Cdk5-mediated JIP1 phosphorylation regulates axonal outgrowth through Notch1 inhibition.

BACKGROUND: Activated Cdk5 regulates a number of processes during nervous system formation, including neuronal differentiation, growth cone stabilization, and axonal growth. Cdk5 phosphorylates its downstream substrates located in axonal growth cones, where the highly expressed c-Jun N-terminal kinase (JNK)-interacting protein1 (JIP1) has been implicated as another important regulator of axonal growth. In addition, stringent control of the level of intracellular domain of Notch1 (Notch1-IC) plays a regulatory role in axonal outgrowth during neuronal differentiation. However, whether Cdk5-JIP1-Notch1 cooperate to regulate axonal outgrowth, and the mechanism of such joint contribution to this pathway, is presently unknown, and here we explore their potential interaction. RESULTS: Our interactome screen identified JIP1 as an interactor of p35, a Cdk5 activator, and we sought to explore the relationship between Cdk5 and JIP1 on the regulation of axonal outgrowth. We demonstrate that JIP1 phosphorylated by Cdk5 at Thr205 enhances axonal outgrowth and a phosphomimic JIP1 rescues the axonal outgrowth defects in JIP1-/- and p35-/- neurons. Axonal outgrowth defects caused by the specific increase of Notch1 in JIP1-/- neurons are rescued by Numb-mediated inhibition of Notch1. Finally, we demonstrate that Cdk5 phosphorylation of JIP1 further amplifies the phosphorylation status of yet another Cdk5 substrate E3-ubiquitin ligase Itch, resulting in increased Notch1 ubiquitination. CONCLUSIONS: Our findings identify a potentially critical signaling axis involving Cdk5-JIP1-Itch-Notch1, which plays an important role in the regulation of CNS development. Future investigation into the way this pathway integrates with additional pathways regulating axonal growth will further our knowledge of normal central nervous system development and pathological conditions.

Valproic Acid-Induced Anxiety and Depression Behaviors are Ameliorated in p39 Cdk5 Activator-Deficient Mice.

Valproic acid (VPA) is a drug used for the treatment of epilepsy, seizures, migraines, and bipolar disorders. Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase activated by p35 or p39 in neurons and plays a role in a variety of neuronal functions, including psychiatric behaviors. We previously reported that VPA suppressed Cdk5 activity by reducing the expression of p35 in cultured cortical neurons, leaving p39 unchanged. In this study, we asked for the role of Cdk5 in VPA-induced anxiety and depression behaviors. Wild-type (WT) mice displayed increased anxiety and depression after chronic administration of VPA for 14 days, when the expression of p35 was decreased. To clarify their relationship, we used p39 knockout (KO) mice, in which p35 is the only Cdk5 activator. When p39 KO mice were treated chronically with VPA, unexpectedly, they exhibited fewer anxiety and depression behaviors than WT mice. The effects were p39 cdk5r2 gene-dosage dependent. Together, these results indicate that Cdk5-p39 plays a specific role in VPA-induced anxiety and depression behaviors.

Cytoplasmic control of Rab family small GTPases through BAG6.

Rab family small GTPases are master regulators of distinct steps of intracellular vesicle trafficking in eukaryotic cells. GDP-bound cytoplasmic forms of Rab proteins are prone to aggregation due to the exposure of hydrophobic groups but the machinery that determines the fate of Rab species in the cytosol has not been elucidated in detail. In this study, we find that BAG6 (BAT3/Scythe) predominantly recognizes a cryptic portion of GDP-associated Rab8a, while its major GTP-bound active form is not recognized. The hydrophobic residues of the Switch I region of Rab8a are essential for its interaction with BAG6 and the degradation of GDP-Rab8a via the ubiquitin-proteasome system. BAG6 prevents the excess accumulation of inactive Rab8a, whose accumulation impairs intracellular membrane trafficking. BAG6 binds not only Rab8a but also a functionally distinct set of Rab family proteins, and is also required for the correct distribution of Golgi and endosomal markers. From these observations, we suggest that Rab proteins represent a novel set of substrates for BAG6, and the BAG6-mediated pathway is associated with the regulation of membrane vesicle trafficking events in mammalian cells.

The LMTK1-TBC1D9B-Rab11A Cascade Regulates Dendritic Spine Formation via Endosome Trafficking.

Dendritic spines are postsynaptic protrusions at excitatory synapses that are critical for proper neuronal synaptic transmission. While lipid and protein membrane components are necessary for spine formation, it is largely unknown how they are recruited to developing spines. Endosomal trafficking is one mechanism that may influence this development. We recently reported that Lemur kinase 1A (LMTK1A), a membrane-bound Ser/Thr kinase, regulates trafficking of endosomes in neurons. LMTK1 has been shown to be a p35 Cdk5 activator-binding protein and a substrate for Cdk5-p35; however, its neuronal function has not been sufficiently studied. Here, we investigate the role of LMTK1 in spine formation. Depletion of LMTK1 increases spine formation, maturation, and density in primary cultured neurons and in mouse brain of either sex. Additionally, expression of kinase-negative LMTK1 stimulates spine formation in primary neurons and in vivo LMTK1 controls spine formation through Rab11, a regulator of recycling endosome trafficking. We identify TBC1D9B, a Rab11A GTPase-activating protein (Rab11A GAP), as a LMTK1 binding protein, and find that TBC1D9B mediates LMTK1 activity on Rab11A. TBC1D9B inactivates Rab11A under the control of LMTK1A. Further, by analyzing the effect of decreased TBC1D9B expression in primary neurons, we demonstrate that TBC1D9B indeed regulates spine formation. This is the first demonstration of the biological function of TBC1D9B. Together, with the regulation of LMTK1 by Cdk5-p35, we propose the Cdk5-LMTK1-TBC1D9B-Rab11A cascade as a novel signaling mechanism regulating endosomal transport for synapse formation and function.SIGNIFICANCE STATEMENT Dendritic spines are postsynaptic specializations essential for synaptic transmission. However, it is not known how critical membrane components are recruited to spines for their formation. Endosomal trafficking is one such mechanism that may mediate this process. Here we investigate regulators of endosomal trafficking and their contribution to spine formation. We identify two novel factors, LMTK1 and TBC1D9B, which regulate spine formation upstream of Rab11A, a small GTPase. LMTK1 is a membrane bound Ser/Thr kinase regulated by Cdk5-p35, and TBC1D9B is a recently identified Rab11 GAP. LMTK1 controls the GAP activity of TBC1D9B on Rab11A, and TBC1D9B mediates the LMTK1 activity on Rab11A. We propose the Cdk5-LMTK1-TBC1D9B-Rab11A cascade as a novel mechanism controlling spine formation and function.

CD2-associated protein (CD2AP) overexpression accelerates amyloid precursor protein (APP) transfer from early endosomes to the lysosomal degradation pathway.

A hallmark of Alzheimer's disease (AD) pathology is the appearance of senile plaques, which are composed of ß-amyloid (Aß) peptides. Aß is produced by sequential cleavages of amyloid precursor protein (APP) by ß- and γ-secretases. These cleavages take place in endosomes during intracellular trafficking of APP through the endocytic and recycling pathways. Genome-wide association studies have identified several risk factors for late-onset AD, one of which is CD2-associated protein (CD2AP), an adaptor molecule that regulates membrane trafficking. Although CD2AP's involvement in APP trafficking has recently been reported, how APP trafficking is regulated remains unclear. We sought to address this question by investigating the effect of CD2AP overexpression or knockdown on the intracellular APP distribution and degradation of APP in cultured COS-7 and HEK293 cells. We found that overexpression of CD2AP increases the localization of APP to Rab7-positive late endosomes, and decreases its localization to Rab5-positive early endosomes. CD2AP overexpression accelerated the onset of APP degradation without affecting its degradation rate. Furthermore, nutrient starvation increased the localization of APP to Rab7-positive late endosomes, and CD2AP overexpression stimulated starvation-induced lysosomal APP degradation. Moreover, the effect of CD2AP on the degradation of APP was confirmed by CD2AP overexpression and knockdown in primary cortical neurons from mice. We conclude that CD2AP accelerates the transfer of APP from early to late endosomes. This transfer in localization stimulates APP degradation by reducing the amount of time before degradation initiation. Taken together, these results may explain why impaired CD2AP function is a risk factor for AD.

Tau isoform expression and phosphorylation in marmoset brains.

Tau is a microtubule-associated protein expressed in neuronal axons. Hyperphosphorylated tau is a major component of neurofibrillary tangles, a pathological hallmark of Alzheimer's disease (AD). Hyperphosphorylated tau aggregates are also found in many neurodegenerative diseases, collectively referred to as "tauopathies," and tau mutations are associated with familial frontotemporal lobar degeneration (FTLD). Previous studies have generated transgenic mice with mutant tau as tauopathy models, but nonhuman primates, which are more similar to humans, may be a better model to study tauopathies. For example, the common marmoset is poised as a nonhuman primate model for investigating the etiology of age-related neurodegenerative diseases. However, no biochemical studies of tau have been conducted in marmoset brains. Here, we investigated several important aspects of tau, including expression of different tau isoforms and its phosphorylation status, in the marmoset brain. We found that marmoset tau does not possess the "primate-unique motif" in its N-terminal domain. We also discovered that the tau isoform expression pattern in marmosets is more similar to that of mice than that of humans, with adult marmoset brains expressing only four-repeat tau isoforms as in adult mice but unlike in adult human brains. Of note, tau in brains of marmoset newborns was phosphorylated at several sites associated with AD pathology. However, in adult marmoset brains, much of this phosphorylation was lost, except for Ser-202 and Ser-404 phosphorylation. These results reveal key features of tau expression and phosphorylation in the marmoset brain, a potentially useful nonhuman primate model of neurodegenerative diseases.

Isoform-independent and -dependent phosphorylation of microtubule-associated protein tau in mouse brain during postnatal development.

Tau is a microtubule (MT)-associated protein that regulates MT dynamics in the axons of neurons. Tau binds to MTs via its C-terminal MT-binding repeats. There are two types of tau, those with three (3R) or four (4R) MT-binding repeats; 4R tau has a stronger MT-stabilizing activity than 3R tau. The MT-stabilizing activity of tau is regulated by phosphorylation. Interestingly, both the isoform and phosphorylation change at the time of neuronal circuit formation during postnatal development; highly phosphorylated 3R tau is replaced with 4R tau, which is less phosphorylated. However, it is not known how the transition of the isoforms and phosphorylation are regulated. Here, we addressed this question using developing mouse brains. Detailed analysis of developing brains revealed that the switch from 3R to 4R tau occurred during postnatal day 9 (P9) to P18 under the same time course as the conversion of phosphorylation from high to low. However, hypothyroidism, which is known to delay brain development, delayed the timing of tau dephosphorylation but not the exchange of isoforms, indicating that isoform switching and phosphorylation are not necessarily linked. Furthermore, we confirmed this finding by using mouse brains that expressed a single isoform of human tau. Human tau, either 3R or 4R, reduced phosphorylation levels during development even though the isoform did not change. We also found that 3R tau and 4R tau were phosphorylated differently in vivo even at the same developmental days. These results show for the first time that the phosphorylation and isoform alteration of tau are regulated differently during mouse development.

Cyclin-dependent kinase 5 promotes proteasomal degradation of the 5-HT1A receptor via phosphorylation.

Serotonin (5-HT) is a major neurotransmitter in mammalian brains and is involved in brain development and psychiatric disorders. The 5-HT1A receptor (5-HT1AR) is a G-protein-coupled receptor (GPCR) associated with an inhibitory G-protein (Gi) with the widest and most abundant expression. It is not known; however, how expression or activity of 5-HTlAR is regulated. We studied here phosphorylation of 5-HT1AR by cyclin-dependent kinase 5 (Cdk5), a neuron-specific membrane-bound Ser/Thr kinase that is activated by binding of the p35 Cdk5 activator. 5-HT1AR was phosphorylated by the Cdk5-p35 complex at Thr314 in the third cytoplasmic loop. The phosphorylation stimulated the degradation of 5-HT1AR by the proteasome, resulting in neutralization of the inhibitory action of 5-HT1AR on intracellular cAMP concentration. These results suggest that Cdk5-p35 modulates 5-HT signaling through phosphorylation-dependent degradation of 5-HTlAR.

Cdk5 Regulation of the GRAB-Mediated Rab8-Rab11 Cascade in Axon Outgrowth.

Neurons communicate with each other through their axons and dendrites. However, a full characterization of the molecular mechanisms involved in axon and dendrite formation is still incomplete. Neurite outgrowth requires the supply of membrane components for surface expansion. Two membrane sources for axon outgrowth are suggested: Golgi secretary vesicles and endocytic recycling endosomes. In non-neuronal cells, trafficking of secretary vesicles from Golgi is regulated by Rab8, a member of Rab small GTPases, and that of recycling endosomes is by Rab11, another member of Rabs. However, whether these vesicles are coordinately or independently transported in growing axons is unknown. Herein, we find that GRAB, a guanine nucleotide exchange factor for Rab8, is a novel regulator of axon outgrowth. Knockdown of GRAB suppressed axon outgrowth of cultured mouse brain cortical neurons. GRAB mediates the interaction between Rab11A and Rab8A, and this activity is regulated by phosphorylation at Ser169 and Ser180 by Cdk5-p35. The nonphosphorylatable GRAB mutant S169/180A promoted axonal outgrowth to a greater extent than did the phosphomimetic GRAB mutant S169/180D. Phosphorylation of GRAB suppressed its guanine nucleotide exchange factor activity and its ability to recruit Rab8A- to Rab11A-positive endosomes. In vivo function of GRAB and its Cdk5-phophorylation were shown in migration and process formation of developing neurons in embryonic mouse brains. These results indicate that GRAB regulates axonal outgrowth via activation and recruitment of Rab8A- to Rab11A-positive endosomes in a Cdk5-dependent manner. SIGNIFICANCE STATEMENT: While axon outgrowth requires membrane supply for surface expansion, the molecular mechanisms regulating the membrane transport in growing axons remain unclear. Here, we demonstrate that GRAB, a guanine nucleotide exchange factor for Rab8, is a novel regulator of axon outgrowth. GRAB promotes the axonal membrane transport by mediating the interaction between Rab11 and Rab8 in neurons. The activity of GRAB is regulated by phosphorylation with Cdk5. We describe an in vivo role for GRAB and its Cdk5 phosphorylation during neuronal migration and process formation in embryonic brains. Thus, the membrane supply for axonal outgrowth is regulated by Cdk5 through the Rab11-GRAB-Rab8 cascade.

Templated Aggregation of TAR DNA-binding Protein of 43 kDa (TDP-43) by Seeding with TDP-43 Peptide Fibrils.

TAR DNA-binding protein of 43 kDa (TDP-43) has been identified as the major component of ubiquitin-positive neuronal and glial inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Aggregation of TDP-43 to amyloid-like fibrils and spreading of the aggregates are suggested to account for the pathogenesis and progression of these diseases. To investigate the molecular mechanisms of TDP-43 aggregation, we attempted to identify the amino acid sequence required for the aggregation. By expressing a series of deletion mutants lacking 20 amino acid residues in the C-terminal region in SH-SY5Y cells, we established that residues 274-313 in the glycine-rich region are essential for aggregation. In vitro aggregation experiments using synthetic peptides of 40 amino acids from this sequence and adjacent regions showed that peptides 274-313 and 314-353 formed amyloid-like fibrils. Transduction of these fibrils induced seed-dependent aggregation of TDP-43 in cells expressing wild-type TDP-43 or TDP-43 lacking nuclear localization signal. These cells showed different phosphorylated C-terminal fragments of TDP-43 and different trypsin-resistant bands. These results suggest that residues 274-353 are responsible for the conversion of TDP-43 to amyloid-like fibrils and that templated aggregation of TDP-43 by seeding with different peptides induces various types of TDP-43 pathologies, i.e. the peptides appear to act like prion strains.

Two Degradation Pathways of the p35 Cdk5 (Cyclin-dependent Kinase) Activation Subunit, Dependent and Independent of Ubiquitination.

Cdk5 is a versatile protein kinase that is involved in various neuronal activities, such as the migration of newborn neurons, neurite outgrowth, synaptic regulation, and neurodegenerative diseases. Cdk5 requires the p35 regulatory subunit for activation. Because Cdk5 is more abundantly expressed in neurons compared with p35, the p35 protein levels determine the kinase activity of Cdk5. p35 is a protein with a short half-life that is degraded by proteasomes. Although ubiquitination of p35 has been previously reported, the degradation mechanism of p35 is not yet known. Here, we intended to identify the ubiquitination site(s) in p35. Because p35 is myristoylated at the N-terminal glycine, the possible ubiquitination sites are the lysine residues in p35. We mutated all 23 Lys residues to Arg (p35 23R), but p35 23R was still rapidly degraded by proteasomes at a rate similar to wild-type p35. The degradation of p35 23R in primary neurons and the Cdk5 activation ability of p35 23R suggested the occurrence of ubiquitin-independent degradation of p35 in physiological conditions. We found that p35 has the amino acid sequence similar to the ubiquitin-independent degron in the NKX3.1 homeodomain transcription factor. An Ala mutation at Pro-247 in the degron-like sequence made p35 stable. These results suggest that p35 can be degraded by two degradation pathways: ubiquitin-dependent and ubiquitin-independent. The rapid degradation of p35 by two different methods would be a mechanism to suppress the production of p25, which overactivates Cdk5 to induce neuronal cell death.

α-Synuclein Fibrils Exhibit Gain of Toxic Function, Promoting Tau Aggregation and Inhibiting Microtubule Assembly.

α-Synuclein is the major component of Lewy bodies and Lewy neurites in Parkinson disease and dementia with Lewy bodies and of glial cytoplasmic inclusions in multiple system atrophy. It has been suggested that α-synuclein fibrils or intermediate protofibrils in the process of fibril formation may have a toxic effect on neuronal cells. In this study, we investigated the ability of soluble monomeric α-synuclein to promote microtubule assembly and the effects of conformational changes of α-synuclein on Tau-promoted microtubule assembly. In marked contrast to previous findings, monomeric α-synuclein had no effect on microtubule polymerization. However, both α-synuclein fibrils and protofibrils inhibited Tau-promoted microtubule assembly. The inhibitory effect of α-synuclein fibrils was greater than that of the protofibrils. Dot blot overlay assay and spin-down techniques revealed that α-synuclein fibrils bind to Tau and inhibit microtubule assembly by depleting the Tau available for microtubule polymerization. Using various deletion mutants of α-synuclein and Tau, the acidic C-terminal region of α-synuclein and the basic central region of Tau were identified as regions involved in the binding. Furthermore, introduction of α-synuclein fibrils into cultured cells overexpressing Tau protein induced Tau aggregation. These results raise the possibility that α-synuclein fibrils interact with Tau, inhibit its function to stabilize microtubules, and also promote Tau aggregation, leading to dysfunction of neuronal cells.

The Effect of Fragmented Pathogenic α-Synuclein Seeds on Prion-like Propagation.

Aggregates of abnormal proteins are widely observed in neuronal and glial cells of patients with various neurodegenerative diseases, and it has been proposed that prion-like behavior of these proteins can account for not only the onset but also the progression of these diseases. However, it is not yet clear which abnormal protein structures function most efficiently as seeds for prion-like propagation. In this study, we aimed to identify the most pathogenic species of α-synuclein (α-syn), the main component of the Lewy bodies and Lewy neurites that are observed in α-synucleinopathies. We prepared various forms of α-syn protein and examined their seeding properties in vitro in cells and in mouse experimental models. We also characterized these α-syn species by means of electron microscopy and thioflavin fluorescence assays and found that fragmented ß sheet-rich fibrous structures of α-syn with a length of 50 nm or less are the most efficient promoters of accumulation of phosphorylated α-syn, which is the hallmark of α-synucleinopathies. These results indicate that fragmented amyloid-like aggregates of short α-syn fibrils are the key pathogenic seeds that trigger prion-like conversion.

Kinase activity of endosomal kinase LMTK1A regulates its cellular localization and interactions with cytoskeletons.

Neurite formation, a fundamental process in neuronal maturation, requires the coordinated regulation of cytoskeletal reorganization and membrane transport. Compared to the understanding of cytoskeletal functions, less is known about the supply of membranes to growing neurites. Lemur kinase 1A (LMTK1A) is an endosomal protein kinase that is highly expressed in neurons. We recently reported that LMTK1A regulates the trafficking of Rab11-positive recycling endosomes in growing axons and dendrites. Here, we used the kinase-negative (kn) mutant to investigate the role of the kinase activity of LMTK1A in its cellular localization and interactions with the cytoskeleton in Neuro2A and PC-12 cells. Kinase activity was required for the localization of LMTK1A in the perinuclear endocytic recycling compartment. Perinuclear accumulation was microtubule dependent, and LMTK1A wild type (wt) localized mainly on microtubules, whereas kn LMTK1A was found in the actin-rich cell periphery. In the neurites of PC-12 cells, LMTK1A showed contrasting distributions depending on the kinase activity, with wt being located in the microtubule-rich shaft and the kn form in the actin-rich tip. Taken together, these results suggest that the kinase activity of LMTK1A regulates the pathway for endosomal vesicles to transfer from microtubules to actin filaments at the tip of growing neurites.

The Abundance of Nonphosphorylated Tau in Mouse and Human Tauopathy Brains Revealed by the Use of Phos-Tag Method.

Tauopathies are neurodegenerative diseases characterized by aggregates of hyperphosphorylated tau. Previous studies have identified many disease-related phosphorylation sites on tau. However, it is not understood how tau is hyperphosphorylated and what extent these sites are phosphorylated in both diseased and normal brains. Most previous studies have used phospho-specific antibodies to analyze tau phosphorylation. These results are useful but do not provide information about nonphosphorylated tau. Here, we applied the method of Phos-tag SDS-PAGE, in which phosphorylated tau was separated from nonphosphorylated tau in vivo. Among heterogeneously phosphorylated tau species in adult mouse brains, the nonphosphorylated 0N4R isoform was detected most abundantly. In contrast, perinatal tau and tau in cold water-stressed mice were all phosphorylated with a similar extent of phosphorylation. In normal elderly human brains, nonphosphorylated 0N3R and 0N4R tau were most abundant. A slightly higher phosphorylation of tau, which may represent the early step of hyperphosphorylation, was increased in Alzheimer disease patients at Braak stage V. Tau with this phosphorylation state was pelleted by centrifugation, and sarkosyl-soluble tau in either Alzheimer disease or corticobasal degeneration brains showed phosphorylation profiles similar to tau in normal human brain, suggesting that hyperphosphorylation occurs in aggregated tau. These results indicate that tau molecules are present in multiple phosphorylation states in vivo, and nonphosphorylated forms are highly expressed among them.

Tau phosphorylation at Alzheimer's disease-related Ser356 contributes to tau stabilization when PAR-1/MARK activity is elevated.

Abnormal phosphorylation of the microtubule-associated protein tau is observed in many neurodegenerative diseases, including Alzheimer's disease (AD). AD-related phosphorylation of two tau residues, Ser262 and Ser356, by PAR-1/MARK stabilizes tau in the initial phase of mismetabolism, leading to subsequent phosphorylation events, accumulation, and toxicity. However, the relative contribution of phosphorylation at each of these sites to tau stabilization has not yet been elucidated. In a Drosophila model of human tau toxicity, we found that tau was phosphorylated at Ser262, but not at Ser356, and that blocking Ser262 phosphorylation decreased total tau levels. By contrast, when PAR-1 was co-overexpressed with tau, tau was hyperphosphorylated at both Ser262 and Ser356. Under these conditions, the protein levels of tau were significantly elevated, and prevention of tau phosphorylation at both residues was necessary to completely suppress this elevation. These results suggest that tau phosphorylation at Ser262 plays the predominant role in tau stabilization when PAR-1/MARK activity is normal, whereas Ser356 phosphorylation begins to contribute to this process when PAR-1/MARK activity is abnormally elevated, as in diseased brains.

Phosphorylation of cyclin-dependent kinase 5 (Cdk5) at Tyr-15 is inhibited by Cdk5 activators and does not contribute to the activation of Cdk5.

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. In contrast to other Cdks that promote cell proliferation, Cdk5 plays a role in regulating various neuronal functions, including neuronal migration, synaptic activity, and neuron death. Cdks responsible for cell proliferation need phosphorylation in the activation loop for activation in addition to binding a regulatory subunit cyclin. Cdk5, however, is activated only by binding to its activator, p35 or p39. Furthermore, in contrast to Cdk1 and Cdk2, which are inhibited by phosphorylation at Tyr-15, the kinase activity of Cdk5 is reported to be stimulated when phosphorylated at Tyr-15 by Src family kinases or receptor-type tyrosine kinases. We investigated the activation mechanism of Cdk5 by phosphorylation at Tyr-15. Unexpectedly, however, it was found that Tyr-15 phosphorylation occurred only on monomeric Cdk5, and the coexpression of activators, p35/p25, p39, or Cyclin I, inhibited the phosphorylation. In neuron cultures, too, the activation of Fyn tyrosine kinase did not increase Tyr-15 phosphorylation of Cdk5. Further, phospho-Cdk5 at Tyr-15 was not detected in the p35-bound Cdk5. In contrast, expression of active Fyn increased p35 in neurons. These results indicate that phosphorylation at Tyr-15 is not an activation mechanism of Cdk5 but, rather, indicate that tyrosine kinases could activate Cdk5 by increasing the protein amount of p35. These results call for reinvestigation of how Cdk5 is regulated downstream of Src family kinases or receptor tyrosine kinases in neurons, which is an important signaling cascade in a variety of neuronal activities.

Extracellular association of APP and tau fibrils induces intracellular aggregate formation of tau.

Alzheimer's disease (AD) is characterized by extracellular amyloid ß (Aß) deposition and intracellular tau aggregation. Many studies have indicated some association between these processes, but it remains unknown how the two pathologies are linked. In this study, we investigated whether expression of amyloid precursor protein (APP) influences extracellular seed-dependent intracellular tau accumulation in cultured cells. Treatment of tau-expressing SH-SY5Y cells with Aß fibrils did not induce intracellular tau aggregation. On the other hand, in cells expressing both tau and APP, treatment with tau fibrils or Sarkosyl-insoluble tau from AD brains induced intracellular tau aggregation. The seed-dependent intracellular tau aggregation was not induced by expression of APP lacking the extracellular domain. The amount of phosphorylated tau aggregates in cultured cells was dose dependently elevated in response to increased levels of APP on the cell membrane. Our results indicate that the extracellular region of APP is involved in uptake of tau fibrils into cells, raising the possibility that APP, but not Aß, influences cell-to-cell spreading of tau pathologies in AD by serving as a receptor of abnormal tau aggregates.

Preferential targeting of p39-activated Cdk5 to Rac1-induced lamellipodia.

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family that plays a role in various neuronal activities including brain development, synaptic regulation, and neurodegeneration. Cdk5 requires the neuronal specific activators, p35 and p39 for subcellular compartmentalization. However, it is not known how active Cdk5 is recruited to F-actin cytoskeleton, which is a Cdk5 target. Here we found p35 and p39 localized to F-actin rich regions of the plasma membrane and investigated the underlying targeting mechanism in vitro by expressing them with Rho family GTPases in Neuro2A cells. Both p35 and p39 accumulated at the cell peripheral lamellipodia and perinuclear regions, where active Rac1 is localized. Interestingly, p35 and p39 displayed different localization patterns as p35 was found more at the perinuclear region and p39 was found more in peripheral lamellipodia. We then confirmed this distinct localization in primary hippocampal neurons. We also determined that the localization of p39 to lamellipodia requires myristoylation and Lys clusters within the N-terminal p10 region. Additionally, we found that p39-Cdk5, but not p35-Cdk5 suppressed lamellipodia formation by reducing Rac1 activity. These results suggest that p39-Cdk5 has a dominant role in Rac1-dependent lamellipodial activity.

Structural basis for the different stability and activity between the Cdk5 complexes with p35 and p39 activators.

Cyclin-dependent kinase 5 (Cdk5) is a brain-specific membrane-bound protein kinase that is activated by binding to the p35 or p39 activator. Previous studies have focused on p35-Cdk5, and little is known regarding p39-Cdk5. The lack of functional understanding of p39-Cdk5 is due, in part, to the labile property of p39-Cdk5, which dissociates and loses kinase activity in nonionic detergent conditions. Here we investigated the structural basis for the instability of p39-Cdk5. p39 and p35 contain N-terminal p10 regions and C-terminal Cdk5 activation domains (AD). Although p35 and p39 show higher homology in the C-terminal AD than the N-terminal region, the difference in stability is derived from the C-terminal AD. Based on the crystal structures of the p25 (p35 C-terminal region including AD)-Cdk5 complex, we simulated the three-dimensional structure of the p39 AD-Cdk5 complex and found differences in the hydrogen bond network between Cdk5 and its activators. Three amino acids of p35, Asp-259, Asn-266, and Ser-270, which are involved in hydrogen bond formation with Cdk5, are changed to Gln, Gln, and Pro in p39. Because these three amino acids in p39 do not participate in hydrogen bond formation, we predicted that the number of hydrogen bonds between p39 and Cdk5 was reduced compared with p35 and Cdk5. Using substitution mutants, we experimentally validated that the difference in the hydrogen bond network contributes to the different properties between Cdk5 and its activators.