The neuronal pentraxin Nptx2 regulates complement activity and restrains microglia-mediated synapse loss in neurodegeneration.

Complement overactivation mediates microglial synapse elimination in neurological diseases such as Alzheimer's disease (AD) and frontotemporal dementia (FTD), but how complement activity is regulated in the brain remains largely unknown. We identified that the secreted neuronal pentraxin Nptx2 binds complement C1q and thereby regulates its activity in the brain. Nptx2-deficient mice show increased complement activity, C1q-dependent microglial synapse engulfment, and loss of excitatory synapses. In a neuroinflammation culture model and in aged TauP301S mice, adeno-associated virus (AAV)-mediated neuronal overexpression of Nptx2 was sufficient to restrain complement activity and ameliorate microglia-mediated synapse loss. Analysis of human cerebrospinal fluid (CSF) samples from a genetic FTD cohort revealed reduced concentrations of Nptx2 and Nptx2-C1q protein complexes in symptomatic patients, which correlated with elevated C1q and activated C3. Together, these results show that Nptx2 regulates complement activity and microglial synapse elimination in the brain and that diminished Nptx2 concentrations might exacerbate complement-mediated neurodegeneration in patients with FTD.

A biomarker-authenticated model of schizophrenia implicating NPTX2 loss of function.

Schizophrenia is a polygenetic disorder whose clinical onset is often associated with behavioral stress. Here, we present a model of disease pathogenesis that builds on our observation that the synaptic immediate early gene NPTX2 is reduced in cerebrospinal fluid of individuals with recent onset schizophrenia. NPTX2 plays an essential role in maintaining excitatory homeostasis by adaptively enhancing circuit inhibition. NPTX2 function requires activity-dependent exocytosis and dynamic shedding at synapses and is coupled to circadian behavior. Behavior-linked NPTX2 trafficking is abolished by mutations that disrupt select activity-dependent plasticity mechanisms of excitatory neurons. Modeling NPTX2 loss of function results in failure of parvalbumin interneurons in their adaptive contribution to behavioral stress, and animals exhibit multiple neuropsychiatric domains. Because the genetics of schizophrenia encompasses diverse proteins that contribute to excitatory synapse plasticity, the identified vulnerability of NPTX2 function can provide a framework for assessing the impact of genetics and the intersection with stress.

All-or-none disconnection of pyramidal inputs onto parvalbumin-positive interneurons gates ocular dominance plasticity.

Disinhibition is an obligatory initial step in the remodeling of cortical circuits by sensory experience. Our investigation on disinhibitory mechanisms in the classical model of ocular dominance plasticity uncovered an unexpected form of experience-dependent circuit plasticity. In the layer 2/3 of mouse visual cortex, monocular deprivation triggers a complete, "all-or-none," elimination of connections from pyramidal cells onto nearby parvalbumin-positive interneurons (Pyr→PV). This binary form of circuit plasticity is unique, as it is transient, local, and discrete. It lasts only 1 d, and it does not manifest as widespread changes in synaptic strength; rather, only about half of local connections are lost, and the remaining ones are not affected in strength. Mechanistically, the deprivation-induced loss of Pyr→PV is contingent on a reduction of the protein neuropentraxin2. Functionally, the loss of Pyr→PV is absolutely necessary for ocular dominance plasticity, a canonical model of deprivation-induced model of cortical remodeling. We surmise, therefore, that this all-or-none loss of local Pyr→PV circuitry gates experience-dependent cortical plasticity.

Cyclin-Dependent Kinase 5-Dependent BAG3 Degradation Modulates Synaptic Protein Turnover.

BACKGROUND: Synaptic protein dyshomeostasis and functional loss is an early invariant feature of Alzheimer's disease (AD), yet the unifying etiological pathway remains largely unknown. Knowing that cyclin-dependent kinase 5 (CDK5) plays critical roles in synaptic formation and degeneration, its phosphorylation targets were reexamined in search of candidates with direct global impacts on synaptic protein dynamics, and the associated regulatory network was also analyzed. METHODS: Quantitative phosphoproteomics and bioinformatics analyses were performed to identify top-ranked candidates. A series of biochemical assays was used to investigate the associated regulatory signaling networks. Histological, electrochemical, and behavioral assays were performed in conditional knockout, small hairpin RNA-mediated knockdown, and AD-related mice models to evaluate the relevance of CDK5 to synaptic homeostasis and functions. RESULTS: Among candidates with known implications in synaptic modulations, BAG3 ranked the highest. CDK5-mediated phosphorylation on S297/S291 (mouse/human) destabilized BAG3. Loss of BAG3 unleashed the selective protein degradative function of the HSP70 machinery. In neurons, this resulted in enhanced degradation of a number of glutamatergic synaptic proteins. Conditional neuronal knockout of Bag3 in vivo led to impairment of learning and memory functions. In human AD and related mouse models, aberrant CDK5-mediated loss of BAG3 yielded similar effects on synaptic homeostasis. Detrimental effects of BAG3 loss on learning and memory functions were confirmed in these mice, and such effects were reversed by ectopic BAG3 reexpression. CONCLUSIONS: Our results highlight that the neuronal CDK5-BAG3-HSP70 signaling axis plays a critical role in modulating synaptic homeostasis. Dysregulation of the signaling pathway directly contributes to synaptic dysfunction and AD pathogenesis.

CDK5-mediated phosphorylation and stabilization of TPX2 promotes hepatocellular tumorigenesis.

BACKGROUND: CDK5, an atypical member of the CDK family, play a significant role in the tumorigenesis of multiple organ, but CDK5 and its substrates in genesis and development of HCC is still unclear. METHODS: Expression of CDK5 in HCC tumor and paired adjacent noncancerous tissues from 90 patients were measured by Western blotting, immunohistochemistry, and real-time PCR. The role of CDK5 in cell function and tumorigenesis was explored in HCC cell lines, ex vivo xenografts and diethylnitrosamine induced HCC model. Furthermore, comparative phosphoproteomic screening identified the oncoprotein TPX2 as a new substrate of CDK5. We also identified the effect of CDK5/P25 interaction blocker tamoxifen on HCC cell growth and migration. RESULTS: CDK5 was increased in HCC tisues and the level of CDK5 was correlated with the severity of HCC based on patient recurrence and 5-year fatality rate. Exogenously expressed CDK5 but not kinase-dead CDK5 promoted proliferation, migration, and invasion of HCC cells. Functional ablation of CDK5 significantly inhibited the exacerbation of HCC cells. Xenograft implantation of HCC cells overexpressing CDK5 promoted tumorigenesis, and genetic knockdown of CDK5 reduced HCC growth and metastasis in vivo. More importantly, heterozygous knockout CDK5 (Cdk5+/-) attenuated HCC tumorigenesis induced by diethylnitrosamine. CDK5-mediated phosphorylation of TPX2 at serine 486 promoted its protein stability. TPX2 silence could restore HCC cell migration capability with overexpression CDK5. Treatment with tamoxifen inhibited cell growth and migration of HCC, demonstrating the role of active CDK5 in HCC. CONCLUSIONS: Our results suggest activation of CDK5 is associated with HCC tumorigenesis. CDK5-mediated phosphorylation and stabilization of TPX2 promotes hepatocellular proliferation and tumorigenicity.

Arc Oligomerization Is Regulated by CaMKII Phosphorylation of the GAG Domain: An Essential Mechanism for Plasticity and Memory Formation.

Arc is a synaptic protein essential for memory consolidation. Recent studies indicate that Arc originates in evolution from a Ty3-Gypsy retrotransposon GAG domain. The N-lobe of Arc GAG domain acquired a hydrophobic binding pocket in higher vertebrates that is essential for Arc's canonical function to weaken excitatory synapses. Here, we report that Arc GAG also acquired phosphorylation sites that can acutely regulate its synaptic function. CaMKII phosphorylates the N-lobe of the Arc GAG domain and disrupts an interaction surface essential for high-order oligomerization. In Purkinje neurons, CaMKII phosphorylation acutely reverses Arc's synaptic action. Mutant Arc that cannot be phosphorylated by CaMKII enhances metabotropic receptor-dependent depression in the hippocampus but does not alter baseline synaptic transmission or long-term potentiation. Behavioral studies indicate that hippocampus- and amygdala-dependent learning requires Arc GAG domain phosphorylation. These studies provide an atomic model for dynamic and local control of Arc function underlying synaptic plasticity and memory.

Cyclin-dependent kinase 5-mediated phosphorylation of chloride intracellular channel 4 promotes oxidative stress-induced neuronal death.

Oxidative stress can cause apoptosis in neurons and may result in neurodegenerative diseases. However, the signaling mechanisms leading to oxidative stress-induced neuronal apoptosis are not fully understood. Oxidative stress stimulates aberrant activation of cyclin-dependent kinase 5 (CDK5), thought to promote neuronal apoptosis by phosphorylating many cell death-related substrates. Here, using protein pulldown methods, immunofluorescence experiments and in vitro kinase assays, we identified chloride intracellular channel 4 (CLIC4), the expression of which increases during neuronal apoptosis, as a CDK5 substrate. We found that activated CDK5 phosphorylated serine 108 in CLIC4, increasing CLIC4 protein stability, and accumulation. Pharmacological inhibition or shRNA-mediated silencing of CDK5 decreased CLIC4 levels in neurons. Moreover, CLIC4 overexpression led to neuronal apoptosis, whereas knockdown or pharmacological inhibition of CLIC4 attenuated H2O2-induced neuronal apoptosis. These results implied that CLIC4, by acting as a substrate of CDK5, mediated neuronal apoptosis induced by aberrant CDK5 activation. Targeting CLIC4 in neurons may therefore provide a therapeutic approach for managing progressive neurodegenerative diseases that arise from neuronal apoptosis.

The roles of Cdk5-mediated subcellular localization of FOXO1 in neuronal death.

Deficiency of cyclin-dependent kinase 5 (Cdk5) has been linked to the death of postmitotic cortical neurons during brain development. We now report that, in mouse cortical neurons, Cdk5 is capable of phosphorylating the transcription factor FOXO1 at Ser249 in vitro and in vivo. Cellular stresses resulting from extracellular stimulation by H2O2 or ß-amyloid promote hyperactivation of Cdk5, FOXO1 nuclear export and inhibition of its downstream transcriptional activity. In contrast, a loss of Cdk5 leads to FOXO1 translocation into the nucleus: a shift due to decreased AKT activity but independent of S249 phosphorylation. Nuclear FOXO1 upregulates transcription of the proapoptotic gene, BIM, leading to neuronal death, which can be rescued when endogenous FOXO1 was replaced by the cytoplasmically localized form of FOXO1, FOXO1-S249D. Cytoplasmic, but not nuclear, Cdk5 attenuates neuronal death by inhibiting FOXO1 transcriptional activity and BIM expression. Together, our findings suggest that Cdk5 plays a novel and unexpected role in the degeneration of postmitotic neurons through modulation of the cellular location of FOXO1, which constitutes an alternative pathway through which Cdk5 deficiency leads to neuronal death.

Cyclin-dependent kinase 5 decreases in gastric cancer and its nuclear accumulation suppresses gastric tumorigenesis.

PURPOSE: As a cyclin-independent atypical CDK, the role of CDK5 in regulating cell proliferation in gastric cancer remains unknown. EXPERIMENTAL DESIGN: Expression of CDK5 in gastric tumor and paired adjacent noncancerous tissues from 437 patients was measured by Western blotting, immunohistochemistry, and real-time PCR. The subcellular translocation of CDK5 was monitored during gastric cancer cell proliferation. The role of nuclear CDK5 in gastric cancer tumorigenic proliferation and ex vivo xenografts was explored. Furthermore, by screening for compounds in the PubChem database that disrupt CDK5 association with its nuclear export facilitator, we identified a small molecular (NS-0011) that inhibits gastric cancer cell growth. RESULTS: CDK5 level was significantly decreased in the majority of gastric tumor tissues, and the reduction of CDK5 correlated with the severity of gastric cancer based on tumor and lymph node metastasis and patient 5-year fatality rate. Nuclear localization of CDK5 was found to be significantly decreased in tumor tissues and gastric cancer cell lines, whereas exogenously expression of nucleus-targeted CDK5 inhibited the proliferation and xenograft implantation of gastric cancer cells. Treatment with the small molecule NS-0011, which increases CDK5 accumulation in the nucleus, suppressed both cancer cell proliferation and xenograft tumorigenesis. CONCLUSIONS: Our results suggest that low CDK5 expression is associated with poor overall survival in patients with gastric cancer, and nuclear accumulation of CDK5 inhibits the proliferation and tumorigenicity of human gastric cancer cells.

CDK5 activator protein p25 preferentially binds and activates GSK3ß.

Glycogen synthase kinase 3ß (GSK3ß) and cyclin-dependent kinase 5 (CDK5) are tau kinases and have been proposed to contribute to the pathogenesis of Alzheimer's disease. The 3D structures of these kinases are remarkably similar, which led us to hypothesize that both might be capable of binding cyclin proteins--the activating cofactors of all CDKs. CDK5 is normally activated by the cyclin-like proteins p35 and p39. By contrast, we show that GSK3ß does not bind to p35 but unexpectedly binds to p25, the calpain cleavage product of p35. Indeed, overexpressed GSK3ß outcompetes CDK5 for p25, whereas CDK5 is the preferred p35 partner. FRET analysis reveals nanometer apposition of GSK3ß:p25 in cell soma as well as in synaptic regions. Interaction with p25 also alters GSK3ß substrate specificity. The GSK3ß:p25 interaction leads to enhanced phosphorylation of tau, but decreased phosphorylation of ß-catenin. A partial explanation for this situation comes from in silico modeling, which predicts that the docking site for p25 on GSK3ß is the AXIN-binding domain; because of this, p25 inhibits the formation of the GSK3ß/AXIN/APC destruction complex, thus preventing GSK3ß from binding to and phosphorylating ß-catenin. Coexpression of GSK3ß and p25 in cultured neurons results in a neurodegeneration phenotype that exceeds that observed with CDK5 and p25. When p25 is transfected alone, the resulting neuronal damage is blocked more effectively with a specific siRNA against Gsk3ß than with one against Cdk5. We propose that the effects of p25, although normally attributed to activate CDK5, may be mediated in part by elevated GSK3ß activity.

Cdk5 levels oscillate during the neuronal cell cycle: Cdh1 ubiquitination triggers proteosome-dependent degradation during S-phase.

When cell cycle re-activation occurs in post-mitotic neurons it places them at increased risk for death. The cell cycle/cell death association has been reported in many neurodegenerative diseases including Alzheimer disease (AD), yet the mechanisms by which a normal neuron suppresses the cycle remain largely unknown. Recently, our laboratory has shown that Cdk5 (cyclin-dependent kinase 5) is a key player in this protective function. When a neuron is under stress, Cdk5 is transported to the cytoplasm; this eliminates its cell cycle suppression activity and the neuron re-enters S-phase. In the current study we show that a similar principle applies during a normal cell cycle. When a neuronal cell enters S phase, Cdk5 is transported to the cytoplasm where it is ubiquitinated by the E3 ligase APC-Cdh1. Ubiquitinated Cdk5 is then rapidly degraded by the proteasome. The ubiquitination site of Cdk5 appears to be in the p35 binding area; in the presence of high levels of p35, the ubiquitination of Cdk5 was blocked, and the degradation in S phase was attenuated. The data suggest an unsuspected role for Cdk5 during the progression of a normal cell cycle and offer new pharmaceutical targets for regulating neuronal cell cycling and cell death.

Phosphoproteins regulated by heat stress in rice leaves.

BACKGROUND: High temperature is a critical abiotic stress that reduces crop yield and quality. Rice (Oryza sativa L.) plants remodel their proteomes in response to high temperature stress. Moreover, phosphorylation is the most common form of protein post-translational modification (PTM). However, the differential expression of phosphoproteins induced by heat in rice remains unexplored. METHODS: Phosphoprotein in the leaves of rice under heat stress were displayed using two-dimensional electrophoresis (2-DE) and Pro-Q Diamond dye. Differentially expressed phosphoproteins were identified by MALDI-TOF-TOF-MS/MS and confirmed by Western blotting. RESULTS: Ten heat-phosphoproteins were identified from twelve protein spots, including ribulose bisphos-phate carboxylase large chain, 2-Cys peroxiredoxin BAS1, putative mRNA binding protein, Os01g0791600 protein, OSJNBa0076N16.12 protein, putative H(+)-transporting ATP synthase, ATP synthase subunit beta and three putative uncharacterized proteins. The identification of ATP synthase subunit beta was further validated by Western-blotting. Four phosphorylation site predictors were also used to predict the phosphorylation sites and the specific kinases for these 10 phosphoproteins. CONCLUSION: Heat stress induced the dephosphorylation of RuBisCo and the phosphorylation of ATP-ß, which decreased the activities of RuBisCo and ATP synthase. The observed dephosphorylation of the mRNA binding protein and 2-Cys peroxiredoxin may be involved in the transduction of heat-stress signaling, but the functional importance of other phosphoproteins, such as H+-ATPase, remains unknown.