Protocols for Characterization of Cdk5 Kinase Activity.

Cyclin-dependent kinases (Cdks) are generally known to be involved in controlling the cell cycle, but Cdk5 is a unique member of this protein family for being most active in post-mitotic neurons. Cdk5 is developmentally important in regulating neuronal migration, neurite outgrowth, and axon guidance. Cdk5 is enriched in synaptic membranes and is known to modulate synaptic activity. Postnatally, Cdk5 can also affect neuronal processes such as dopaminergic signaling and pain sensitivity. Dysregulated Cdk5, in contrast, has been linked to neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Despite primarily being implicated in neuronal development and activity, Cdk5 has lately been linked to non-neuronal functions including cancer cell growth, immune responses, and diabetes. Since Cdk5 activity is tightly regulated, a method for measuring its kinase activity is needed to fully understand the precise role of Cdk5 in developmental and disease processes. This article includes methods for detecting Cdk5 kinase activity in cultured cells or tissues, identifying new substrates, and screening for new kinase inhibitors. Furthermore, since Cdk5 shares homology and substrate specificity with Cdk1 and Cdk2, the Cdk5 kinase assay can be used, with modification, to measure the activity of other Cdks as well. © 2021 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Measuring Cdk5 activity from protein lysates Support Protocol 1: Immunoprecipitation of Cdk5 using Dynabeads Alternate Protocol: Non-radioactive protocols to measure Cdk5 kinase activity Support Protocol 2: Western blot analysis for the detection of Cdk5, p35, and p39 Support Protocol 3: Immunodetection analysis for Cdk5, p35, and p39 Support Protocol 4: Genetically engineered mice (+ and - controls) Basic Protocol 2: Identifying new Cdk5 substrates and kinase inhibitors.

Analyzing the Metabolic Phenotype of Bone Marrow-Derived Dendritic Cells by Assessing Their Oxygen Consumption and Extracellular Acidification.

Dendritic cells (DCs) are the bridge between innate and T cell-dependent adaptive immunity, and are promising therapeutic targets for cancer and immune-mediated disorders. In the recent past, DCs have gained significant interest to manipulate them for the treatment of cancer and immune-mediated disorders. This can be achieved by differentiating them into either immunogenic or tolerogenic DCs (TolDCs), by modulating their metabolic pathways, including glycolysis, oxidative phosphorylation, and fatty acid metabolism, to orchestrate their desired function. For immunogenic DCs, this maturation shifts the metabolic profile to a glycolytic metabolic state and leads to the use of glucose as a carbon source, whereas TolDCs prefer oxidative phosphorylation (OXPHOS) and fatty acid oxidation for their energy resource.Understanding the metabolic regulation of DC subsets and functions at large not only will improve our understanding of DC biology and immune regulation, but can also open up opportunities for treating immune-mediated ailments and cancers by tweaking endogenous T-cell responses through DC-based immunotherapies. Here we describe a method to analyze this dichotomous metabolic reprogramming of the DCs for generating reliable and effective DC cell therapy products. We, hereby, report how to measure the OXPHOS and glycolysis level of DCs. We focus on the metabolic reprogramming of TolDCs using a pharmacological nuclear factor (erythroid-derived 2)-like-2 factor (Nrf2) activator as an example to illustrate the metabolic profile of TolDCs.

Nrf2-mediated metabolic reprogramming of tolerogenic dendritic cells is protective against aplastic anemia.

Aplastic anemia (AA) is a rare disease characterized by immune-mediated suppression of bone marrow (BM) function resulting in progressive pancytopenia. Stem cell transplant and immunosuppressive therapies remain the major treatment choices for AA patients with limited benefit and undesired side effects. Here, we report for the first time the therapeutic utility of Nrf2-induced metabolically reprogrammed tolerogenic dendritic cells (TolDCs) in the suppression of AA in mice. CDDO-DFPA-induced Nrf2 activation resulted in a TolDC phenotype as evidenced by induction of IL-4, IL-10, and TGF-ß and suppression of TNFα, IFN-γ, and IL-12 levels in Nrf2+/+ but not Nrf2-/- DCs. Cellular metabolism holds the key to determining DC immunogenic or tolerogenic cell fate. Although immature and LPS-induced (mature) Nrf2+/+ and Nrf2-/- DCs exhibited similar patterns of oxidative phosphorylation (OXPHOS) and glycolysis, only Nrf2+/+ DCs partially restored OXPHOS and reduced glycolysis during CDDO-DFPA-induced Nrf2 activation. These results were further confirmed by altered glucose uptake and lactate production. We observed significantly enhanced HO-1 and reduced iNOS/NO production in Nrf2+/+ compared to Nrf2-/- DCs, suggesting Nrf2-dependent TolDC induction is linked to suppression of the inhibitory effect of NO on OXPHOS. Furthermore, Nrf2-/- DCs demonstrated higher antigen-specific T cell proliferation. Lastly, TolDC administration improved hematopoiesis and survival in AA murine model, with decreased Th17 and increased Treg cells. Concomitantly, immunohistochemical analysis of AA patient BM biopsies displayed higher DCs, T cells, and iNOS expression accompanied with lower Nrf2 and HO-1 expression when compared to normal subjects. These results provide new insight into the therapeutic utility of metabolically reprogrammed TolDCs by CDDO-DFPA induced Nrf2 signaling in the treatment of AA.

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells.

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating naïve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Curcumin Ameliorates Neuroinflammation, Neurodegeneration, and Memory Deficits in p25 Transgenic Mouse Model that Bears Hallmarks of Alzheimer's Disease.

Several studies have indicated that neuroinflammation is indeed associated with neurodegenerative disease pathology. However, failures of recent clinical trials of anti-inflammatory agents in neurodegenerative disorders have emphasized the need to better understand the complexity of the neuroinflammatory process in order to unravel its link with neurodegeneration. Deregulation of Cyclin-dependent kinase 5 (Cdk5) activity by production of its hyperactivator p25 is involved in the formation of tau and amyloid pathology reminiscent of Alzheimer's disease (AD). Recent studies show an association between p25/Cdk5 hyperactivation and robust neuroinflammation. In addition, we recently reported the novel link between the p25/Cdk5 hyperactivation-induced inflammatory responses and neurodegenerative changes using a transgenic mouse that overexpresses p25 (p25Tg). In this study, we aimed to understand the effects of early intervention with a potent natural anti-inflammatory agent, curcumin, on p25-mediated neuroinflammation and the progression of neurodegeneration in p25Tg mice. The results from this study showed that curcumin effectively counteracted the p25-mediated glial activation and pro-inflammatory chemokines/cytokines production in p25Tg mice. Moreover, this curcumin-mediated suppression of neuroinflammation reduced the progression of p25-induced tau/amyloid pathology and in turn ameliorated the p25-induced cognitive impairments. It is widely acknowledged that to treat AD, one must target the early-stage of pathological changes to protect neurons from irreversible damage. In line with this, our results demonstrated that early intervention of inflammation could reduce the progression of AD-like pathological outcomes. Moreover, our data provide a rationale for the potential use of curcuminoids in the treatment of inflammation associated neurodegenerative diseases.

A unique tolerizing dendritic cell phenotype induced by the synthetic triterpenoid CDDO-DFPA (RTA-408) is protective against EAE.

Tolerogenic dendritic cells (DCs) have emerged as relevant clinical targets for the treatment of multiple sclerosis and other autoimmune disorders. However, the pathways essential for conferring the tolerizing DC phenotype and optimal methods for their induction remain an intense area of research. Triterpenoids are a class of small molecules with potent immunomodulatory activity linked to activation of Nrf2 target genes, and can also suppress the manifestations of experimental autoimmune encephalomyelitis (EAE). Here we demonstrate that DCs are a principal target of the immune modulating activity of triterpenoids in the context of EAE. Exposure of DCs to the new class of triterpenoid CDDO-DFPA (RTA-408) results in the induction of HO-1, TGF-ß, and IL-10, as well as the repression of NF-κB, EDN-1 and pro-inflammatory cytokines IL-6, IL-12, and TNFα. CDDO-DFPA exposed DCs retained expression of surface ligands and capacity for antigen uptake but were impaired to induce Th1 and Th17 cells. TGF-ß was identified as the factor mediating suppression of T cell proliferation by CDDO-DFPA pretreated DCs, which failed to passively induce EAE. These findings demonstrate the potential therapeutic utility of CDDO-DFPA in the treatment and prevention of autoimmune disorders, and its capacity to induce tolerance via modulation of the DC phenotype.

Cyclin-dependent kinase 5 activity is required for allogeneic T-cell responses after hematopoietic cell transplantation in mice.

Molecular intermediates in T-cell activation pathways are crucial targets for the therapy and prevention of graft-versus-host disease (GVHD) following allogeneic hematopoietic cell transplantation (allo-HCT). We recently identified an essential role for cyclin-dependent kinase 5 (Cdk5) in T-cell activation and effector function, but the contribution of Cdk5 activity to the development of GVHD has not been explored. Using an established, preclinical, murine, GVHD model, we reveal that Cdk5 activity is increased in key target organs early after allo-HCT. We then generated chimeric mice (Cdk5+/+C or Cdk5-/-C) using hematopoietic progenitors from either embryonic day 16.5 Cdk5+/+ or Cdk5-/- embryos to enable analyses of the role of Cdk5 in GVHD, as germ line Cdk5 gene deletion is embryonically lethal. The immunophenotype of adult Cdk5-/-C mice is identical to control Cdk5+/+C mice. However, transplantation of donor Cdk5-/-C bone marrow and T cells dramatically reduced the severity of systemic and target organ GVHD. This phenotype is attributed to decreased T-cell migration to secondary lymphoid organs (SLOs), reduced in vivo proliferation within these organs, and fewer cytokine-producing donor T cells during GVHD development. Moreover, these defects in Cdk5-/- T-cell function are associated with altered CCR7 signaling following ligation by CCL19, a receptor:ligand interaction critical for T-cell migration into SLOs. Although Cdk5 activity in donor T cells contributed to graft-versus-tumor effects, pharmacologic inhibition of Cdk5 preserved leukemia-free survival. Collectively, our data implicate Cdk5 in allogeneic T-cell responses after HCT and as an important new target for therapeutic intervention.

Cdk5 disruption attenuates tumor PD-L1 expression and promotes antitumor immunity.

Cancers often evade immune surveillance by adopting peripheral tissue- tolerance mechanisms, such as the expression of programmed cell death ligand 1 (PD-L1), the inhibition of which results in potent antitumor immunity. Here, we show that cyclin-dependent kinase 5 (Cdk5), a serine-threonine kinase that is highly active in postmitotic neurons and in many cancers, allows medulloblastoma (MB) to evade immune elimination. Interferon-γ (IFN-γ)-induced PD-L1 up-regulation on MB requires Cdk5, and disruption of Cdk5 expression in a mouse model of MB results in potent CD4(+) T cell-mediated tumor rejection. Loss of Cdk5 results in persistent expression of the PD-L1 transcriptional repressors, the interferon regulatory factors IRF2 and IRF2BP2, which likely leads to reduced PD-L1 expression on tumors. Our finding highlights a central role for Cdk5 in immune checkpoint regulation by tumor cells.

Cyclin-dependent kinase 5 represses Foxp3 gene expression and Treg development through specific phosphorylation of Stat3 at Serine 727.

Cyclin-dependent kinase 5 (Cdk5) is known as a unique member of the cyclin-dependent family of serine/threonine kinases. Previously, we demonstrated Cdk5 to be an important regulator of T cell function and that disruption of Cdk5 expression ameliorates T cell mediated neuroinflammation. Here, we show a novel role of Cdk5 in the regulation of Foxp3 expression in murine CD4(+) T cells. Our data indicate that disruption of Cdk5 activity in T cells abrogates the IL-6 suppression of Foxp3 expression. This effect is achieved through Cdk5 phosphorylation of the signal transducer and activator of transcription 3 (Stat3) specifically at Serine 727 in T cells, and we show this post-translational modification is required for proper Stat3 DNA binding to the Foxp3 gene on the enhancer II region. Taken together, our data point to an essential role for Cdk5 in the differentiation of T cells as it regulates Foxp3 gene expression through phosphorylation of Stat3.

Cdk5 controls IL-2 gene expression via repression of the mSin3a-HDAC complex.

Cyclin-dependent kinase 5 (Cdk5) is a unique member of a family of serine/threonine cyclin-dependent protein kinases. We previously demonstrated disruption of Cdk5 gene expression in mice impairs T-cell function and ameliorates T-cell-mediated neuroinflammation. Here, we show Cdk5 modulates gene expression during T-cell activation by impairing the repression of gene transcription by histone deacetylase 1 (HDAC1) through specific phosphorylation of the mSin3a protein at serine residue 861. Disruption of Cdk5 activity in T-cells enhances HDAC activity and binding of the HDAC1/mSin3a complex to the IL-2 promoter, leading to suppression of IL-2 gene expression. These data point to essential roles for Cdk5 in regulating gene expression in T-cells and transcriptional regulation by the co-repressor mSin3a.

Specific inhibition of p25/Cdk5 activity by the Cdk5 inhibitory peptide reduces neurodegeneration in vivo.

The aberrant hyperactivation of Cyclin-dependent kinase 5 (Cdk5), by the production of its truncated activator p25, results in the formation of hyperphosphorylated tau, neuroinflammation, amyloid deposition, and neuronal death in vitro and in vivo. Mechanistically, this occurs as a result of a neurotoxic insult that invokes the intracellular elevation of calcium to activate calpain, which cleaves the Cdk5 activator p35 into p25. It has been shown previously that the p25 transgenic mouse as a model to investigate the mechanistic implications of p25 production in the brain, which recapitulates deregulated Cdk5-mediated neuropathological changes, such as hyperphosphorylated tau and neuronal death. To date, strategies to inhibit Cdk5 activity have not been successful in targeting selectively aberrant activity without affecting normal Cdk5 activity. Here we show that the selective inhibition of p25/Cdk5 hyperactivation in vivo, through overexpression of the Cdk5 inhibitory peptide (CIP), rescues against the neurodegenerative pathologies caused by p25/Cdk5 hyperactivation without affecting normal neurodevelopment afforded by normal p35/Cdk5 activity. Tau and amyloid pathologies as well as neuroinflammation are significantly reduced in the CIP-p25 tetra transgenic mice, whereas brain atrophy and subsequent cognitive decline are reversed in these mice. The findings reported here represent an important breakthrough in elucidating approaches to selectively inhibit the p25/Cdk5 hyperactivation as a potential therapeutic target to reduce neurodegeneration.

Cdk5: An Emerging Kinase in Pain Signaling.

Pain is an important survival mechanism for an organism. It can turn into severe mental and physical disorder however, if the molecular and/or cellular pathways involved in pain signaling are altered. Chronic pain is characterized by an altered pain perception that includes allodynia (a response to a normally non-noxious stimulus) and hyperalgesia (an exaggerated response to a normally noxious stimulus). Past few years of pain research has been mainly focused on precise understanding of the molecular and cellular nociceptive signatures altered during chronic pain, so that more effective pain relievers can be developed. The importance of protein kinases in normal cellular homeostasis and disease pathogenesis has evolved rapidly in the past few decades. The recent advancement defining the role of multiple protein kinases in regulating neuronal plasticity and pain sensitization has gained enough attention of pharmaceutical industry to develop specific and selective kinase inhibitors as analgesics. Cyclin-dependent kinase 5 (Cdk5) is one such emerging kinase in pain biology. We will discuss here the recent advancement and therapeutic potential of Cdk5 in pain signaling.

Cdk5/p25-induced cytosolic PLA2-mediated lysophosphatidylcholine production regulates neuroinflammation and triggers neurodegeneration.

The deregulation of cyclin-dependent kinase 5 (Cdk5) by p25 has been shown to contribute to the pathogenesis in a number of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). In particular, p25/Cdk5 has been shown to produce hyperphosphorylated tau, neurofibrillary tangles as well as aberrant amyloid precursor protein processing found in AD. Neuroinflammation has been observed alongside the pathogenic process in these neurodegenerative diseases, however the precise mechanism behind the induction of neuroinflammation and the significance in the AD pathogenesis has not been fully elucidated. In this report, we uncover a novel pathway for p25-induced neuroinflammation where p25 expression induces an early trigger of neuroinflammation in vivo in mice. Lipidomic mass spectrometry, in vitro coculture and conditioned media transfer experiments show that the soluble lipid mediator lysophosphatidylcholine (LPC) is released by p25 overexpressing neurons to initiate astrogliosis, neuroinflammation and subsequent neurodegeneration. Reverse transcriptase PCR and gene silencing experiments show that cytosolic phospholipase 2 (cPLA2) is the key enzyme mediating the p25-induced LPC production and cPLA2 upregulation is critical in triggering the p25-mediated inflammatory and neurodegenerative process. Together, our findings delineate a potential therapeutic target for the reduction of neuroinflammation in neurodegenerative diseases including AD.

Triterpenoid modulation of IL-17 and Nrf-2 expression ameliorates neuroinflammation and promotes remyelination in autoimmune encephalomyelitis.

Inflammatory cytokines and endogenous anti-oxidants are variables affecting disease progression in multiple sclerosis (MS). Here we demonstrate the dual capacity of triterpenoids to simultaneously repress production of IL-17 and other pro-inflammatory mediators while exerting neuroprotective effects directly through Nrf2-dependent induction of anti-oxidant genes. Derivatives of the natural triterpene oleanolic acid, namely CDDO-trifluoroethyl-amide (CDDO-TFEA), completely suppressed disease in a murine model of MS, experimental autoimmune encephalomyelitis (EAE), by inhibiting Th1 and Th17 mRNA and cytokine production. Encephalitogenic T cells recovered from treated mice were hypo-responsive to myelin antigen and failed to adoptively transfer the disease. Microarray analyses showed significant suppression of pro-inflammatory transcripts with concomitant induction of anti-inflammatory genes including Ptgds and Hsd11b1. Finally, triterpenoids induced oligodendrocyte maturation in vitro and enhanced myelin repair in an LPC-induced non-inflammatory model of demyelination in vivo. These results demonstrate the unique potential of triterpenoid derivatives for the treatment of neuroinflammatory disorders such as MS.

Cyclin-dependent kinase 5 activity is required for T cell activation and induction of experimental autoimmune encephalomyelitis.

Cyclin-dependent kinase 5 (Cdk5) is a ubiquitously expressed serine/threonine kinase. However, a requirement for Cdk5 has been demonstrated only in postmitotic neurons where there is abundant expression of its activating partners p35 and/or p39. Although hyperactivation of the Cdk5-p35 complex has been found in a variety of inflammatory neurodegenerative disorders, the potential contribution of nonneuronal Cdk5-p35 activity has not been explored in this context. We describe a previously unknown function of the Cdk5-p35 complex in T cells that is required for induction of experimental autoimmune encephalomyelitis (EAE). T cell receptor (TCR) stimulation leads to a rapid induction of Cdk5-p35 expression that is required for T lymphocyte activation. Chimeric mice lacking Cdk5 gene expression in hematopoietic tissues (Cdk5(-/-C)) are resistant to induction of EAE, and adoptive transfer of either Cdk5(-/-C) or p35(-/-) encephalitogenic lymphocytes fails to transfer disease. Moreover, our data reveal a novel mechanism involving Cdk5-mediated phosphorylation of the actin modulator coronin 1a on threonine 418. Cdk5-deficient lymphocytes lack this posttranslational modification of coronin 1a and exhibit defective TCR-induced actin polarization and reduced migration toward CCL-19. These data define a distinct role for Cdk5 in lymphocyte biology and suggest that inhibition of this kinase may be beneficial in the treatment of T cell-mediated inflammatory disorders.

Phosphorylation of p27Kip1 at Thr187 by cyclin-dependent kinase 5 modulates neural stem cell differentiation.

Cyclin-dependent kinase 5 (Cdk5) plays a key role in the development of the mammalian nervous system; it phosphorylates a number of targeted proteins involved in neuronal migration during development to synaptic activity in the mature nervous system. Its role in the initial stages of neuronal commitment and differentiation of neural stem cells (NSCs), however, is poorly understood. In this study, we show that Cdk5 phosphorylation of p27(Kip1) at Thr187 is crucial to neural differentiation because 1) neurogenesis is specifically suppressed by transfection of p27(Kip1) siRNA into Cdk5(+/+) NSCs; 2) reduced neuronal differentiation in Cdk5(-/-) compared with Cdk5(+/+) NSCs; 3) Cdk5(+/+) NSCs, whose differentiation is inhibited by a nonphosphorylatable mutant, p27/Thr187A, are rescued by cotransfection of a phosphorylation-mimicking mutant, p27/Thr187D; and 4) transfection of mutant p27(Kip1) (p27/187A) into Cdk5(+/+) NSCs inhibits differentiation. These data suggest that Cdk5 regulates the neural differentiation of NSCs by phosphorylation of p27(Kip1) at theThr187 site. Additional experiments exploring the role of Ser10 phosphorylation by Cdk5 suggest that together with Thr187 phosphorylation, Ser10 phosphorylation by Cdk5 promotes neurite outgrowth as neurons differentiate. Cdk5 phosphorylation of p27(Kip1), a modular molecule, may regulate the progress of neuronal differentiation from cell cycle arrest through differentiation, neurite outgrowth, and migration.

Cdk5-mediated phosphorylation of delta-catenin regulates its localization and GluR2-mediated synaptic activity.

Cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation plays an important role in proper synaptic function and transmission. Loss of Cdk5 activity results in abnormal development of the nervous system accompanied by massive disruptions in cortical migration and lamination, therefore impacting synaptic activity. The Cdk5 activator p35 associates with delta-catenin, the synaptic adherens junction protein that serves as part of the anchorage complex of AMPA receptor at the postsynaptic membrane. However, the implications of Cdk5-mediated phosphorylation of delta-catenin have not been fully elucidated. Here we show that Cdk5-mediated phosphorylation of delta-catenin regulates its subcellular localization accompanied by changes in dendritic morphogenesis and synaptic activity. We identified two Cdk5 phosphorylation sites in mouse delta-catenin, serines 300 and 357, and report that loss of Cdk5 phosphorylation of delta-catenin increased its localization to the membrane. Furthermore, mutations of the serines 300 and 357 to alanines to mimic nonphosphorylated delta-catenin resulted in increased dendritic protrusions accompanied by increased AMPA receptor subunit GluR2 localization at the membrane. Consistent with these observations, loss of Cdk5 phosphorylation of delta-catenin increased the AMPA/NMDA ratio. This study reveals how Cdk5 phosphorylation of the synaptic mediator protein delta-catenin can alter its localization at the synapse to impact neuronal synaptic activity.

Conditional deletion of neuronal cyclin-dependent kinase 5 in developing forebrain results in microglial activation and neurodegeneration.

Neuronal migration disorders are often identified in patients with epilepsy refractory to medical treatment. The prolonged or repeated seizures are known to cause neuronal death; however, the mechanism underlying seizure-induced neuronal death remains to be elucidated. An essential role of cyclin-dependent kinase 5 (Cdk5) in brain development has been demonstrated in Cdk5(-/-) mice, which show neuronal migration defects and perinatal lethality. Here, we show the consequences of Cdk5 deficiency in the postnatal brain by generating Cdk5 conditional knockout mice, in which Cdk5is selectively eliminated from neurons in the developing forebrain. The conditional mutant mice were viable, but exhibited complex neurological deficits including seizures, tremors, and growth retardation. The forebrain not only showed disruption of layering, but also neurodegenerative changes accompanied by neuronal loss and microglial activation. The neurodegenerative changes progressed with age and were accompanied by up-regulation of the neuronal tissue-type plasminogen activator, a serine protease known to mediate microglial activation. Thus age-dependent neurodegeneration in the Cdk5 conditional knockout mouse brain invoked a massive inflammatory reaction. These findings indicate an important role of Cdk5 in inflammation, and also provide a mouse model to examine the possible involvement of inflammation in the pathogenesis of progressive cognitive decline in patients with neuronal migration disorders.

Molecular Roles of Cdk5 in Pain Signaling.

Injury and inflammation trigger activation of several critical cellular pathways in nociceptive signaling in the peripheral nervous system, but their precise molecular mechanisms have not been clearly defined. Cyclin-dependent kinase 5 (Cdk5), a serine/threonine kinase, is mainly expressed in the post-mitotic neurons, and has many important roles in the development, functions and pathophysiology of diseases of the nervous system. Although many functional roles of Cdk5 have been identified in neurons, its precise role in pain signaling has not been well determined. Experimental inflammation in the hind paws of mice resulted in increased mRNA and protein levels of Cdk5 and its activator p35, as well as the Cdk5 activity in nociceptive neurons (Pareek et al., 2006). Furthermore, we also identified that Cdk5 phosphorylates transient receptor potential vanilloid 1 (TRPV1), a key receptor that modulates agonist-induced calcium influx in the neurons (Pareek et al., 2007). We subsequently demonstrated that inflammation triggers increase in Cdk5 activity through activation of early growth response 1 (Egr-1) and p35 expression by tumor necrosis factor alpha (TNF-α) (Utreras et al., 2009). These findings suggest that Cdk5 plays an important role in pain signaling and therefore Cdk5 and its activators are potentially important drug targets for development of novel analgesics to treat neuropathic pain.

Neurofilament tail phosphorylation: identity of the RT-97 phosphoepitope and regulation in neurons by cross-talk among proline-directed kinases.

As axons myelinate, establish a stable neurofilament network, and expand in caliber, neurofilament proteins are extensively phosphorylated along their C-terminal tails, which is recognized by the monoclonal antibody, RT-97. Here, we demonstrate in vivo that RT-97 immunoreactivity (IR) is generated by phosphorylation at KSPXK or KSPXXXK motifs and requires flanking lysines at specific positions. extracellular signal regulated kinase 1,2 (ERK1,2) and pERK1,2 levels increase in parallel with phosphorylation at the RT-97 epitope during early postnatal brain development. Purified ERK1,2 generated RT-97 on both KSP motifs on recombinant NF-H tail domain proteins, while cdk5 phosphorylated only KSPXK motifs. RT-97 epitope generation in primary hippocampal neurons was regulated by extensive cross-talk among ERK1,2, c-Jun N-terminal kinase 1,2 (JNK1,2) and cdk5. Inhibition of both ERK1,2 and JNK1,2 completely blocked RT-97 generation. Cdk5 influenced RT-97 generation indirectly by modulating JNK activation. In mice, cdk5 gene deletion did not significantly alter RT-97 IR or ERK1,2 and JNK activation. In mice lacking the cdk5 activator P35, the partial suppression of cdk5 activity increased RT-97 IR by activating ERK1,2. Thus, cdk5 influences RT-97 epitope generation partly by modulating ERKs and JNKs, which are the two principal kinases regulating neurofilament phosphorylation. The regulation of a single target by multiple protein kinases underscores the importance of monitoring other relevant kinases when the activity of a particular one is blocked.