Cdk5-mediated phosphorylation and neuronal functions

点击翻译按钮获取中文摘要

Cdk5 and nociception: In general nociceptive (painful) neural signals originate from sensory neurons in peripheral tissues and are transmitted to second-order neurons in the spinal cord, which then convey the message to specific nuclei in the brain for perception of pain. Nociception results from the activation of molecular and cellular mechanisms in damaged tissue, sensory neurons, and the spinal cord. Many cellular pathways have been implicated in nociceptive signaling, but their precise molecular mechanisms have not been clearly defined. Abnormalities in molecular pathways underlying nociceptive processes may result in chronic pain conditions. It follows that a detailed characterization of these pathways is necessary for developing effective strategies to treat pain. Signal transduction mechanisms activated by nociceptive stimuli have not been fully characterized. Recent advances, especially the cloning and characterization of the capsaicin receptor (also known as vanilloid receptor 1, VR1, TRPV1) remarkably improved our understanding of signal transduction in nociceptive neurons. TRPV1 is a member of the transient receptor potential (TRP) family. It is a polymodal ligand-gated ion channel that is expressed in small-diameter sensory neurons (C-fibers) and activated by heat, protons, anandamide, and leukotrienes. TRPV1 knockout mice show reduced thermal hyperalgesia following inflammation and reduced nociceptive responsiveness in a model of bone cancer pain. TRPV1 has been connected to a broad spectrum of physiological conditions and diseases such as thermal hyperalgesia, allodynia, inflammatory bowel disease, Crohn's disease, vulvodynia, osteoarthritis, pancreatitis, gastroesophageal reflux disease, bladder disease, cystitis, and asthma. There is mounting evidence that TRPV1 is subject to multiple interacting levels of control. Phosphorylation of TRPV1 is critical for its function in response to nociceptive stimuli. We previously described the expression and specific activity of Cdk5/p35 in TRPV1-positive cells of dorsal root ganglia (DRG) and trigeminal ganglia (TG) and the modulation of Cdk5 activity in response to peripheral inflammatory pain. We also found that p35 knockout mice, which have significantly reduced Cdk5 activity, behaviorally mimic the TRPV1 knockout pain phenotype. Cdk5, a neuron-specific, proline-directed serine/threonine kinase is activated by 2 noncyclin activators, p35 and/or p39. Cdk5 phosphorylates serine and threonine immediately upstream of a proline residue. In addition to an absolute requirement for proline in the +1 position, Cdk5 shows a marked preference for a basic residue in the +3 position and phosphorylates the consensus sequence (S/T)PX(K/H/R). Several neuronal and nonneuronal substrates are phosphorylated by Cdk5, and the list of new substrates is increasing. Earlier studies showed that Cdk5 knockout and p35/p39 double-knockout mice are embryonically lethal with neuronal migration defects, whereas p35 knockout mice show inverted neuronal layering with a concurrent attenuated response to noxious heat; conversely mice overexpressing p35 were hyperalgesic. Subsequently, we analyzed TRPV1 for potential phosphorylation by Cdk5. We report that Cdk5 can directly phosphorylate TRPV1 at threonine 407, and this in turn modulates agonist-induced calcium influx. We initially found that inhibiting Cdk5 activity resulted in attenuation of capsaicin-induced calcium influx in cultured DRG neurons, and this attenuation was reversible. These observations suggest that Cdk5-mediated phosphorylation of TRPV1 is important for capsaicin-mediated calcium influx through this receptor. Since germline Cdk5 knockout mice are embryonically lethal and p35 knockout mice present various neuronal disorders, we generated primary nociceptor-specific Cdk5 conditional knockout (Cdk5-CoKO) mice to identify the precise role of Cdk5 in primary afferent pain signaling. In the basal state, the conditional knockout mice showed significant hypoalgesia, confirming the direct role of normal Cdk5 activity in primary afferent. Collectively, our findings describe a novel molecular mechanism for the functional regulation of TRPV1 by Cdk5 and provide further insights into the role of Cdk5 in the pain pathway. Cdk5 and brain development: Analysis of functional roles of Cdk5 in postnatal brain development has been hampered because of perinatal lethality of Cdk5-/- mice and the compensated phenotype of p35-/- mice. To study the role of Cdk5 in postnatal development of the midbrain-hindbrain (MHB), we generated MHB-specific Cdk5 conditional knockout mice by crossing Cdk5 floxed mice with Wnt1-Cre mice. Wnt1-Cre-mediated Cdk5 conditional knockout (WCOKO) mice have lower body weight than controls, an ataxic gait, and early postnatal lethality. Histological analysis indicated a smaller cerebellum with defective migration of the Purkinje cells. A detailed analysis of WCOKO mice showed a complete lack of inward migration of the granule cells. In addition, we also identified a complete absence of the superior colliculus in Cdk5-/- mice and its abnormal development in WCOKO mice. These results indicate that Cdk5 plays important roles in mouse MHB development.