Kathryn M. Albers - US grants

This application combines the expertise of three established laboratories in sensory biology to investigate the cellular, molecular and physiological aspects of age-related changes in the somatosensory system. The experiments address the goals of the Program Announcement (99-123) titled The Aging Senses: Relationships Among Multiple Sensory Systems. The long-term goal of this research is to identify the cellular mechanisms that lead to degeneration of sensory neurons and their end organs and determine whether modulating the level of trophic support provided by the skin can alleviate the onset and progression of age-related deficits in sensation. Sensations of touch, pain and temperature are transmitted by a variety of morphologically and electrophysiologically distinct mechanosensory endings located in the skin. These endings are comprised of sensory neurons and specialized end organs, e.g., Merkel complexes, Meissner corpuscles, that undergo degeneration as a consequence of aging. The loss of innervation causes reduced tactile sensitivity that can have significant impact on job performance and the quality of life for elderly individuals. Studies have shown that atrophy in the PNS is characterized by decreased neuron sensitivity and reduction in the integrity and/or number of peripheral terminals. We hypothesize these degenerative changes are related to growth factor signaling, in particular for the neurotrophins NT3 and BDNF. Studies of transgenic mice that overexpress NT3 or BDNF in the skin have shown they act in very specific manners to enhance sensory ending development, maintenance and physiological properties. In this project we propose to identify how NT3 and BDNF affect the morphological, cellular and physiological properties of sensory neurons using an ex vivo skin/nerve/spinal cord preparation. This preparation will be used to identify the physiological properties and chemical phenotype of cutaneous neurons of young and old mice and mice that overexpress either NT3 or BDNF in the skin. In this way we will define differences in sensitivity and response properties in relation to neuron phenotype and age, and determine how trophic factors alter these properties. To explore why neurons lose sensitivity in the aging system, we will analyze the expression of channel receptor proteins thought to mediate mechanosensation in physiologically characterized neurons of young and old mice and determine whether their expression is modulated in response to elevation in NT3 or BDNF expression. The experimental design will use immunocytochemistry, immunoblotting, RT-PCR and in situ hybridization to analyze RNA and protein expression, an ex vivo skin/nerve/spinal cord preparation for physiological analysis and transgenic mice that either constitutively or inducibly express increased levels of NT3 nr BDMF in the skin.

DESCRIPTION (provided by applicant): Understanding the mechanisms of pain signaling and control of pain has become a national health priority as evidenced by the recent mandate from Congress that established 2001-2010 as the "Decade of Pain Control and Research." To understand how pain develops and is maintained, we are studying the development, phenotype and physiologic properties of primary sensory neurons that project to the skin and respond to painful stimuli (nociception). The role of target-derived artemin in nociceptive system development and function will be determined using a multifaceted approach that includes behavior, anatomy, electrophysiology and molecular biology to define how artemin dependent neurons function in perception and processing of pain stimuli. An established transgenic expression system and ex vivo nerve-skin preparation that allows for comprehensive analysis of individual sensory neurons will be used in this analysis. The long-term objective of this research is to understand how growth factors regulate the development and properties of neurons that transmit painful stimuli. The Specific Aims are to: 1) Test the hypothesis that skin-derived artemin is a survival and differentiation factor for specific types of nociceptor neurons, 2) Test the hypothesis that artemin responsive neurons have a comprehensive phenotype (CP) distinct from NGF-dependent and GDNFdependent nociceptor neurons, and 3) Test the hypothesis that enhanced levels of artemin modulates behavioral response properties following inflammatory and neuropathic pain stimuli. These experiments will use anatomical and immunohistochemical analysis, ELISAs, reverse transcriptase PCR assays, electrophysiological recording and characterization of single afferents and behavioral testing to define the role of the artemin growth factor in development of sensory neurons and the role of these neurons in pain transmission.

Description: Enhanced expression of the transcription factor Sox11 was identified in developing sensory neurons of trigeminal and dorsal root ganglia of transgenic mice that overexpress neurotrophic growth factors in the skin. This expression suggested that Sox11 transcriptionally regulate genes involved in the enhanced neuron survival and axon projections exhibited by cutaneous sensory neurons in these animals. In adult DRG Sox11 was expressed at a low level but showed a significant increase following peripheral nerve crush. The high level of Sox11 expression during development and following adult neuron injury suggests Sox11 modulates a specific set of genes that have essential roles in neuron survival and axon growth. The experiments of this proposal will begin to define putative targets of Sox11 action and determine how its expression in adult neurons modulates their survival, axonal growth and response properties. Three specific aims are proposed. Aim 1 will use luciferase reporter assays to test if identified target genes involved in survival and axon growth are modulated by Sox11 expression. Aim 2 will examine if the level of Sox11 expression in DRG cultures or following in vivo nerve injury correlates with the rate and quality of anatomical and functional recovery. In these studies we will manipulate Sox11 level using two approaches. To decrease expression in DRG neurons we will use a RNAi approach. To increase expression of Sox11 we will use non-replicating, neurotropic HSV viral vectors. Changes in Sox11 level may alter expression of genes involved in afferent sensitivity and pain signaling which could lead to behavioral sensitivity. Aim 3 will test this possibility by measuring behavioral responses to applied thermal and mechanical stimuli. Relevance to Public Health: Impaired recovery following nerve injury can have significant effects on the quality of life and productivity of an individual due to abnormal nerve function or persistent pain following injury. Improved understanding of the cellular and molecular mechanisms that underlie survival and functional recovery of neurons following traumatic injury is required for design of effective strategies for recovery.

DESCRIPTION (provided by applicant): Diverse sets of sensory afferents that transmit temperature, touch and painful stimuli innervate the epidermal layer of the skin. Although stimulus detection was thought to be afferent specific, production of growth factors (NGF), neuromodulators (e.g., ATP, ACh, CGRP) and activation of ion channels (e.g., TRP, Na+) by epidermal keratinocytes suggests the epidermis also impacts sensory signaling. To examine nerve-epithelial communication we first developed optogenetic mouse models in which light activated channelrhodopsin (ChR2) is targeted to peripheral neurons using a peripherin promoter driving Cre recombinase. Light stimulation of the skin of peripherin-ChR2 mice elicits a robust nocifensive behavioral response and electrophysiological assays using a skin-nerve-ganglia and spinal cord ex vivo preparation showed preferential activation of C-fiber nociceptors. Thus, blue-laser light penetrates the epidermis and sensory neuron specific gene promoters drive in vivo expression of ChR2 at levels that depolarize peripheral nerve terminals. Interestingly, light activation of some neurons did not elicit response properties identical to thoe obtained using direct mechanical or thermal stimulation of the skin. In this R21 application we therefore propose to test the hypothesis that the difference between light-stimulation and direct mechanical or thermal stimulation of skin is due to factors released by the skin in response to mechanical or thermal manipulation. To test this hypothesis we isolated optogenetic mouse strains in which ChR2 is targeted to epidermal keratinocytes using the K14 keratin gene promoter. Preliminary analysis of these mice using an ex vivo skin-nerve-ganglia-spinal cord preparation show that light stimulation of keratinocytes evokes changes in behavioral and electrophysiologic response properties. We also found that different subtypes of cutaneous afferents are activated and that combined stimulation of the skin using light and mechanical or thermal stimuli evokes greater activation of individual fibers. In Aim 1 we will further characterie K14-ChR2 mice at anatomical, behavioral and biochemical levels. Aim 2 experiments will determine how light activation of keratinocytes affects response properties of functionally defined subsets of cutaneous sensory neurons and determine how this activation compares to mechanical and/or thermal stimulation of the skin. We will use the data generated in this R21 pilot study to design future studies that will investigate neuromodulator release mechanisms and determine if neuromodulators released from keratinocytes activate specific sensory afferent subtypes.

? DESCRIPTION (provided by applicant): The transduction of cutaneous stimuli has been previously thought to be solely a function of sensory fibers. It is now recognized that production of growth factors and neuroactivators (e.g., NGF, ATP, ACh, glutamate) by epidermal keratinocytes can have a profound effect on this process. To unravel these complex interactions and advance our understanding of the mechanisms regulating neural-keratinocyte communication, we developed optogenetic mouse models in which light activated channelrhodopsin (ChR2) is targeted to cutaneous sensory neurons. Light stimulation of the skin of these mice was found to elicit a robust nocifensive behavioral response. Electrophysiological analysis of this activation using a skin-nerve-ganglia and spinal cord ex vivo preparation showed preferential activation of C-fiber nociceptors. Thus, blue-laser light penetrates the epidermis and activates ChR2 at levels that depolarize peripheral nerve terminals. Interestingly, light activation of some neurons did not elicit response properties identical to those obtained using direct mechanical or thermal stimulation of the skin. We hypothesized this lack of a full response reflected a missing stimulus from the skin. We therefore isolated mice in which ChR2 was targeted exclusively to K14 keratin expressing keratinocytes. Remarkably, light stimulation of keratinocytes expressing ChR2 evoked changes in behavioral and electrophysiologic response properties of cutaneous sensory neurons. We also found that different subtypes of cutaneous afferents are activated at different levels suggesting heterogeneity in skin-neural communication. Using these new genetic models we propose three specific aims to advance these findings: Aim 1 experiments will examine how light-induced release of neuroactivators (e.g., ATP) from ChR2- expressing keratinocytes activates subtypes of primary sensory afferents. Aim 2 will determine how light activation of ChR2 or halorhodopsin expressed by subtypes of sensory afferents or keratinocytes affects afferent response properties. We will also determine how this activation compares to mechanical and/or thermal stimulation of the skin. Aim 3 experiments will determine the contribution of changes in keratinocytes and sensory neurons to thermal and mechanical hyperalgesia in a model of inflammatory pain. These studies will determine if hyperalgesia is caused by changes in primary afferents, skin keratinocytes or both. The ability to control activation of either keratinocytes or sensory afferents will provide new insights into how the skin and sensory nervous system communicate under normal and inflamed conditions.

Abstract The skin is a highly innervated organ and contains numerous sensory afferent nerve fibers that respond to a diverse array of stimuli ranging from gentle touch to noxious, painful stimuli. There are also many skin-resident immune cells including several subsets of dendritic cells that direct effector responses from skin-resident innate and adaptive T cells. During C. albicans skin infection, cutaneous nociceptors (unmyelinated pain- sensing neurons) are able to directly sense C. albicans and are required for the resulting cascade of down- stream Type 17 immune events resulting in efficient clearance of C. albicans. In addition, in models of psoriasis-like skin inflammation, nociceptors are required for cutaneous Type 17 inflammation. A key step in this cascade is the interaction of nociceptors with cutaneous dendritic cells that drive both innate Type 17 inflammation and are required for the development of adaptive Th17 cells that provide long-term protection. This could explain why in human patients, loss of nerve innervation or spinal cord injury results in clearance of psoriatic lesions in denervated limbs as well as increased rates of superficial C. albicans infection. Thus, in both humans and mice, cutaneous nerves play a critical role in regulating the Type 17 immunity in the skin during both infection and autoimmunity. Despite this progress, a large number of important unanswered questions remain on how the nervous and immune systems communicate in response to immune challenges. The goal of this project is to define the mechanism(s) of neuronal activation and resulting neuronal influence on immune responses in the setting of host defense and autoimmunity. In particular, we will test how nociceptors are activated and the effect of isolated nociceptor activation. By dissecting these pathways in detail we expect to create a rational use for novel pharmacological agents that target peripheral nerve function in the treatment of skin diseases. We hypothesize that the nociceptive subset of cutaneous afferents identified by expression of TRPV1 directly recognize C. albicans and Imiquimod through engagement of the TLR pathway. We will test this hypothesis by comparing responses of dorsal root ganglion neurons isolated from mice with genetic defects in the TLR pathway to mutant strains of C. albicans in vitro. We will also determine whether activation of nociceptors in vivo is sufficient for the establishment of Type 17 immunity using optogenetics. Finally, using mice with genetic ablation of nociceptors, we will test whether nociception is required for the development of adaptive Th17 cells in vivo.

SUMMARY Visceral pain is notoriously difficult to treat, often persisting long after the precipitating injury/disease is no longer evident. In this application we will explore a novel, multicellular peripheral circuit that we hypothesize explains many of the intractable features of chronic, visceral pain. We now know that epithelial-neuronal communication is widespread, with numerous epithelial cell types releasing neuroactive substances (e.g., ATP, ACh, 5HT, glutamate). This is particularly apparent in the colon where we have found that channelrhodopsin (ChR2) -induced activation of colon epithelial cells produces high frequency bursting of colon extrinsic primary afferent neurons (ExPAN?s), phenocopying physiologic stimuli and inducing robust behavioral responses (visceromotor responses (VMR), a validated assay of hypersensitivity). Building on these findings, new surprising data indicate colon epithelium also receives functional input from sympathetic neurons; activation of sympathetic projections to the colon induces large, phase-locked calcium signals in the epithelium. Closing the loop, we found that activation of ExPAN?s via colorectal distension (CRD) induces calcium signals in the post-ganglionic sympathetic neurons projecting to the colon, and that ChR2- induced activation of ExPAN?s induces cFos expression in these same neurons. That this multicellular circuit plays a role in visceral pain is supported further by preliminary data that shows that inflammation (acute and/or chronic) is correlated with increased signaling in all portions of this circuit. Thus, the goal of the proposed experiments is to test the hypothesis that persistent visceral hypersensitivity is due, at least in part, to amplification in an epithelial-ExPAN-sympathetic circuit such that it is possible to treat pain by breaking any limb of this feed-forward circuit (Fig.1). This hypothesis will be tested in 3 aims: Aim 1: Determine if persistent hypersensitivity induced in a model of IBD (DSS (dextran sulfate sodium)) is due to increased epithelial signaling and/or ExPAN excitability, Aim 2: Determine if DSS-induced inflammation increases the ability of ExPANs to activate sympathetic neurons in prevertebral sympathetic ganglion (PrSG) directly (via synapses in PrSG) or indirectly (via a spinal cord circuit) and, Aim 3 Determine the ability of sympathetic neurons to drive activity in epithelial cells in naïve mice and in the DSS model of IBD.

Abstract The skin is a highly innervated organ and contains numerous sensory afferent neurons that respond to a diverse array of stimuli including touch, pruritogens (inducing itch) and noxious agents (inducing pain). There are also many skin-resident immune cells including dendritic cells and mast cells that make direct contact with sensory neurons. It has very recently become appreciated that cutaneous sensory neurons and skin-resident innate immune cells work synergistically to initiate local inflammation and host defense. We have previously shown that TRPV1+ neurons that sense pain are necessary and sufficient for cutaneous innate Type-17 inflammation and host defense against C. albicans. Thus, a neuron subset associated with painful stimuli is necessary and sufficient to drive a Type-17 immune response which is the optimal immune response against extracellular pathogens that can cause painful stimuli. We now propose to test the immunologic potential of nonpeptidergic sensory neurons are that can be divided based on single cell RNAseq into at least 3 subsets: NP1, NP2, and NP3. The overall goal of this proposal is to understand the unique contribution of individual subsets of nonpeptidergic sensory neurons to the modulation of skin immunity. Exploring this neuro- immune interaction will better define the cellular circuits driving inflammation and allow for the use of agonists/antagonists that target neuron subsets in order to modulate specific types of cutaneous immune responses without global immunosuppression. Specifically, we hypothesize that NP1 sensory nerves function to suppress mast cell activity. We further hypothesize that the NP2 and NP3 subsets that communicate itch sensation in response to pruritogens also participate in the development of Type-2 immune responses analogous to TRPV1+ neurons and Type-17 immune responses. The potential to modulate Th2 and mast cell function makes these pathways potential key for allergic disease pathogenesis.

Hirschsprung?s disease (HSCR) is a birth defect where the enteric nervous system (ENS) is absent from the distal bowel. Most HSCR patients have reduced activity of RET and many have reduced EDNRB (endothelin receptor 3) activity. Although only the distal colon is aganglionic (missing neurons and glia) in 80% of children with HSCR, proximal bowel regions (and the rest of the GI tract) innervated by enteric neurons harbor the same mutated genes. Since RET and EDNRB are needed for many aspects of ENS development, it seems likely enteric neurons in ganglionic regions of the colon and their connections (hereafter referred to as the ENS connectome) are abnormal. Consistent with this hypothesis, HSCR surgery to remove aganglionic bowel and reconnect ?normal bowel? to the anal verge does not alleviate all HSCR symptoms; up to 50% of children have ongoing issues post-surgery including bowel distension, inflammation, explosive diarrhea, blood in the stool, lethargy and poor feeding. The long-term goal of this research project is to determine how mutations in HSCR-related genes affect the entire ENS and contribute to GI tract dysfunction. The short-term goals are: 1) to identify the gene-expression changes downstream of mutations in RET and EDNRB that occur in HSCR patients and in mice in that contain HSCR-relevant mutations, and 2) Perform anatomical and functional studies in HSCR mouse models to determine how these gene defects negatively impact the enteric nervous system. The overall hypothesis guiding these experiments is that for the innervated portion of the colon, different HSCR mutations produce defects in motility resulting from specific changes in communication between unique subsets of neurons (in the myenteric plexus and autonomic nervous system (ANS)), ICC, and glia. Aim 1: Conduct pooled and single cell RNA-Seq analysis on enteric neurons of wild type and HSCR mice models; compare murine data with RNA-Seq analysis from HSCR patients and controls. Aim 2: Determine the effect of HSCR-associated mutations on ENS/ICC/glia communication. Aim 3: Examine extrinsic parasympathetic and sympathetic drive of myenteric neurons, ICC, glia and associated smooth muscle contractions in HSCR mouse models. Impact: Transcriptomics indicate that the different HSCR mutations will negatively affect the function of different cell types in the ENS and ANS (neurons, glia and ICC). The consequence of the involvement of multiple cell types is that patients may appear similar symptomatically, but the underlying cause, and hence appropriate treatment, may be very different. This research program is designed to identify mutation-specific mechanisms of disease as a basis for development of patient-specific treatments.