Characterization of Molecular Pathways in Chronic Pain Conditions

National Institute Of Dental & Craniofacial Research

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Abstract

Our lab has acquired DRGs from patients that have been impacted with chronic pain, which affects over 100 million people in the United States. Chronic pain can be difficult to treat with most patients finding even opioids to be ineffective. To study the cellular mechanisms that can lead to chronic pain, we acquired DRGs from patients exhibiting painful diabetic neuropathy (DPN). DPN commonly 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 DPN. 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 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. Again, mouse models will be used to validate some of the findings discovered in the human studies. Through our past work with genetically engineered mice, our lab has shown that the activity of the key neuronal enzyme cyclin dependent kinase 5 (Cdk5) is important in regulating of pain hypersensitivity. Cdk5 activity has been shown to have important roles in neurodevelopment and neurophysiology, but aberrant kinase activity has been implicated in neurodegenerative diseases and in cancer. Because of Cdk5's important role in neurological functions such as neurotransmitter release, behavior, and addiction, we wanted to see if Cdk5 activity was also involved in pain. We demonstrated in mice that inflammation causes increased Cdk5 enzymatic activity in nociceptors, both within DRG neurons that innervate the periphery as well as within TG neurons that respond to orofacial inflammation. We then tested the pain responses of genetically engineered mice exhibiting either Cdk5 hyperactivity or decreased Cdk5 activity. Our behavior testing showed that Cdk5 activity modulates thermo-, mechano-, and chemo- nociception in mice, where increased Cdk5 activity promotes hyperalgesia to various noxious stimuli while decreased Cdk5 activity conversely results in hypoalgesia. Essentially, inflammation can induce Cdk5 activity that will, in turn, promote increased pain hypersensitivity, while inhibitors of Cdk5 may have analgesic properties, as suggested in our behavioral studies. Our lab has next identified three key pain transducing ion channels that are affected by Cdk5 activity. Cdk5 phosphorylates both TRPV1, a thermosensitive ion channels that is activated by noxious heat and acidity, along with another TRP channel, TRPA1, which functions as a chemosensor to detect the presence of noxious chemicals in the environment such as the pungent plant defensive compounds found in mustard oil and cinnamon. In addition, Cdk5 also phosphorylates P2X2a, an ion channel that detects cell damage. By phosphorylating these pain transducing ion channels, Cdk5 then modulates their activity that, in turn, plays a part in causing nociceptor hypersensitivity to heat, noxious chemicals, and tissue injury. To further our pain studies involving Cdk5, we also wanted to visualize and record the nociceptor firing of TG neurons of mice in response to painful stimuli. 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. Currently, we are using a genetically encoded calcium indicator that is transgenically expressed only in pain sensing neurons. Then, we image neuronal responses to both noxious (i.e., heat) and non-noxious (i.e., light brush) stimuli in our genetically engineered mice. This technique is based on a modified green fluorescent protein that will only fluoresce in the presence of calcium. 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 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 complete Freunds adjuvant (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. 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. Our lab will continue to develop and analyze new mouse models to examine pain. We also plan to use data acquired from human patients with chronic pain to identify new avenues of pain signaling research. Lastly, we intend to examine if inhibiting Cdk5 activity may have a therapeutic effect that will attenuate chronic pain.