RPS23RG1 modulates tau phosphorylation and axon outgrowth through regulating p35 proteasomal degradation

Tauopathies are a group of neurodegenerative diseases characterized by hyperphosphorylation of the microtubule-binding protein, tau, and typically feature axon impairment and synaptic dysfunction. Cyclin-dependent kinase5 (Cdk5) is a major tau kinase and its activity requires p35 or p25 regulatory subunits. P35 is subjected to rapid proteasomal degradation in its membrane-bound form and is cleaved by calpain under stress to a stable p25 form, leading to aberrant Cdk5 activation and tau hyperphosphorylation. The type Ib transmembrane protein RPS23RG1 has been implicated in Alzheimer’s disease (AD). However, physiological and pathological roles for RPS23RG1 in AD and other tauopathies are largely unclear. Herein, we observed retarded axon outgrowth, elevated p35 and p25 protein levels, and increased tau phosphorylation at major Cdk5 phosphorylation sites in Rps23rg1 knockout (KO) mice. Both downregulation of p35 and the Cdk5 inhibitor roscovitine attenuated tau hyperphosphorylation and axon outgrowth impairment in Rps23rg1 KO neurons. Interestingly, interactions between the RPS23RG1 carboxyl-terminus and p35 amino-terminus promoted p35 membrane distribution and proteasomal degradation. Moreover, P301L tau transgenic (Tg) mice showed increased tau hyperphosphorylation with reduced RPS23RG1 levels and impaired axon outgrowth. Overexpression of RPS23RG1 markedly attenuated tau hyperphosphorylation and axon outgrowth defects in P301L tau Tg neurons. Our results demonstrate the involvement of RPS23RG1 in tauopathy disorders, and implicate a role for RPS23RG1 in inhibiting tau hyperphosphorylation through homeostatic p35 degradation and suppression of Cdk5 activation. Reduced RPS23RG1 levels in tauopathy trigger aberrant Cdk5-p35 activation, consequent tau hyperphosphorylation, and axon outgrowth impairment, suggesting that RPS23RG1 may be a potential therapeutic target in tauopathy disorders.Subject terms: Neural ageing, Neurological disorders     

Drosophila lacking the Cdk5 activator, p35, display defective axon guidance, age-dependent behavioral deficits and reduced lifespan

The cyclin-dependent kinase Cdk5 has attracted a great deal of attention both because of its roles in cell migration and axon patterning, and the extensive data implicating it in adult-onset neurodegeneration in mammals. Both the kinase activity and the biological effects of Cdk5 are absolutely dependent on association with an activating subunit, called p35. We show here that Drosophila lacking the Cdk5 activator, D-p35, display a wide range of defects in embryonic axon patterning. We further show that, while viable and fertile, p35 mutant adults display progressive, age-dependent loss of motor function and have a significantly shortened lifespan.     

GTP-dependent secretion from neutrophils is regulated by Cdk5

We have previously shown evidence for the existence of a calcium-independent, GTP-regulated mechanism of secretion from neutrophils, but this secretory mechanism remains to be fully elucidated. Cyclin-dependent kinase 5 (Cdk5), the various substrates of which include Munc18 and synapsin 1, has been implicated in neuronal secretion. Although the Cdk5 activator, p35, and Cdk5-p35 activity are primarily associated with neurons, we report here that p35 also exists in neutrophils and that an active Cdk5-p35 complex is present in these cells. Cdk5-p35 activity in human neutrophils is mostly localized in secretory granules, which show an increase in Cdk5-p35 level and activity upon GTP stimulation. The potent Cdk5 inhibitor, roscovitine, completely blocks GTP-stimulated granule Cdk5 activity, which accompanies lactoferrin secretion from neutrophil-specific granules. Roscovitine also inhibits GTP-induced lactoferrin secretion and surface localization of the secretion markers, CD63 and CD66b, to a certain extent. Furthermore, neutrophils from wild-type mice treated with roscovitine and neutrophils from p35(-/-) mice exhibit comparable surface expression levels of both CD63 and CD66b upon GTP stimulation. Although our data suggest that other molecules control GTP-induced secretion from neutrophils, it is clear that Cdk5-p35 is required to elicit the maximum GTP-induced secretory response. Our observation that multiple proteins in neutrophil granules serve as specific substrates of Cdk5 further supports the premise that the kinase is a key component of the GTP-regulated secretory apparatus in neutrophils.     

Regulation of N-cadherin-mediated adhesion by the p35-Cdk5 kinase

BACKGROUND: The p35-Cdk5 kinase has been implicated in a variety of functions in the central nervous system (CNS), including axon outgrowth, axon guidance, fasciculation, and neuronal migration during cortical development. In p35(-/-) mice, embryonic cortical neurons are unable to migrate past their predecessors, leading to an inversion of cortical layers in the adult cortex. RESULTS: In order to identify molecules important for p35-Cdk5-dependent function in the cortex, we screened for p35-interacting proteins using the two-hybrid system. In this study, we report the identification of a novel interaction between p35 and the versatile cell adhesion signaling molecule beta-catenin. The p35 and beta-catenin proteins interacted in vitro and colocalized in transfected COS cells. In addition, the p35-Cdk5 kinase was associated with a beta-catenin-N-cadherin complex in the cortex. In N-cadherin-mediated aggregation assays, inhibition of Cdk5 kinase activity using the Cdk5 inhibitor roscovitine led to the formation of larger aggregates of embryonic cortical neurons. This finding was recapitulated in p35(-/-) cortical neurons, which aggregated to a greater degree than wild-type neurons. In addition, introduction of active p35-Cdk5 kinase into COS cells led to a decreased beta-catenin-N-cadherin interaction and loss of cell adhesion. CONCLUSIONS: The association between p35-Cdk5 and an N-cadherin adhesion complex in cortical neurons and the modulation of N-cadherin-mediated aggregation by p35-Cdk5 suggests that the p35-Cdk5 kinase is involved in the regulation of N-cadherin-mediated adhesion in cortical neurons.     

Identification of the N-terminal functional domains of Cdk5 by molecular truncation and computer modeling

Cyclin dependent kinase (Cdk) 5, an atypical member of the Cdk family, plays a fundamental role in the development of the nervous system, and may also be involved in the pathogenesis of certain neurodegenerative diseases. Further, Cdk5 is activated by the specific regulatory proteins p39, p35, or p25 rather than cyclins, and in contrast to other members of the Cdk family is not involved in the progression of the cell cycle. A three-dimensional computer model of Cdk5-p25-ATP has been generated previously [Chou et al., Biochem Biophys Res Commun 1999;259:420-428], providing a structural basis for the study of the mechanisms of Cdk5 activation. To assess the predicted ATP and p25 binding domains at the N-terminal of Cdk5, two mutants of Cdk5 were prepared in which amino acids 9-15 (Delta9-15) or 9-47 (Delta9-47) were deleted. The results of these studies clearly demonstrate that an N-terminal loop and the PSSALRE helix are indispensable for Cdk5-p25 interactions, and amino acids 9-15 are necessary for ATP binding but are not involved in Cdk5-p25 interactions. Predicted models of Delta9-15 Cdk5 and Delta9-47 Cdk5 were generated, and were used to interpret the experimental data. The experimental and molecular modeling results confirm and extend specific aspects of the original predicted computer model, and may provide useful information for the design of highly selective inhibitors of Cdk5, which could be used in the treatment of certain neurodegenerative conditions.     

Phosphorylation of Pak1 by the p35/Cdk5 kinase affects neuronal morphology

The small GTPase Rac and its effectors, the Pak1 and p35/Cdk5 kinases, have been assigned important roles in regulating cytoskeletal dynamics in neurons. Our previous work revealed that the neuronal p35/Cdk5 kinase associates with Pak1 in a RacGTP-dependent manner, causing hyperphosphorylation and down-regulation of Pak1 kinase activity. We have now demonstrated direct phosphorylation of Pak1 on threonine 212 by the p35/Cdk5 kinase. In neuronal growth cones, Pak1 phosphorylated on Thr-212 localized to actin and tubulin-rich areas, suggesting a role in regulating growth cone dynamics. The expression of a non-phosphorylatable Pak1 mutant (Pak1A212) induced dramatic neurite disorganization. We also observed a strong association between p35/Cdk5 and the Pak1 C-terminal kinase domain. Overall, our data show that in neurons, membrane-associated, active Pak1 is regulated by the p35/Cdk5 kinase both by association and phosphorylation, which is essential for the proper regulation of the cytoskeleton during neurite outgrowth and remodeling.     

A 24-Residue Peptide (p5), Derived from p35, the Cdk5 Neuronal Activator,Specifically Inhibits Cdk5-p25 Hyperactivity and Tau Hyperphosphorylation

The activity of Cdk5-p35 is tightly regulated in the developing and mature nervous system. Stress-induced cleavage of the activator p35 to p25 and a p10 N-terminal domain induces deregulated Cdk5 hyperactivity and perikaryal aggregations of hyperphosphorylated Tau and neurofilaments, pathogenic hallmarks in neurodegenerative diseases, such as Alzheimer disease and amyotrophic lateral sclerosis, respectively. Previously, we identified a 125-residue truncated fragment of p35 called CIP that effectively and specifically inhibited Cdk5-p25 activity and Tau hyperphosphorylation induced by Aβ peptides in vitro, in HEK293 cells, and in neuronal cells. Although these results offer a possible therapeutic approach to those neurodegenerative diseases assumed to derive from Cdk5-p25 hyperactivity and/or Aβ induced pathology, CIP is too large for successful therapeutic regimens. To identify a smaller, more effective peptide, in this study we prepared a 24-residue peptide, p5, spanning CIP residues Lys²⁴⁵–Ala²⁷⁷. p5 more effectively inhibited Cdk5-p25 activity than did CIP in vitro. In neuron cells, p5 inhibited deregulated Cdk5-p25 activity but had no effect on the activity of endogenous Cdk5-p35 or on any related endogenous cyclin-dependent kinases in HEK293 cells. Specificity of p5 inhibition in cortical neurons may depend on the p10 domain in p35, which is absent in p25. Furthermore, we have demonstrated that p5 reduced Aβ(1–42)-induced Tau hyperphosphorylation and apoptosis in cortical neurons. These results suggest that p5 peptide may be a unique and useful candidate for therapeutic studies of certain neurodegenerative diseases.     

Microtubule association of the neuronal p35 activator of Cdk5

Cdk5 and its neuronal activator p35 play an important role in neuronal migration and proper development of the brain cortex. We show that p35 binds directly to alpha/beta-tubulin and microtubules. Microtubule polymers but not the alpha/beta-tubulin heterodimer block p35 interaction with Cdk5 and therefore inhibit Cdk5-p35 activity. p25, a neurotoxin-induced and truncated form of p35, does not have tubulin and microtubule binding activities, and Cdk5-p25 is inert to the inhibitory effect of microtubules. p35 displays strong activity in promoting microtubule assembly and inducing formation of microtubule bundles. Furthermore, microtubules stabilized by p35 are resistant to cold-induced disassembly. In cultured cortical neurons, a significant proportion of p35 localizes to microtubules. When microtubules were isolated from rat brain extracts, p35 co-assembled with microtubules, including cold-stable microtubules. Together, these findings suggest that p35 is a microtubule-associated protein that modulates microtubule dynamics. Also, microtubules play an important role in the control of Cdk5 activation.     

The Cdk5-p35 kinase associates with the Golgi apparatus and regulates membrane traffic

We show here that an active Cdk5-p35 kinase is present in Golgi membranes, where it associates with a detergent-insoluble fraction containing actin. In addition, Cdk5-p35-dependent phosphorylation of α-PAK immunoreactive protein species was detected in Golgi membranes, as well as an interaction with the small GTPase, Cdc42. Moreover, antisense oligonucleotide suppression of Cdk5 or p35 in young cultured neurons, as well as inhibition of Cdk5 activity with olomoucine, blocks the formation of membrane vesicles from the Golgi apparatus. Taken together, these results show a novel subcellular localization of this kinase and suggest a role for Cdk5-p35 in membrane traffic during neuronal process outgrowth.     

The protein kinase Cdk5. Structural aspects, roles in neurogenesis and involvement in Alzheimer's pathology.

A set of different protein kinases have been involved in tau phosphorylations, including glycogen synthase kinase 3beta (GSK3 beta), MARK kinase, MAP kinase, the cyclin-dependent kinase 5 (Cdk5) system and others. The latter system include the catalytic component Cdk5 and the regulatory proteins p35, p25 and p39. Cdk5 and its neuron-specific activator p35 are essential molecules for neuronal migration and for the laminar configuration of the cerebral cortex. Recent evidence that the Cdk5/p35 complex concentrates at the leading edge of axonal growth cones, together with the involvement of this system in the phosphorylation of neuronal microtubule-asociated proteins (MAPs), provide further support to the role of this protein kinase in regulating axonal extension in developing brain neurons. Although the aminoacid sequence of p35 has little similarity with those of normal cyclins, studies have shown that its activation domain may adopt a conformation of the cyclin-folded structure. The computed structure for Cdk5 is compatible with experimental data obtained from studies on the Cdk5/p35 complex, and has allowed predictions on the protein interacting domains. This enzyme exhibits a wide cell distribution, even though a regulated Cdk5 activity has been shown only in neuronal cells. Cdk5 has been characterized as a proline-directed Ser/Thr protein kinase, that contributes to phosphorylation of human tau on Ser202, Thr205, Ser235 and Ser404. Cdk5 is active in postmitiotic neurons, and it has been implicated in cytoskeleton assembly and its organization during axonal growth. In addition to tau and other MAPs, Cdk5 phosphorylates the high molecular weight neurofilament proteins at their C-terminal domain. Moreover, nestin, a protein that regulates cytoskeleton organization of neuronal and muscular cells during development of early embryos, and several other regulatory proteins appear to be substrates of Cdk5 and are phosphorylated by this kinase. Studies also suggest, that in addition to Cdk5 involvement in neuronal differentiation, its activity is induced during myogenesis, however, the mechanisms of how this activity is regulated during muscular differentiation has not yet been elucidated. Recent studies have shown that the beta-amyloid peptide (A beta) induces a deregulation of Cdk5 in cultured brain cells, and raises the question on the possible roles of this tau-phosphorylating protein kinase in the sequence of molecular events leading to neuronal death triggered by A beta. In this context, there are evidence that Cdk5 is involved in tau hyperphosphorylation promoted by A beta in its fibrillary form. Cdk5 inhibitors protect hippocampal neurons against both tau anomalous phosphorylations and neuronal death. The links between the studies on the Cdk5/p35 system in normal neurogenesis and its claimed participation in neurodegeneration, provide the framework to understand the regulatory relevance of this kinase system, and changes in its regulation that may be implicated in disturbances such as those occurring in Alzheimer disease.     

Phosphorylation of AATYK1 by Cdk5 Suppresses Its Tyrosine Phosphorylation

Apoptosis-associated tyrosine kinase 1 (AATYK1), a novel serine/threonine kinase that is highly expressed in the brain, is involved in neurite extension and apoptosis of cerebellar granule neurons; however, its precise function remains unknown. In this study, we investigated the interaction of AATYK1A with Cyclin-dependent kinase 5 (Cdk5)/p35, a proline-directed protein kinase that is predominantly expressed in neurons. AATYK1A bound to the p35 activation subunit of Cdk5 in cultured cells and in mouse brains and colocalized with p35 on endosomes in COS-7 cells. AATYK1A was phosphorylated at Ser34 by Cdk5/p35 in vitro, in cultured neurons and in mouse brain. In PC12D cells, Ser34 phosphorylation increased after treatment with nerve growth factor and phosphorylated AATYK1A accumulated in growth cones of PC12D cells. Ser34 phosphorylation suppressed the tyrosine phosphorylation of AATYK1A by Src family kinases. These results suggest a possibility that AATYK1A plays a role in early to recycling endosomes and its function is regulated by phosphorylation with Cdk5 or Src-family kinases.     

Protein-protein interactions in Cdk5 regulation and function

Cdk5 is a unique member of the cyclin-dependent kinase (Cdk) family of small protein kinases. In association with its neuron-specific activator p35 or p39, Cdk5 displays many regulatory properties distinct from other Cdks. A growing body of evidence has suggested that Cdk5-p35 has important implications in a variety of neuronal activities occurring in the central nervous system. In brain, Cdk5-p35 appears to exist as large molecular complexes with other proteins, and protein-protein interactions appear to be a molecular principle for Cdk5-p35 to conduct its physiological functions. Over the past decade, a number of proteins have been identified to associate with Cdk5-p35. While the majority of these proteins mediate their interaction with Cdk5 through p35, implying that p35 may act not only as an activator of Cdk5 but also as an adaptor to associate Cdk5 with its regulators and physiological targets, a small group of other proteins are found to link directly with Cdk5. In addition, Cdk5 has been found to phosphorylate a diverse list of substrates, further implicating its regulatory roles in a wide range of cellular processes. In this review, we present an updated inventory of the interacting proteins of Cdk5-p35 kinase and its substrates as well as a discussion on the implicated effects of these interactions.     

Robust neuronal symmetry breaking by Ras-triggered local positive feedback

Neuronal polarity is initiated by a symmetry-breaking event whereby one out of multiple minor neurites undergoes rapid outgrowth and becomes the axon [1]. Axon formation is regulated by phosphatidylinositol 3-kinase (PI3K)-related signaling elements [2-10] that drive local actin [11] and microtubule reorganization [3, 12], but the upstream signaling circuit that causes symmetry breaking and guarantees the formation of a single axon is not known. Here, we use live FRET imaging in hippocampal neurons and show that the activity of the small GTPase HRas, an upstream regulator of PI3K, markedly increases in the nascent axonal growth cone upon symmetry breaking. This local increase in HRas activity results from a positive feedback loop between HRas and PI3K, locally reinforced by vesicular transport of HRas to the axonal growth cone. Recruitment of HRas to the axonal growth cone is paralleled by a decrease in HRas concentration in the remaining neurites, suggesting that competition for a limited pool of HRas guarantees that only one axon forms. Mathematical modeling demonstrates that local positive feedback between HRas and PI3K, coupled to recruitment of a limited pool of HRas, generates robust symmetry breaking and formation of a single axon in the absence of extrinsic spatial cues.     

Cyclin-dependent protein kinase 5 (Cdk5) and the regulation of neurofilament metabolism.

Cyclin-dependent kinase 5 (Cdk5), a complex of Cdk5 and its activator p35 (Cdk5/p35), phosphorylates diverse substrates which have multifunctional roles in the nervous system. During development, it participates in neuronal differentiation, migration, axon outgrowth and synaptogenesis. Cdk5, acting together with other kinases, phosphorylates numerous KSPXK consensus motifs in diverse cytoskeletal protein target molecules, including neurofilaments, and microtubule associated proteins, tau and MAPs. Phosphorylation regulates the dynamic interactions of cytoskeletal proteins with one another during all aspects of neurogenesis and axon radial growth. In this review we shall focus on Cdk5 and its regulation as it modulates neurofilament metabolism in axon outgrowth, cytoskeletal stabilization and radial growth. We suggest that Cdk5/p35 forms compartmentalized macromolecular complexes of cytoskeletal substrates, other neuronal kinases, phosphatases and activators ('phosphorylation machines') which facilitate the dynamic molecular interactions that underlie these processes.     

Activation of latent cyclin-dependent kinase 5 (Cdk5)-p35 complexes by membrane dissociation

Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase of increasingly recognized importance in a large number of fields, ranging from neuronal migration to synaptic plasticity and neurodegeneration. However, little is known about its mechanism of activation beyond its requirement for binding to p35 or p39. We have examined membrane interactions as one method of regulating the Cdk5-p35 complex. The kinase activity of Cdk5-p35 is low when it is bound to membranes. The Cdk5-p35 found in rat brain extract associates with membranes in two ways. Approximately 75% of complexes associate with membranes via ionic interactions only, and the remaining 25% associate with membranes via ionic interactions together with lipidic interactions. Solubilization with detergent or high-salt solution activates Cdk5-p35 several fold, and this activation is reversible. Therefore, membrane interactions represent a novel mechanism for the regulation of Cdk5-p35 kinase activity.     

Impairment of hippocampal long-term depression and defective spatial learning and memory in p35 mice

Cdk5 (cyclin-dependent kinase 5) activity is dependent upon association with one of two neuron-specific activators, p35 or p39. Genetic deletion of Cdk5 causes perinatal lethality with severe defects in corticogenesis and neuronal positioning. p35(-/-) mice are viable with milder histological abnormalities. Although substantial evidence implicates Cdk5 in synaptic plasticity, its role in learning and memory has not been evaluated using mutant mouse models. We report here that p35(-/-) mice have deficiencies in spatial learning and memory. Close examination of hippocampal circuitry revealed subtle histological defects in CA1 pyramidal cells. Furthermore, p35(-/-) mice exhibit impaired long-term depression and depotentiation of long-term potentiation in the Schaeffer collateral CA1 pathway. Moreover, the Cdk5-dependent phosphorylation state of protein phosphatase inhibitor-1 was increased in 4-week-old mice due to increased levels of p39, which co-localized with inhibitor-1 and Cdk5 in the cytoplasm. These results demonstrate that p35-dependent Cdk5 activity is important to learning and synaptic plasticity. Deletion of p35 may shift the substrate specificity of Cdk5 due to compensatory expression of p39.     

Neuronal migration.

Like other motile cells, neurons migrate in three schematic steps, namely leading edge extension, nuclear translocation or nucleokinesis, and retraction of the trailing process. In addition, neurons are ordered into architectonic patterns at the end of migration. Leading edge extension can proceed at the extremity of the axon, by growth cone formation, or from the dendrites, by formation of dendritic tips. Among both categories of leading edges, variation seems to be related to the rate of extension of the leading process. Leading edge extension is directed by microfilament polymerization following integration of extracellular cues and is regulated by Rho-type small GTPases. In humans, mutations of filamin, an actin-associated protein, result in heterotopic neurons, probably due to defective leading edge extension. The second event in neuron migration is nucleokinesis, a process which is critically dependent on the microtubule network, as shown in many cell types, from slime molds to vertebrates. In humans, mutations in the PAFAH1B1 gene (more commonly called LIS1) or in the doublecortin (DCX) gene result in type 1 lissencephalies that are most probably due to defective nucleokinesis. Both the Lis1 and doublecortin proteins interact with microtubules, and two Lis1-interacting proteins, Nudel and mammalian NudE, are components of the dynein motor complex and of microtubule organizing centers. In mice, mutations of Cdk5 or of its activators p35 and p39 result in a migration phenotype compatible with defective nucleokinesis, although an effect on leading edge formation is also likely. The formation of architectonic patterns at the end of migration requires the integrity of the Reelin signalling pathway. Other known components of the pathway include members of the lipoprotein receptor family, the intracellular adaptor Dab1, and possibly integrin alpha 3 beta 1. Defective Reelin leads to poor lamination and, in humans, to a lissencephaly phenotype different from type 1 lissencephaly. Although the action of Reelin is unknown, it may trigger some recognition-adhesion among target neurons. Finally, pattern formation requires the integrity of the external limiting membrane, defects of which lead to overmigration of neurons in meninges and to human type 2 lissencephaly.     

Identification of nuclear import mechanisms for the neuronal Cdk5 activator

The activation of Cdk5 by p35 plays a pivotal role in a multitude of nervous system activities ranging from neuronal differentiation to degeneration. A fraction of Cdk5 and p35 localizes in the nucleus where Cdk5-p35 exerts its functions via protein phosphorylation, and p35 displays a dynamic localization between the cytoplasm and the nucleus. Here, we examined the nuclear import properties of p35. In nuclear import assays, p35 was actively transported into the nuclei of digitonin-permeabilized HeLa cells and cortical neurons by cytoplasmic carrier-mediated mechanisms. Importin-beta, importin-5, and importin-7 were identified to import p35 into the nuclei via a direct interaction with it. An N-terminal region of p35 was defined to interact with the above importins, serving as a nuclear localization signal. Finally, we show that the nuclear localization of p35 does not require the association of Cdk5. Furthermore, Cdk5 and importin-beta/5/7 are mutually exclusive in binding to p35. These results suggest that p35 employs pathways distinct from that used by Cdk5 for transport to the nucleus.     

The role of microtubules in growth cone turning at substrate boundaries

To understand the role of microtubules in growth cone turning, we observed fluorescently labeled microtubules in neurons as they encountered a substrate boundary. Neurons growing on a laminin-rich substrate avoided growing onto collagen type IV. Turning growth cones assumed heterogeneous morphologies and behaviors that depended primarily in their extent of adhesion to the substrate. We grouped these behaviors into three categories-sidestepping, motility, and growth-mediated reorientation. In sidestepping and motility-mediated reorientation, the growth cone and parts of the axon were not well attached to the substrate so the acquisition of an adherent lamella caused the entire growth cone to move away from the border and consequently reoriented the axon. In these cases, since the motility of the growth cone dominates its reorientation, the microtubules were passive, and reorientation occurred without significant axon growth. In growth-mediated reorientation, the growth cone and axon were attached to the substrate. In this case, microtubules reoriented within the growth cone to stabilize a lamella. Bundling of the reoriented microtubules was followed by growth cone collapse to form new axon, and further, polarized lamellipodial extension. These observations indicate that when the growth cone remains adherent to the substrate during turning, the reorientation and bundling of microtubules is an important, early step in growth cone turning.     

Cdk5 regulates STAT3 activation and cell proliferation in medullary thyroid carcinoma cells

The biological behaviors of thyroid cancer are varied, and the pathological mechanisms remain unclear. Some reports indicated an apparent aggregation of amyloid accompanying medullary thyroid carcinoma (MTC). Amyloid aggregation in neurodegeneration leads to hyperactivation of Cdk5 and subsequent neuronal death. Based on the connection with amyloid, the role of Cdk5 in MTC is worthy of investigation. Initially, the expression of Cdk5 and its activator, p35, in MTC cell lines was identified. Cdk5 inhibition by specific inhibitors or short interfering RNA decreased the proliferation of MTC cell lines, which reveals the importance of Cdk5 in MTC cell growth. Although p35 cleavage has been considered as an important element in neurodegeneration, it seems that p35 cleavage was not a major cause in Cdk5 activity-dependent MTC cell proliferation because neither Cdk5 activity nor cell growth was affected by the inhibition of p35 cleavage. Clearance of amyloid by antibody neutralization indicated that MTC cell proliferation was supported by calcitonin-derived extracellular amyloid and subsequent Her2 and Cdk5 activation. Significantly, the STAT3 pathway was involved in Cdk5-dependent proliferation of MTC cells through Ser-727 phosphorylation. In addition, Cdk5 inhibition reduced nuclear distributions of both the Cdk5-p35 complex and phospho-STAT3 in MTC cells. Finally, Cdk5 inhibition retarded tumor formation in vivo accompanying the reduction of phospho-STAT3. Our findings suggest the first demonstration of a novel and specific role for Cdk5 kinase in supporting the proliferation of the medullary thyroid carcinoma cells and could shed light on a new field for diagnosis and therapy of thyroid cancer.