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Cyclin dependent kinase 5 (Cdk5) is a key neuronal kinase whose activity can modulate thermo-, mechano-, and chemo-nociception. Cdk5 can modulate nociceptor firing by phosphorylating pain transducing ion channels like the transient receptor potential vanilloid 1 (TRPV1), a thermoreceptor that is activated by noxious heat, acidity, and capsaicin. TRPV1 is phosphorylated by Cdk5 at threonine-407 (T407), which then inhibits Ca2+ dependent desensitization. To explore the in vivo implications of Cdk5-mediated TRPV1 phosphorylation on pain perception, we engineered a phospho-null mouse where we replaced T407 with alanine (T407A). The T407A point mutation did not affect the expression of TRPV1 in nociceptors of the dorsal root ganglia and trigeminal ganglia (TG). However, behavioral tests showed that the TRPV1T407A knock-in mice have reduced aversion to oral capsaicin along with a trend towards decreased facial displays of pain after a subcutaneous injection of capsaicin into the vibrissal pad. In addition, the TRPV1T407A mice display basal thermal hypoalgesia with increased paw withdrawal latency while tested on a hot plate. These results indicate that phosphorylation of TRPV1 by Cdk5 can have important consequences on pain perception, as loss of the Cdk5 phosphorylation site reduced capsaicin- and heat-evoked pain behaviors in mice. [ABSTRACT FROM AUTHOR]

Abstract Background We have previously reported that cyclin-dependent kinase 5 (Cdk5) participates in the regulation of nociceptive signaling. Through activation of the ERK1/2 pathway, Tumor Necrosis Factor-α (TNF-α) induces expression of Egr-1. This results in the sustained and robust expression of p35, a coactivator of Cdk5, in PC12 cells, thereby increasing Cdk5 kinase activity. The aim of our present study was to test whether resveratrol, a polyphenolic compound with known analgesic activity, can regulate Cdk5/p35 activity. Results Here we used a cell-based assay in which a p35 promoter-luciferase construct was stably transfected in PC12 cells. Our studies demonstrate that resveratrol inhibits p35 promoter activity and also blocks the TNF-α mediated increase in Cdk5 activity in PC12 cells. Resveratrol also inhibits p35 expression and blocks the TNF-α mediated increase in Cdk5 activity in DRG neurons. In the presence of resveratrol, the MEK inhibitor decreased p35 promoter activity, whereas the inhibitors of p38 MAPK, JNK and NF-κB increased p35 promoter activity, indicating that these pathways regulate p35 expression differently. The TNF-α-mediated increase in Egr-1 expression was decreased by resveratrol treatment with a concomitant reduction in p35 expression and protein levels, resulting in reduced Cdk5 kinase activity. Conclusions We demonstrate here that resveratrol regulates p35 promoter activity in PC12 cells and DRG neurons. Most importantly, resveratrol blocks the TNF-α-mediated increase in p35 promoter activity, thereby reducing p35 expression and subsequent Cdk5 kinase activity. This new molecular mechanism adds to the known analgesic effects of resveratrol and confirms the need for identifying new analgesics based on their ability to inhibit Cdk5 activity for effective treatment of pain.

Cdk5 is a key neuronal kinase necessary for proper brain development, which has recently been implicated in modulating nociception. Conditional deletion of Cdk5 in pain-sensing neurons attenuates pain responses to heat in both the periphery and orofacial regions. Cdk5 activity is regulated by binding to the activators p35 and p39, both of which possess a cyclin box. Our previous examination of the nociceptive role of the well-characterized Cdk5 activator p35 using mice that either lack or overexpress this regulatory subunit demonstrated that Cdk5/p35 activity affects mechanical, chemical, and thermal nociception. In contrast, the nociceptive role of Cdk5's other less-studied activator p39 is unknown. Here, we report that the knockout of p39 in mice did not affect orofacial and peripheral nociception. The lack of any algesic response to nociceptive stimuli in the p39 knockout mice contrasts with the hypoalgesic effects that result from the deletion of p35. Our data demonstrate different and nonoverlapping roles of Cdk5 activators in the regulation of orofacial as well as peripheral nociception with a crucial role for Cdk5/p35 in pain signaling. [ABSTRACT FROM AUTHOR]

Background Cyclin-dependent kinase 5 (Cdk5) is essential for brain development and function, and its deregulated expression is implicated in some of neurodegenerative diseases. We reported earlier that the forebrain-specific Cdk5 conditional knockout (cKO) mice displayed an early lethality associated with neuroinflammation, increased expression of the neuronal tissue-type plasminogen activator (tPA), and neuronal migration defects. Methods In order to suppress neuroinflammation in the cKO mice, we first treated these mice with pioglitazone, a PPARγ agonist, and analyzed its effects on neuronal loss and longevity. In a second approach, to delineate the precise role of tPA in neuroinflammation in these mice, we generated Cdk5 cKO; tPA double knockout (dKO) mice. Results We found that pioglitazone treatment significantly reduced astrogliosis, microgliosis, neuronal loss and behavioral deficit in Cdk5 cKO mice. Interestingly, the dKO mice displayed a partial reversal in astrogliosis, but they still died at early age, suggesting that the increased expression of tPA in the cKO mice does not contribute significantly to the pathological process leading to neuroinflammation, neuronal loss and early lethality. Conclusion The suppression of neuroinflammation in Cdk5 cKO mice ameliorates gliosis and neuronal loss, thus suggesting the potential beneficial effects of the PPARγ agonist pioglitazone for the treatment for neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

The mechanisms underlying facial pain are still incompletely understood, posing major therapeutic challenges. Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase involved in pain signaling. However, the regulatory roles of Cdk5 in facial pain signaling and the possibility of therapeutic intervention at the level of mouse trigeminal ganglion primary neurons remain elusive. In this study, we use optimized intravital imaging to directly compare trigeminal neuronal activities after mechanical, thermal, and chemical stimulation. We then test whether facial inflammatory pain in mice could be alleviated by the Cdk5 inhibitor peptide TFP5. We demonstrate regulation of total Ca2+ intensity by Cdk5 activity using transgenic and knockout mouse models. In mice with vibrissal pad inflammation, application of TFP5 specifically decreases total Ca2+ intensity in response to noxious stimuli. It also alleviates inflammation-induced allodynia by inhibiting activation of trigeminal peripheral sensory neurons. Cdk5 inhibitors may provide promising non-opioid candidates for pain treatment. [Display omitted] • Characterization of calcium signaling in trigeminal ganglion using in vivo live imaging • Cdk5 activity regulates trigeminal peripheral neuronal pain signaling • Elevated Cdk5 activity increases the proportion of polymodal nociceptors • A Cdk5 inhibitor alleviates inflammation-induced trigeminal hyperalgesia signaling By using intravital live imaging to record mouse trigeminal ganglion neuronal activities in response to facial pain, Hu et al. demonstrate that Cdk5 regulates primary sensory neuronal pain signaling. In addition, a Cdk5 peptide inhibitor can suppress inflammation-induced pain signaling at the peripheral nervous system level. [ABSTRACT FROM AUTHOR]

Background: Odontoblasts are specialized cells that form dentin and they are believed to be sensors for tooth pain. Transforming growth factor-β1 (TGF-β1), a pro-inflammatory cytokine expressed early in odontoblasts, plays an important role in the immune response during tooth inflammation and infection. TGF-β1 is also known to participate in pain signaling by regulating cyclin-dependent kinase 5 (Cdk5) in nociceptive neurons of the trigeminal and dorsal root ganglia. However, the precise role of TGF-β1 in tooth pain signaling is not well characterized. The aim of our present study was to determine whether or not in odontoblasts Cdk5 is functionally active, if it is regulated by TGF-β1, and if it affects the downstream pain receptor, transient receptor potential vanilloid-1 (TRPV1). Results: We first determined that Cdk5 and p35 are indeed expressed in an odontoblast-enriched primary preparation from murine teeth. For the subsequent analysis, we used an odontoblast-like cell line (MDPC-23) and found that Cdk5 is functionally active in these cells and its kinase activity is upregulated during cell differentiation. We found that TGF-β1 treatment potentiated Cdk5 kinase activity in undifferentiated MDPC-23 cells. SB431542, a specific inhibitor of TGF-β1 receptor 1 (Tgfbr1), when co-administered with TGF-β1, blocked the induction of Cdk5 activity. TGF-β1 treatment also activated the ERK1/2 signaling pathway, causing an increase in early growth response-1 (Egr-1), a transcription factor that induces p35 expression. In MDPC-23 cells transfected with TRPV1, Cdk5-mediated phosphorylation of TRPV1 at threonine-407 was significantly increased after TGF-β1 treatment. In contrast, SB431542 co-treatment blocked TRPV1 phosphorylation. Moreover, TGF-β1 treatment enhanced both proton- and capsaicin-induced Ca2+ influx in TRPV1-expressing MDPC-23 cells, while co-treatment with either SB431542 or roscovitine blocked this effect. Conclusions: Cdk5 and p35 are expressed in a murine odontoblast-enriched primary preparation of cells from teeth. Cdk5 is also functionally active in odontoblast-like MDPC-23 cells. TGF-β1 sensitizes TRPV1 through Cdk5 signaling in MDPC-23 cells, suggesting the direct involvement of odontoblasts and Cdk5 in dental nociceptive pain transduction. [ABSTRACT FROM AUTHOR]

Background Cyclin-dependent kinase 5 (Cdk5) is a unique member of the serine/threonine kinase family. This kinase plays an important role in neuronal development and deregulation of its activity leads to neurodegenerative disorders. Cdk5 also serves an important function in the regulation of nociceptive signaling. Our previous studies revealed that the expression of Cdk5 and its activator, p35, is upregulated in nociceptive neurons during peripheral inflammation. The aim of the present study was to characterize the involvement of Cdk5 in orofacial pain. Since mechanical hyperalgesia is the distinctive sign of many orofacial pain conditions, we adapted an existing orofacial stimulation test to assess the behavioral responses to mechanical stimulation in the trigeminal region of the transgenic mice with either reduced or increased Cdk5 activity. Results Mice overexpressing or lacking p35, an activator of Cdk5, showed altered phenotype in response to noxious mechanical stimulation in the trigeminal area. Mice with increased Cdk5 activity displayed aversive behavior to mechanical stimulation as indicated by a significant decrease in reward licking events and licking time. The number of reward licking/facial contact events was significantly decreased in these mice as the mechanical intensity increased. By contrast, mice deficient in Cdk5 activity displayed mechanical hypoalgesia. Conclusions Collectively, our findings demonstrate for the first time the important role of Cdk5 in orofacial mechanical nociception. Modulation of Cdk5 activity in primary sensory neurons makes it an attractive potential target for the development of novel analgesics that could be used to treat multiple orofacial pain conditions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

At the lateral wall of the lateral ventricles in the adult rodent brain, neuroblasts form an extensive network of elongated cell aggregates called chains in the subventricular zone and migrate toward the olfactory bulb. The molecular mechanisms regulating this migration of neuroblasts are essentially unknown. Here, we report a novel role for cyclin-dependent kinase 5 (Cdk5), a neuronal protein kinase, in this process. Using in vitro and in vivo conditional knock-out experiments, we found that Cdk5 deletion impaired the chain formation, speed, directionality, and leading process extension of the neuroblasts in a cell-autonomous manner. These findings suggest that Cdk5 plays an important role in neuroblast migration in the postnatal subventricular zone. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinase 5 (cdk5) is a serine/threonine kinase activated by associating with its neuron-specific activators p35 and p39. Analysis of cdk5-/- and p35-/- mice has demonstrated that both cdk5 and p35 are essential for neuronal migration, axon pathfinding and the laminar configuration of the cerebral cortex, suggesting that the cdk5-p35 complex may play a role in neuron survival. However, the targets of cdk5 that regulate neuron survival are unknown. Here, we show that cdk5 directly phosphorylates c-Jun N-terminal kinase 3 (JNK3) on Thr131 and inhibits its kinase activity, leading to reduced c-Jun phosphorylation. Expression of cdk5 and p35 in HEK293T cells inhibits c-Jun phosphorylation induced by UV irradiation. These effects can be restored by expression of a catalytically inactive mutant form of cdk5. Moreover, cdk5-deficient cultured cortical neurons exhibit increased sensitivity to apoptotic stimuli, as well as elevated JNK3 activity and c-Jun phosphorylation. Taken together, these findings show that cdk5 may exert its role as a key element by negatively regulating the c-Jun N-terminal kinase/stress-activated protein kinase signaling pathway during neuronal apoptosis. [ABSTRACT FROM AUTHOR]

The mammalian cerebral cortex consists of six layers that are generated via coordinated neuronal migration during the embryonic period. Recent studies identified specific phases of radial migration of cortical neurons. After the final division, neurons transform from a multipolar to a bipolar shape within the subventricular zone-intermediate zone (SVZ-IZ) and then migrate along radial glial fibres. Mice lacking Cdk5 exhibit abnormal corticogenesis owing to neuronal migration defects. When we introduced GFP into migrating neurons at E14.5 by in utero electroporation, we observed migrating neurons in wild-type but not in Cdk5-/- embryos after 3-4 days. Introduction of the dominant-negative form of Cdk5 into the wild-type migrating neurons confirmed specific impairment of the multipolar-to-bipolar transition within the SVZ-IZ in a cell-autonomous manner. Cortex-specific Cdk5 conditional knockout mice showed inverted layering of the cerebral cortex and the layer V and callosal neurons, but not layer VI neurons, had severely impaired dendritic morphology. The amount of the dendritic protein Map2 was decreased in the cerebral cortex of Cdk5-deficient mice, and the axonal trajectory of cortical neurons within the cortex was also abnormal. These results indicate that Cdk5 is required for proper multipolar-to-bipolar transition, and a deficiency of Cdk5 results in abnormal morphology of pyramidal neurons. In addition, proper radial neuronal migration generates an inside-out pattern of cerebral cortex formation and normal axonal trajectories of cortical pyramidal neurons. [ABSTRACT FROM AUTHOR]

We reported earlier that TNF-α, a proinflammatory cytokine implicated in many inflammatory disorders causing orofacial pain, increases the activity of Cdk5, a key kinase involved in brain development and function and recently found to be involved in pain signaling. To investigate a potential mechanism underlying inflammatory pain in trigeminal ganglia (TGs), we engineered a transgenic mouse model (TNF) that can conditionally overexpresses TNF-α upon genomic recombination by Cre recombinase. TNF mice were bred with Nav1.8-Cre mouse line that expresses the Cre recombinase in sensory neurons to obtain TNF-α:Nav1.8-Cre (TNF-α cTg) mice. Although TNF-α cTg mice appeared normal without any gross phenotype, they displayed a significant increase in TNF-α levels after activation of NFκB signaling in the TG. IL-6 and MCP-1 levels were also increased along with intense immunostaining for Iba1 and GFAP in TG, indicating the presence of infiltrating macrophages and the activation of satellite glial cells. TNF-α cTg mice displayed increased trigeminal Cdk5 activity, and this increase was associated with elevated levels of phospho-T407-TRPV1 and capsaicin-evocated Ca influx in cultured trigeminal neurons. Remarkably, this effect was prevented by roscovitine, an inhibitor of Cdk5, which suggests that TNF-α overexpression induced sensitization of the TRPV1 channel. Furthermore, TNF-α cTg mice displayed more aversive behavior to noxious thermal stimulation (45°C) of the face in an operant pain assessment device as compared with control mice. In summary, TNF-α overexpression in the sensory neurons of TNF-α cTg mice results in inflammatory sensitization and increased Cdk5 activity; therefore, this mouse model would be valuable for investigating the mechanism of TNF-α involved in orofacial pain. [ABSTRACT FROM AUTHOR]