Characterization of Molecular Pathways in Chronic Pain Conditions

National Institute Of Dental & Craniofacial Research

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Abstract

Our lab acquired DRGs from donors with chronic pain, specifically individuals with familiar disorders of diabetic painful neuropathy (DPN) and rheumatoid arthritis (RA). Although, we only have a small number of samples, our studies provide an initial understanding about the development of chronic pain in humans. Diabetic painful neuropathy, a chronic neuropathic pain condition: Neuropathic pain is thought to account for about a fifth of the cases of chronic pain. Neuropathic pain tends to be more refractory than non-neuropathic pain to conventional analgesics, such as non-steroidal anti-inflammatory drugs and opioids. In particular, the most common form of neuropathic pain arises from Type 2 diabetes mellitus (T2DM), where metabolic imbalances such as hyperglycemia causes damage in sensory neurons. To gain a better understanding of neuropathic pain, our lab was foremost in obtaining hDRGs from individuals with painful diabetic neuropathy (DPN) for biochemical study. DPN is considered a length-dependent neuropathy, where distal extremities such as the hands and feet are predominantly affected by pain, so we analyzed L4, L5, and S1 DRGs as these ganglia contain the soma of the primary afferent neurons innervating the foot. Overall, using our DRG samples, we were able to use a multiomic approach to analyzing DPN, including transcriptomic, metabolomic, and proteomic analysis. In 2022, we published two papers covering our findings, one on the transcriptome while the other focused on the metabolic and proteomic changes occurring due to DPN. With our transcriptomic studies, gene pathway analysis of the differentially expressed genes (DEGs) has expanded our understanding of the etiology of DPN by showing that there is an upregulation of genes in response to oxidative stress, a decrease in key mitochondrial genes, and an upregulated gene signature related to the processes of cell death. While there is neuronal loss observed in our DRG studies, it must be noted that the remaining neurons can be driven to hyperexcitability by damage signals from neighboring injured neurons as well as the subsequent immune response needed to clear cellular debris, meaning there can be ongoing pain signaling despite the reduced number of nociceptive neurons. We have essentially identified several candidate genes that may to contribute to neuropathy and/or pain by regulating pH, neuronal excitability, ER stress, etc. 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. Over two decades, our lab has been involved in understanding the role of candidate genes associated with craniofacial development and disease through either gene ablation or overexpression. In addition to above mentioned Cdk5 mouse models, we had previously developed mouse models to understand other aspects of oral health. Although we are currently no longer working directly on these mouse models, we still provide consultations and collaborate on appropriate research projects involving these transgenic and knockout lines.