransmitter receptors at the postsynaptic site, and dendritic spine remodeling (Kessels & Malinow 2009). Cdk5 is involved in synaptic plasticity through its functions at both presynaptic and postsynaptic sites (see also the following “Membrane trafficking and signaling at synapse” section).

In hippocampal slices, a low-frequency stimulation of the Schaeffer collaterals of CA3 pyramidal neurons induces LTD at the CA3-CA1 synapses, whereas a 100-Hz tetanus stimulation induces LTP. p35 knockout mice show impairment of LTD, but not LTP, in the hippocampal CA3-CA1 synapses, and defects in spatial learning and memory, as evaluated by use of the Morris water maze (Ohshima et al. 2005). When rat hippocampal slices are treated with Roscovitine, an inhibitor for CDKs, including Cdk5, LTP is reduced in CA1 neurons (Li et al. 2001). These differences may be due to the side-effects of Roscovitine on voltage-dependent calcium channels and/or a compensatory increase of p39 in p35-deficient mice.

In contrast to 100-Hz-tetanus, theta-burst stimulation induces a late-phase LTP (L-LTP), a long-lasting form of synaptic plasticity, which requires the activity-dependent release and synthesis of brain-derived neurotrophic factor (BDNF) (Patterson et al. 2001). BDNF promotes NMDA receptor-mediated cytoskeletal changes at postsynaptic sites during L-LTP (Rex et al. 2007). Cdk5 is reported to phosphorylate a BDNF receptor, TrkB, at Ser478, and this phosphorylation is required for the theta-burst stimulation-induced L-LTP in the hippocampal CA3-CA1 synapses and spatial memory (Lai et al. 2012). On the other hand, a weak theta-burst stimulation (two bursts of four pulses at 100 Hz), which is insufficient to induce LTP in wild type mice, can induce LTP in p35-deficient mice, suggesting that p35-deficient mice have a lower threshold for LTP-induction (Wei et al. 2005).

While conditional knockout of Cdk5 in the adult brain, by use of an inducible Cre-ERT driven by the prion promoter, enhances hippocampal LTP and spatial and context-dependent fear memory formation (Hawasli et al. 2007), forebrain-specific Cdk5 conditional knockout mice, generated by use of αCaMKII-Cre, show impaired spatial learning and contextual fear memory as well as hyperactivity and reduced anxiety (Su et al. 2013). Furthermore, hippocampal Cdk5 is involved in the extinction of contextual fear memory (Hawasli et al. 2007; Sananbenesi et al. 2007). This suggests that Cdk5 may have various synaptic roles in different brain regions. In fact, Cdk5 also plays important roles in the expression of acetylcholine receptor (AChR) at neuromuscular junctions (NMJs) (Fu et al. 2001), and negatively regulates dopamine release and dopamine-mediated postsynaptic signaling in the striatum (Bibb et al. 1999; Chergui et al. 2004).

Neurodegenerative diseases

Neurodegenerative diseases, such as Alzheimer's, Parkinson's and Huntington diseases and amyotrophic lateral sclerosis (ALS), are characterized by a progressive loss of specific neuronal populations. The pathological hallmarks of Alzheimer's disease are extracellular senile plaque and intracellular neurofibrillary tangles (NFTs), whose major components are an amyloid-beta 42 peptide (Aβ42), derived from amyloid precursor protein (APP), and a hyperphosphorylated tau, respectively. Cdk5 has been identified as a tau kinase and also reported to control Aβ production (Cheung & Ip 2012; Shukla et al. 2012).

In pathological conditions, p35 is cleaved into p25, in which the N-terminal region of p35 is truncated (Patrick et al. 1999) (Fig. 3). This cleavage is mediated by Calpain (Kusakawa et al. 2000; Lee et al. 2000). Because membrane association of p35 largely depends on myristoylation at its N-terminal region, p35 and p25 exhibit different subcellular localization (Asada et al. 2008). Furthermore, the p25 protein is much more stable than p35, and the two proteins show different substrate specificities. Cdk5/p25 strongly phosphorylates tau and another microtubule-associated protein, MAP1B, both of which are found in NFTs, in contrast to Cdk5/p35 which hardly phosphorylates them (Patrick et al. 1999; Kawauchi et al. 2005) (Fig. 3). However, other reports show that Cdk5 negatively controls another tau kinase, glycogen synthase kinase 3 beta (GSK3β) (Plattner et al. 2006; Wen et al. 2008). Cooperation and/or balance between Cdk5 and GSK3β kinase activities may play important roles in tau hyperphosphorylation in brains with Alzheimer's disease.

Figure 3.

Cyclin-dependent kinase-5 (Cdk5) substrates in brains with neurodegenerative diseases. In brains with neurodegenerative diseases, p35 is cleaved into a more stable isoform, p25. Cdk5/p25 exhibits different substrate specificities from Cdk5/p35. See text for detail.

Transgenic mice overexpressing p25 in postnatal forebrains, exhibit hyperphosphorylation and abnormal accumulation of endogenous tau proteins, neuronal loss and NFT formation (Cruz et al. 2003). Transgenic mice expressing both p25 and P301L-mutated human tau (found in Frontotemporal dementia with Parkinsonism linked to chromosome 17 [FTDP-17] patients), also show increased tau phosphorylation and aggregation and NFT formation (Noble et al. 2003). Furthermore, it has been reported that p25-overexpressing mice enhance the production of Aβ (Wen et al. 2008).

Cdk5/p25 is involved in other neurodegenerative diseases. Missense mutations in Cu/Zn superoxide dismutase 1 (SOD1) are found in familial ALS patients. Transgenic mice with G37R-mutated SOD1, an ALS model, show abnormal perikaryal accumulation of neurofilaments (Patzke & Tsai 2002). In the G37R-SOD1-transgenic mice, p25 production is elevated and Cdk5/p25 is colocalized with the perikaryal neurofilaments (Nguyen et al. 2001). In addition, the correlation between the enhanced Cdk5 activity and shortened longevity is observed. It is also known that Cdk5/p25 plays important roles in the degeneration of dopaminergic neurons in Parkinson's disease (Smith et al. 2003). Interestingly, however, Cdk5 is reported to have protective roles in the neuropathology of Huntington disease (Luo et al. 2005; Anne et al. 2007; Kaminosono et al. 2008).

Molecular and cellular functions of Cdk5

Microtubule regulation

Microtubules consist of α- and β-tubulin heterodimers and contribute to various cellular events, including morphological change, axon and dendrite formation, and intracellular transport. Microtubule regulation plays important roles in cortical neuronal migration because mutations in tubulins and several microtubule-associated proteins, such as Lis1 and Dcx, are found in human lissencephaly, a neuronal migration disorder (Reiner et al. 1993; Des Portes et al. 1998; Gleeson et al. 1998; Keays et al. 2007).

Suppression of Cdk5 kinase activity leads to over-stabilization of microtubules, resulting in decreased microtubule dynamics, and this dysregulation of microtubule stability/dynamics disturbs cortical neuronal migration (Kawauchi et al. 2005). Cdk5 is known to phosphorylate many microtubule-regulatory proteins (Contreras-Vallejos et al. 2014); Cdk5 phosphorylates Dcx at Ser297 (Tanaka et al. 2004) and a Lis1-binding protein, Ndel1 (previously referred to as Nudel) (Niethammer et al. 2000; Sasaki et al. 2000), suggesting that Cdk5 functions as an upstream regulator of lissencephaly-associated proteins (Fig. 4). Dcx promotes microtubule polymerization, stability and bundling activity (Francis et al. 1999; Gleeson et al. 1999; Horesh et al. 1999). Cdk5-mediated phosphorylation of Dcx negatively regulates its microtubule-binding affinity (Tanaka et al. 2004). Ndel1 and Lis1 are key regulators for the dynein complex, a minus end-directed microtubule motor (Yamada et al. 2008). Because dynein-mediated motor activity has essential roles in nuclear movement of the migrating neurons (Tsai & Gleeson 2005; Tsai et al. 2007) and as Cdk5 is required for the locomotion mode of migration (Nishimura et al. 2010), this shows that one of the important downstream events of Cdk5 is control of the dynein complex during the locomotion mode of neuronal migration.

Figure 4.

Cyclin-dependent kinase-5 (Cdk5) substrates in developing brains. Cdk5 phosphorylates many substrate molecules to control neurogenesis, neuronal migration and neurite elongation. See text for detail.

As described above, Cdk5 is known to phosphorylate MAP1B, but this phosphorylation is less prominent in developing cortical neurons. In fact, Cdk5/p35 does not phosphorylate MAP1B in vitro, whereas Cdk5/p25 strongly phosphorylates MAP1B (Kawauchi et al. 2005). The different substrate specificities between Cdk5/p25 and Cdk5/p35 may contribute to the determination of the physiological and pathological conditions in brains.

Focal adhesion kinase (FAK) is a major protein localized at focal adhesions, cell–extracellular matrix (ECM) adhesions, and is involved in actin organization in non-neuronal cells (Kawauchi 2012). In contrast, FAK is colocalized with the perinuclear microtubules in cortical neurons (Xie et al. 2003). Cdk5 phosphorylates FAK at Ser732, which is required for the microtubule organization around nuclei and has a role in the microtubule-dependent nuclear elongation during neuronal migration (Xie et al. 2003) (Fig. 4).

Actin organization

Although many studies focus on the relationship between Cdk5 and microtubule regulation, Cdk5 is also colocalized with F-actin in the growth cone of cultured neurons (Nikolic et al. 1996). Furthermore, both p39 and p35 are reported to interact with F-actin (Humbert et al. 2000; He et al. 2011).

Cyclin-dependent kinase-5 phosphorylates a CDK-inhibitor protein, p27kip1, at Ser10, and this phosphorylation stabilizes the p27kip1 protein through prevention of its proteasome-dependent protein degradation (Kawauchi et al. 2006) (Fig. 4). Although a major function of p27kip1 is to promote cell cycle exit, recent reports have indicated that p27kip1 has alternative functions. In non-neuronal cells, p27kip1 suppresses the activity of a small GTPase, RhoA, through interfering with the binding of guanine nucleotide exchange factors (activators) for RhoA (Besson et al. 2004). Cdk5 and p27kip1 promote the activity of an actin-binding protein, Cofilin, via the suppression of RhoA and its downstream kinase, Rho kinase/ROCK, in cortical neurons (Kawauchi et al. 2006), although it is unclear whether p27kip1 directly or indirectly regulates RhoA activity in neurons. Rho kinase/ROCK is known to phosphorylate LIM kinase, which negatively regulates the actin-binding activity of Cofilin through the phosphorylation at Ser3 (Maekawa et al. 1999; Mizuno 2013), and Ser3-phosphorylation of Cofilin has a role in cortical neuronal migration (Kawauchi et al. 2006). Furthermore, in vivo knockdown of p27kip1 decreases the amount of F-actin in multipolar processes of migrating neurons, and suppression of Cdk5 or p27kip1 perturbs multipolar process formation and neuronal migration (Kawauchi et al. 2006). Interestingly, Cerebral cavernous malformation 3 (CCM3, also known as PDCD10), a causative gene product for an autosomal dominant cerebrovascular disorder, is reported to be an upstream candidate molecule of the Cdk5-27kip1-Cofilin pathway (Louvi et al. 2014).

A recent study has indicated that p27kip1 is associated with microtubules, and is required for proper migration of cortical interneurons, which migrate tangentially from the ganglionic eminence to the cortical plate (Godin et al. 2012). Because Cdk5/p35 is involved in the tangential migration of cortical neurons (Rakic et al. 2009), Cdk5-mediated regulation of p27kip1 may regulate cortical interneurons as well as excitatory projection neurons.

Neurabin-I is a neuron-specific actin-binding protein. Cdk5 phosphorylates Neurabin-I and Neurabin-II (also known as Spinophilin) in vitro, but Cdk5-mediated phosphorylation of Neurabin-I, but not Neurabin-II, controls the interaction with F-actin and neuronal morphologies (Futter et al. 2005; Causeret et al. 2007) (Fig. 4). A recent report has shown that Cdk5 phosphorylates another F-actin-binding protein, Drebrin that regulates neuritogenesis and spine morphologies, and this phosphorylation relieves the intramolecular interaction of Drebrin, resulting in the promotion of its F-actin binding (Worth et al. 2013; Tanabe et al. 2014). Thus, Cdk5 regulates actin organization in several ways.

Intermediate filaments

Cyclin-dependent kinase-5 is known to control at least two types of intermediate filaments, neurofilaments and nestin. As described above, Cdk5 phosphorylates neurofilament proteins and Cdk5-mediated phosphorylation of neurofilaments is associated with the pathology of neurodegenerative diseases, such as ALS (Nguyen et al. 2001). Furthermore, Cdk5 phosphorylates Nestin at Thr316 (Sahlgren et al. 2003). During the AChR clustering at NMJs, Nestin promotes Cdk5/p35 activity, but overexpression of Thr316-mutated Nestin decreases the Cdk5/p35 activity and dispersion of AChR in response to ACh, suggesting the existence of a feedback loop between Cdk5/p35 and Nestin (Yang et al. 2011).

Cell adhesion

Cell adhesion is classified into cell–cell adhesions and cell–ECM adhesions, whose major adhesion molecules are Cadherins and Integrins, respectively (Kawauchi 2012). Cadherin-mediated cell–cell adhesions and Integrin-mediated cell–ECM adhesions are differentially regulated by the intracellular binding partners, Catenins and Talin, respectively, although some molecules, such as Vinculin and its binding protein, Vinexin, are localized at both cell–cell and cell–ECM adhesions (Kioka et al. 1999; Kawauchi et al. 2001; Peng et al. 2011).

p35 has been reported to bind to β-Catenin in cortical neurons, and Cdk5/p35 phosphorylates β-Catenin (Kwon et al. 2000; Kesavapany et al. 2001) (Fig. 4). Pharmacological inhibition of Cdk5 enhances N-cadherin-mediated cell–cell adhesions, suggesting that Cdk5 negatively regulates N-cadherin (Kwon et al. 2000). Because the reconstitution of N-cadherin-mediated adhesion complex is required for radial fiber-dependent neuronal migration (Kawauchi et al. 2010; Shikanai et al. 2011), Cdk5 may control neuronal migration partly through the regulation of cell–cell adhesion dynamics, in addition to controlling dynein and microtubule. Interestingly, p35 downregulates the precursor form, but not mature form, of E-cadherin via the enhancement of its lysosomal degradation in a Cdk5-independent manner (Lin et al. 2008).

Overexpression of Cdk5 in N/N1003A rabbit lens epithelial cells slightly decreases the early step of the attachment to a fibronectin, a major ECM component, but greatly enhances the late phase of the adhesion, when Vinculin-positive focal adhesions are observed (Negash et al. 2002). In addition, pharmacological inhibition of Cdk5 in HaCaT keratinocytes suppresses Integrin-mediated cell-ECM adhesions, but enhances cell aggregation (Nakano et al. 2005), suggesting that Cdk5 positively and negatively controls Integrin-mediated cell–ECM and Cadherin-mediated cell–cell adhesions, respectively.

Importantly, it has been reported that Cdk5 is involved in the regulation of the Calpain-mediated focal adhesion turnover via the phosphorylation of the Talin head domain, one of two cleavage products by Calpain protease (Huang et al. 2009). Cdk5 phosphorylates Ser425 on the Talin head domain and this phosphorylation interferes with the binding between Talin and Smurf1, an E3 ubiquitin ligase, suppressing the ubiquitination and degradation of Talin (Huang et al. 2009) (Fig. 4). Expression of a Ser425-mutated Talin head promotes focal adhesion turnover and suppresses cell migration (Huang et al. 2009). Although a recent report indicates that Integrin is required for the final phase of neuronal migration (Sekine et al. 2012), it is still unclear whether Cdk5 also controls Integrin-mediated adhesion in neurons.

Cell cycle exit and differentiation (neurogenesis)

The progenitors of excitatory neurons are classified into several types, including radial glial cells in the VZ and basal progenitors (or intermediate progenitors) in the subventricular zone (SVZ) (Kawauchi et al. 2013). Cdk5 functions as a molecular switch from progenitor proliferation to neuronal differentiation and migration. Furthermore, a few cells in the cortical plate of Cdk5-deficient cortex express cell cycle markers, Cyclin-A, Cyclin-D, Ki-67 and PCNA, suggesting that Cdk5 plays a part in maintaining a quiescent state in neurons (Cicero & Herrup 2005).

Cyclin-dependent kinase-5 stabilizes p27kip1 through phosphorylation at Ser10 (Kawauchi et al. 2006) (Fig. 4). Because p27kip1 has multiple functions in the promotion of cell cycle exit, neuronal differentiation and migration (Kawauchi et al. 2006; Nguyen et al. 2006), the stabilization of p27kip1 plays important roles in the switch from progenitor into differentiation states. The regulation of neuronal differentiation by p27kip1 requires its phosphorylation at Thr187, and this phosphorylation is mediated, at least in part, by Cdk5 in cortical neurons (Zheng et al. 2010) (Fig. 4).

Cyclin-dependent kinase-5-mediated phosphorylation of Dixdc1, a binding partner of a schizophrenia-related protein, Disc1, promotes the interaction between Ndel1 and Disc1, which positively regulate neuronal migration, whereas non-phosphorylated Dixdc1 is involved in neural progenitor proliferation (Singh et al. 2010) (Fig. 4). Furthermore, a recent report has indicated that while Axin promotes basal progenitor production through interaction with GSK3β, Cdk5 phosphorylates Axin at Thr485, which triggers neuronal differentiation (Fang et al. 2013) (Fig. 4).

Membrane trafficking and signaling at synapse

Cyclin-dependent kinase-5 is known to function at both presynaptic and postsynaptic sites (Fig. 5A,B). At the presynapse, Cdk5 is involved in determination of the recycling pool size of synaptic vesicles (Kim & Ryan 2010; Marra et al. 2012). Cdk5 inhibition promotes the conversion of the resting vesicle pool into the recycling vesicle pool at active presynaptic terminals, and unmasks the inactive presynapse, suggesting that Cdk5 negatively regulates neurotransmitter release (Kim & Ryan 2010) (Fig. 5B). On the other hand, Cdk5 phosphorylates the pore-forming subunit of an N-type calcium channel, CaV2.2, and increases the number of docked vesicles at presynaptic terminals, resulting in the facilitation of neurotransmitter release (Su et al. 2012) (Fig. 5C). This suggests that Cdk5 differentially regulates multiple steps of neurotransmitter release.

Figure 5.

Cyclin-dependent kinase-5 (Cdk5) functions in synapse formation and plasticity. (A) Neurexin and Neuroligin play important roles in synapse formation. Cdk5 phosphorylates a Neurexin-binding intracellular protein, CASK, and is involved in synapse formation. (B) Cdk5 regulates both presynaptic and postsynaptic proteins, and thereby synaptic plasticity, such as long-term potentiation (LTP) and depression (LTD), during which the cell surface levels of AMPA receptors are increased or decreased, respectively. (C) Cdk5 phosphorylates many presynaptic and postsynaptic proteins. See text for detail.

Cyclin-dependent kinase-5 is known to control both exocytosis and endocytosis of synaptic vesicles. The assembly and disassembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) have pivotal roles in the vesicle fusion during exocytosis (Hong & Lev 2014). Cdk5 phosphorylates Munc18a, a regulator for the SNARE complex, and promotes the dissociation of the Munc18a/Syntaxin1a complex, a component of the SNARE complex (Fletcher et al. 1999) (Fig. 5C).

During endocytosis, Cdk5 phosphorylates Dynamin1 and Amphiphysin1 and these proteins are de-phosphorylated by Calcineurin, a Calcium-sensitive phosphatase (Floyd et al. 2001; Tan et al. 2003; Tomizawa et al. 2003) (Fig. 5B,C). Cdk5-mediated phosphorylation of these proteins is reported to negatively regulate their functions in endocytosis. However, it is known that inhibition of Cdk5 as well as Calcineurin eventually blocks the uptake of an endocytic marker, FM1-43, suggesting that the phosphorylation cycle, mediated by Cdk5 and Calcineurin, plays important roles in proper regulation of endocytosis in cells.

At postsynaptic sites, Cdk5 phosphorylates several substrates, including NR2A (GluN2A) and NR2B (GluN2B) (subunits of an NMDA receptor) (Li et al. 2001; Plattner et al. 2014), PSD-95 (a major scaffold protein at postsynaptic density) (Morabito et al. 2004), DARPP-32 (a regulator for dopamine signaling) (Bibb et al. 1999) and Dopamine D2 receptor (Jeong et al. 2013) (Fig. 5C). PSD-95 links NMDA receptors and cytoskeletal and signaling proteins at the postsynapse. Phosphorylation of the N-terminal domain of PSD-95 by Cdk5 prevents the multimerization of PSD-95 and its dependent clustering of NMDA receptors (Morabito et al. 2004).

Phosphorylation of NR2A at Ser1232 by Cdk5 is associated with ischemia-mediated cell death of hippocampal CA1 pyramidal neurons (Wang et al. 2003) (Fig. 5C). Cdk5 also controls the cell surface levels of NR2B through regulation of the binding between NR2B and AP-2, an adapter protein involved in clathrin-mediated endocytosis (Zhang et al. 2008). Cdk5 enhances the endocytosis and degradation of NR2B and thereby suppresses NMDA receptor-mediated current in hippocampus (Hawasli et al. 2007) (Fig. 5B). Interestingly, Cyclin-E is highly expressed not only in proliferating cells but also adult brains, and negatively controls the synaptic functions of Cdk5 (Odajima et al. 2011) (Fig. 5B). Conditional knockout of Cyclin-E in adult brains results in the reduction of cell surface expression of NMDA receptors and impairment of LTP in hippocampal CA1 pyramidal neurons.

In addition to its function in mature synapses, Cdk5 is suggested to have roles in synaptogenesis. Synapse formation is controlled by several synaptic cell adhesion molecules and secreted molecules, such as presynaptic Neurexins and postsynaptic Neuroligins (Craig & Kang 2007; Yuzaki 2011). CASK, a MAGUK family member, interacts with Neurexins (Hata et al. 1996), and is a substrate of Cdk5. Cdk5-mediated phosphorylation of CASK is required for the recruitment of CASK to presynaptic terminals and synapse formation (Samuels et al. 2007) (Fig. 5A). Taken together with the involvement of Cdk5 in the EphA4-mediated dendritic spine remodeling (Fu et al. 2007) (Fig. 5B), it appears that Cdk5 is a central regulator for synapse formation and plasticity.

Nuclear functions and cell cycle events

In adult brains, mature neurons maintain a quiescent state. Although some cell cycle-related proteins, such as Cyclin-E and p27kip1, have alternative functions, other than cell cycle, in post-mitotic neurons (Kawauchi et al. 2013), cell cycle progression itself is inhibited in neurons. Re-activation of the cell cycle machinery in mature neurons is known to be a trigger of neuronal cell death (Yang & Herrup 2007). It has been reported that nuclear Cdk5 suppresses the re-activated neuronal cell cycle events through binding to p35, but not p25 or p39 (Zhang et al. 2010a).

In the forebrains of CaMKII-promoter-driven p25 transgenic mice, the expression of genes involved in cell cycle and DNA damage response are upregulated before neuronal death (Kim et al. 2008). Cdk5/p25 inhibits the activity of histone deacetylase 1 (HDAC1) that protects against DNA damage and neurotoxicity, resulting in the induction of neuronal death (Kim et al. 2008). Cdk5 phosphorylates apurinic/apyrimidinic endonuclease 1 (Ape1), resulting in the suppression of its apurinic/apyrimidinic endonuclease activity; this phosphorylation is observed in post-mortem brains of patients with Parkinson's and Alzheimer's diseases (Huang et al. 2010). Cdk5 also functions as a downstream of DNA damage responses (Tian et al. 2009). DNA damage-dependent activation of Cdk5 induces the phosphorylation of Ser794 on ataxia-telangiectasia mutated (ATM), a central regulator of DNA damage response (Tian et al. 2009). Cdk5 promotes DNA damage-induced cell cycle re-entry of post-mitotic neurons and neuronal cell death.

Conclusion

An unconventional CDK, Cdk5, regulates various aspects of brain development and function. (i) Cdk5 promotes neuronal differentiation and maintains the post-mitotic state of cortical neurons. (ii) Cdk5 regulates multiple steps of neuronal migration in the developing cerebral cortex and cerebellum. (iii) Cdk5 controls axon elongation and dendrite arborization. (iv) Cdk5 phosphorylates several presynaptic and postsynaptic proteins and thereby regulates synaptic plasticity and high-ordered brain functions, such as memory formation and extinction. In addition, Cdk5 is closely associated with several neurodegenerative diseases. Such multiple functionality of Cdk5 is based on the fact that Cdk5 phosphorylates numerous substrate molecules involved in many cellular events. Cdk5 is known as a key regulator for microtubule organization, but recent advances have revealed that Cdk5 is also required for the regulation of cell adhesion, membrane trafficking, actin cytoskeleton, cell cycle exit and differentiation. Future studies to identify upstream regulators of Cdk5 in each situation may explain how this huge variety of functions is conferred on Cdk5.

Acknowledgments

The author thanks Dr Ruth T. Yu (Salk Institute, USA) for critical reading of the manuscript, and Dr Wataru Kakegawa (Keio University, Japan) and Dr Mikio Hoshino (NCNP, Japan) for helpful comments on synaptic plasticity and cerebellar development, respectively.

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