Michael J. Caterina - US grants

DESCRIPTION (provided by applicant): Acute and chronic pain represent significant and under-treated health problems in the U.S. due, in part, to our incomplete understanding of the mechanisms by which the peripheral nervous system detects noxious stimuli. TRPV1 is a heat-gated ion channel required for some, but not all aspects of heat-evoked pain. Three related channels, TRPV2, TRPV3, and TRPV4, can also be activated by elevated temperatures and therefore may contribute to the detection of painful heat. TRPV2 is activated at very high temperatures (>52 degrees C) and expressed most highly in a subset of sensory neurons. TRPV3 and TRPV4 are activated at temperatures >32 degrees C. In the skin, TRPV3 and TRPV4 expression is most prominent in epithelial keratinocytes, raising the possibility that these cells participate in an indirect mode of thermosensation involving TRPV3 and TRPV4. TRPV2 and TRPV4 can also be activated by cell swelling, suggesting that they may participate in mechanosensation. This proposal is aimed at achieving the following goals: (1) To determine whether and how TRPV2 and TRPV4 contribute to the detection of painful and nonpainful thermal and mechanical stimuli. (2) To determine whether keratinocyte TRPV3 and TRPV4 contribute to noxious and/or innocuous thermosensation and mechanosensation. (3) To determine how keratinocytes communicate the presence of thermal stimuli to adjacent sensory neurons. To achieve these goals, TRPV2 and TRPV4 null mutant mice will be analyzed for acute responsiveness to mechanical and thermal stimuli and for enhancement of thermo- or mechanosensitivity following inflammation or nerve injury. Wild-type or dominant negative forms of TRPV3 or TRPV4 will be overexpressed selectively in keratinocytes and the effects on thermosensation evaluated behaviorally. Finally, biochemical electrophysiological, fluorescent calcium imaging, and behavioral methods will be used to identify molecules through which heat-exposed keratinocytes communicate with nearby sensory neurons.

[unreadable] DESCRIPTION (provided by applicant): The capsaicin receptor, TRPV1, is one of four cation channels of the transient receptor potential vanilloid (TRPV) subfamily that can be activated by warm or painfully hot temperatures. Several of these channels (TRPV1, TRPV3, TRPV4) have been shown to participate in heat-evoked pain sensation in mice. We recently found that heat-evoked activation of TRPV3 results in a biphasic current response that reflects an initial channel block by divalent cations and subsequent removal of that block by persistent stimulation. This loss of block is dependent on channel density, proceeds synchronously, once initiated, and is associated with increased permeability to the large organic cation N-methyl-d-glucamine (NMDG). Dynamic ion selectivity in TRPV channels could have implications for neurotransmitter release, pain sensation, and capsaicin neurbtoxicity, among other processes. We therefore propose to test the hypotheses that these changes in TRPV3 function reflect progressive heat-evoked dilation of the channel pore, and that TRPV1 also exhibits pore dilation in response to strong chemical or thermal activation. In addition, we will determine whether pore dilation occurs in native TRPV1 and TRPV3 expressed in sensory neurons and keratinocytes, respectively. Using electrophysiological and fluorescence microscopy approaches, we will examine the mechanistic basis of TRPV1 and TRPV3 pore dilation and explore the relationship between agonist-evoked pore dilation and known or presumed mechanisms of TRPV1 and TRPV3 regulation, such as phosphorylation and phospholipid binding. Finally, we will seek to identify amino acids that are selectively important for agonist-evoked pore dilation in TRPV1 and TRPV3 via systematic mutagenesis of residues within the domains adjacent to the selectivity filter. Mutants arising from this screen will be further examined for their abilities to mediate potential "downstream" effects of pore dilation, including changes in cell morphology, cell death, and neurotransmitter/cytokine release in native and non-native cellular contexts. These studies will allow us to determine whether and how the ion selectivities of TRPV1 and TRPV3 are regulated and may provide a rational basis for the development of either anti-dilation antagonists or dilation- promoting agonists, for the treatment of chronic pain. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Inadequate treatment of pain imposes an enormous burden on society, and is due, in part, to a limited understanding of biological events that underlie the transition from acute to chronic pain. Barriers to a better understanding of this transition have included an inability to selectively and efficiently visualize or manipulate specifc sensory neuron populations and the very low throughput of available methods to monitor nociceptor function at the cellular level. In this collaborative proposal, we have teamed together a developmental neurobiologist and several pain biologists to synergistically overcome both of these barriers. Using newly- developed molecular-genetic strategies, we will selectively label each of the four major subpopulations of low- threshold mechanoreceptive (LTMR) neurons, as well as peptidergic and MrgprD-expressing nonpeptidergic nociceptors, in mice. This technology will be combined with selective expression of the genetically encoded calcium indicator, GCAMP3, in mouse peripheral sensory neurons to permit direct visualization of neuronal activity in the skin and DRG. In our first two aims, these tools will allow us to efficiently and with high spatiotemporal resolution monitor changes in the anatomy and function of unambiguously defined nociceptor and LTMR populations during the development of neuropathic pain. These changes will be correlated with corresponding behavioral changes, monitored using classical and newly developed assays of thermal and mechanical sensitivity. In Aim 3, we will selectively ablate two LTMR populations that are candidate participants in pain and define their respective contributions to the establishment, maintenance, and manifestation of neuropathic mechanical hypersensitivity. Together, these studies will provide us with an unprecedented view of the dynamic processes associated with the transition to chronic pain and define cellular targets for the development of improved analgesic therapies. PUBLIC HEALTH RELEVANCE: Chronic pain affects millions of people and is often difficult to treat. This is due, in part, to our poor understanding of why some people, but not others, transition to a state of chronic pain after nerve injury. In this proposal, we will take advantage f new technology to directly visualize specific classes of nerves in the skin to understand how changes in their structure and function after nerve injury enhances the perception of pain. This will allow the rational development of improved therapies to treat chronic pain.

DESCRIPTION (provided by applicant): Chronic pain is a significant public health problem for which current therapies are inadequate. The development of improved treatments for pain will require a better understanding of the fundamental mechanisms by which nociceptive neurons are activated or inhibited. A growing body of evidence suggests that activation of skin keratinocytes by thermal, chemical, or mechanical stimuli could result in activation or inhibition of adjacent epidermal nocieptor terminals, and that such signaling might exhibit specificity among basal vs. superficial keratinocyte layers. A major hurdle to addressing these possibilities definitively is the lack of a means of selectively stimulating keratinocytes without simultaneously stimulating nociceptors, themselves. In this proposal, we outline a plan to selectively express, within transgenic mouse keratinocytes, ion channel receptors (TRPV1 and channelrhodopsin) that can be uniquely activated by chemical and/or light stimuli. Activation of these receptors in the intact mouse will allow us to determine whether keratinocyte stimulation is sufficient to trigger pain-related behaviors and signaling to the spinal cord. Furthermore, by precisely regulating the expression patterns of these channels in discrete epidermal layers, we will be able to establish how the consequences of signaling from keratinocytes differ across the basal to apical spectrum of the epidermis. Success in these aims will provide us with a clearer understanding of the role of keratinocytes in the initiation and control of pain, as well as powerfl new tools that will in the future allow us to guide the rational development of improved therapies for pain.

Neuropathic pain is a poorly treated medical condition of enormous public health importance. One mechanism by which nerve injury leads to neuropathic pain is by altering the expression of numerous proteins that regulate neuronal excitability. MicroRNAs (miRNAs) are key modulators of protein synthesis, and nerve injury alters the neuronal expression of many individual miRNAs. miRNAs function by reducing the stability and/or translation of target messenger RNAs (mRNAs) that contain binding sequences with partial complementarity to the miRNAs. A single miRNA can thereby repress the synthesis of many proteins. However, miRNAs themselves are coordinately regulated by upstream control pathways. One such pathway involves Lin28a and Lin28b, RNA binding proteins that selectively and coordinately suppress the biogenesis of the Let-7 family of miRNAs, which are amongst the most abundant miRNAs in differentiated tissues. Since many Let-7 miRNA targets encode proteins involved in growth and regeneration, increased Lin28 signaling promotes pro-growth programs of protein synthesis. Indeed, Let-7 miRNAs have been implicated as possible regulators of axon growth and nerve regeneration. Yet, the directions and cell type specificity of these effects remain unclear, as does the potential involvement of Lin28. Furthermore, the possibility that the Lin28/Let-7 pathway contributes to the severity or duration of neuropathic pain remains entirely unexplored. Here, we outline plans to directly tackle these questions, through a multidisciplinary approach. In Aim 1, we will map the spatiotemporal patterns and cell type specificity of changes in the expression of Let-7 miRNAs and Lin28 in three complementary mouse models of neuropathic pain. In Aim 2, we will utilize mouse genetics and adeno- associated virus mediated gene transfer, along with behavioral and in vivo electrophysiological approaches, to increase or ablate Let-7 miRNAs or Lin28 protein expression selectively in peripheral neurons or glia, to assess the functional importance of the Lin28/Let-7 pathway to the initiation, maintenance, and reversal of neuropathic pain. Finally, in Aim 3, we will combine bioinformatics and proteomic approaches to define the programs of altered protein expression following nerve injury that depend upon the Lin28/Let-7 pathway, and that might underlie the contributions of this pathway to neuropathic pain. Together, these studies will help to define the roles of the Lin28/Let-7 pathway in neuropathic pain and provide important information regarding when, where, and in what cell type this master regulatory pathway might be therapeutically targeted to alleviate pain.