Molecular Roles of Cdk5 in Neuronal Functions and Pain Signaling

National Institute Of Dental & Craniofacial Research

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Abstract

Our lab has been involved in creating engineered mouse models that mimic aspects of known painful orofacial disorders in order to understand pain signaling. Inflammation, in particular, is often the root cause in many instances of orofacial pain. We, therefore, have used mouse models where we inject inflammatory agents such as carrageenan and complete Freunds adjuvant (CFA) into the mouse vibrissal pad. These mice show impaired masticatory function as measured by slower gnawing times, which can be an indicator of orofacial pain. In these mice, we have then detected increases of Cdk5 activity in the nociceptors of the TG and have studied aspects of activated immune responses in the central nervous system. To further our studies of orofacial pain, we wanted to visualize and record the nociceptor firing of TG neurons in response to painful stimuli. We first performed calcium imaging using dissociated cultured neurons from mice with genetically altered Cdk5 activity. As mentioned, we identified the pain transducing receptors TRPV1, TRPA1, and P2X2a as substrates of Cdk5, and, when activated, these ion channels open to allow both Na+ and Ca2+ to enter into a cell. This, in turn, causes the neuron to depolarize and leads to an action potential. The entry of Ca2+ into the cell can be fluorescently detected using calcium indicators. Unlike patch clamp recordings of an individual neuron, we were able to see how multiple neurons respond to a noxious stimulus. In this way, we were able to determine that Cdk5 activity can affect the number of neurons that fire upon application of mustard oil, an agonist of TRPA1. Currently, we are using genetically encoded calcium indicators to fluorescently track the firing of sensory TG neurons in vivo. This technique doesnt use a dye, but, instead, is based on a modified green fluorescent protein that will only fluoresce in the presence of calcium. The genetically encoded calcium indicator is expressed in primary afferent neurons and an epifluorescence microscope is positioned above the TG. This allows us to record neuronal responses to brush, heat, and capsaicin when applied facially onto a mouse. In particular, the Cdk5 substrate TRPV1 is activated by both heat and capsaicin, so this technique allows us to now visualize how modifying Cdk5 activity in mice may then affect the activation kinetics of this receptor in vivo. As such, mice with engineered Cdk5 hyperactivity showed both stronger neuronal responses and higher numbers of activated neurons to orofacial application of heat and capsaicin, while mice with a 80-90% decrease in Cdk5 activity showed the opposite effects. We then used the inflammatory agent CFA to induce Cdk5 activity and determine if a peptide inhibitor can reduce nociceptor activity. TFP5 is a 24 amino acid peptide that has been shown to reduce Cdk5 hyperactivity back to normal physiological levels. Either applied directly onto the TG or injected in mice, TFP5 was able to reduce neuronal hyperexcitability caused by injection of the inflammatory agent CFA. Additional mouse models of orofacial pain are being planned for future imaging experiments. To measure the pain sensitivity in our mouse models, our lab uses a battery of behavioral tests that record a mouses aversion to specific noxious stimuli. We are able to measure the behavior of our mice in response to painful stimuli both in the periphery as well as within the orofacial region. Initially, we relied on peripheral pain testing that mostly records reflex reactions such as paw and tail withdrawal to noxious thermal or mechanical stimuli. Currently, our lab also employs devices that measure orofacial pain, which are typically based on a conflict/reward paradigm. This allows the animals to make a choice whether to withdraw from an aversive stimulus or endure pain to achieve a reward. These behavioral tests additionally provide investigator-independent measurement of pain using automatically recorded behavior of the observed animals, so they incur less stress, and their behavior can be measured repeatedly in a non-biased fashion. Some of our orofacial pain testing involves a conflict/reward model where the mouse must stick their snout between either abrasive wires or thermodes set at noxious hot or cold temperatures to gain access to a desired sugary solution. We can also measure the drinking behavior of mice to water spiked with agonists to pain transducing ion channels, including capsaicin and mustard oil. Since orofacial pain can affect mastication, our lab has additionally acquired a device that measures gnawing behavior. Beyond just using mouse models to examine aspects of pain signaling, our lab has also acquired DRGs from patients that have been impacted with chronic pain, which affects over 100 million people in the United States. Chronic pain is generally defined as any pain lasting for more than three months. Chronic pain can be difficult to treat with most patients finding even opioids to be ineffective, which are presently the only major drug class used to specifically affect neuronal firing within the nervous system to alleviate pain signaling. For example, we acquired DRGs from patients exhibiting painful diabetic neuropathy, which results from systemic metabolic imbalances such as hyperglycemia that can be toxic to neurons. Pain is often considered the most bothersome symptom for patients with diabetic neuropathy, and is commonly described as sharp, burning, and tingling. DRGs from individuals with painful diabetic neuropathy underwent transcriptomic and histological evaluation to identify possible pathological mechanisms that could be producing pain hypersensitivity. The DRG itself is heterogeneous and is comprised of not just nociceptors, but also of accompanying glial, perivascular, and resident immune cells, where our transcriptomic analysis would identify gene expression changes within these cell types that may participate in the progression of painful neuropathy. An upregulation of many inflammatory markers was detected in the individuals with painful diabetic neuropathy. Some of these inflammatory pathways can have excitatory effects on DRG neurons that could possibly contribute to ongoing pain. Skin biopsies from patients with diabetic neuropathy often show that nociceptive neurons start dying-back even at early stages in the disease, and our transcriptomic analysis likewise shows an associated loss in neuronally related gene expression in the DRGs of individuals experiencing neuropathic pain. Sections of the DRGs were next histologically examined to look for any pathology that would corroborate our transcriptomic findings of increased inflammation combined with the decreased expression of neuronal genes. Indeed, neuronal loss was detected in 4 out of the 5 DPN subjects that ranged from mild to severe. Immunostaining also detected increases in the number of macrophages in the DRG of those with painful diabetic neuropathy. Macrophages are immune cells that, when activated, produce inflammatory mediators that can contribute to neuropathic pain. Further biochemical analyses will be conducted using these DRGs in order to better identify cellular changes that lead to pain hypersensitivity in patients with diabetic neuropathy. In addition, other painful disorders will be examined using DRGs from chronic pain patients. Lastly, mouse models be used to validate some of the findings discovered in the human studies. In summary, our lab has been interested in understanding various aspects of chronic pain, from developing new mouse models mimicking these painful conditions, to identifying a key neuronal enzyme that can modulate pain sensitivity, and with an analysis of gene expression changes from the DRGs of patients with chronic pain conditions.