Ultrastructure of dorsal root ganglia.

Dorsal root ganglia (DRG) contains thousands of sensory neurons that transmit information about our external and internal environment to the central nervous system. This includes signals related to proprioception, temperature, and nociception. Our understanding of DRG has increased tremendously over the last 50 years and has established the DRG as an active participant in peripheral processes. This includes interactions between neurons and non-neuronal cells such as satellite glia cells and macrophages that contribute to an increasingly complex cellular environment that modulates neuronal function. Early ultrastructural investigations of the DRG have described subtypes of sensory neurons based on differences in the arrangement of organelles such as the Golgi apparatus and the endoplasmic reticulum. The neuron-satellite cell complex and the composition of the axon hillock in DRG have also been investigated, but, apart from basic descriptions of Schwann cells, ultrastructural investigations of other cell types in DRG are limited. Furthermore, detailed descriptions of key components of DRG, such as blood vessels and the capsule that sits at the intersection of the meninges and the connective tissue covering the peripheral nervous system, are lacking to date. With rising interest in DRG as potential therapeutic targets for aberrant signalling associated with chronic pain conditions, gaining further insights into DRG ultrastructure will be fundamental to understanding cell-cell interactions that modulate DRG function. In this review, we aim to provide a synopsis of the current state of knowledge on the ultrastructure of the DRG and its components, as well as to identify areas of interest for future studies.

Regulator of Calcineurin 1 helps coordinate whole-body metabolism and thermogenesis.

Increasing non-shivering thermogenesis (NST), which expends calories as heat rather than storing them as fat, is championed as an effective way to combat obesity and metabolic disease. Innate mechanisms constraining the capacity for NST present a fundamental limitation to this approach, yet are not well understood. Here, we provide evidence that Regulator of Calcineurin 1 (RCAN1), a feedback inhibitor of the calcium-activated protein phosphatase calcineurin (CN), acts to suppress two distinctly different mechanisms of non-shivering thermogenesis (NST): one involving the activation of UCP1 expression in white adipose tissue, the other mediated by sarcolipin (SLN) in skeletal muscle. UCP1 generates heat at the expense of reducing ATP production, whereas SLN increases ATP consumption to generate heat. Gene expression profiles demonstrate a high correlation between Rcan1 expression and metabolic syndrome. On an evolutionary timescale, in the context of limited food resources, systemic suppression of prolonged NST by RCAN1 might have been beneficial; however, in the face of caloric abundance, RCAN1-mediated suppression of these adaptive avenues of energy expenditure may now contribute to the growing epidemic of obesity.

Inhibition of motor neuron death in vitro and in vivo by a p75 neurotrophin receptor intracellular domain fragment.

The p75 neurotrophin receptor (p75(NTR); also known as NGFR) can mediate neuronal apoptosis in disease or following trauma, and facilitate survival through interactions with Trk receptors. Here we tested the ability of a p75(NTR)-derived trophic cell-permeable peptide, c29, to inhibit p75(NTR)-mediated motor neuron death. Acute c29 application to axotomized motor neuron axons decreased cell death, and systemic c29 treatment of SOD1(G93A) mice, a common model of amyotrophic lateral sclerosis, resulted in increased spinal motor neuron survival mid-disease as well as delayed disease onset. Coincident with this, c29 treatment of these mice reduced the production of p75(NTR) cleavage products. Although c29 treatment inhibited mature- and pro-nerve-growth-factor-induced death of cultured motor neurons, and these ligands induced the cleavage of p75(NTR) in motor-neuron-like NSC-34 cells, there was no direct effect of c29 on p75(NTR) cleavage. Rather, c29 promoted motor neuron survival in vitro by enhancing the activation of TrkB-dependent signaling pathways, provided that low levels of brain-derived neurotrophic factor (BDNF) were present, an effect that was replicated in vivo in SOD1(G93A) mice. We conclude that the c29 peptide facilitates BDNF-dependent survival of motor neurons in vitro and in vivo.

Peptidergic nerve fibers in the urethra: Morphological and neurochemical characteristics in female mice of reproductive age.

BACKGROUND: Peptidergic nerve fibers provide important contributions to urethral function. Urethral innervation of female mice is not well documented. AIMS: To determine the distribution and projection sites of nerve fibers immunoreactive for vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP), and neuropeptide Y (NPY) in the urethra of wild-type control mice and compare innervation characteristics between the proximal and distal urethra of young nullipara and older multipara mice. Furthermore, to identify the location and neurochemical coding of the spinal afferent nerve endings in the urethra, whose sensory neurons reside in lumbosacral dorsal root ganglia (DRG). METHODS: Multiple labeling immunohistochemistry of urethral sections of nulliparous (6-8 weeks old), and multiparous (9-12 months old) mice, and anterograde axonal tracing from L5-S2 (DRG) in vivo. RESULTS: Abundant VIP-, CGRP-, SP-, and NPY-immunoreactive nerve fibers were identified in the adventitia, muscularis, and lamina propria of proximal and distal segments of the urethra. A proportion of fibers were closely associated with blood vessels, glands, and cells immunoreactive for PGP9.5. The epithelium contained abundant nerve fibers immunoreactive for CGRP and/or SP. Epithelial innervation was increased in the distal urethra of multipara mice. Abundant fibers were traced from L5-S2 DRG to all urethral regions. CONCLUSIONS: We present the first identification of spinal afferent endings in the urethra. Peptidergic nerve fibers, including multiple populations of spinal afferents, provide rich innervation of the female mouse urethra. The morphology of fibers in the epithelium and other regions suggests multiple nerve-cell interactions impacting on urethral function.

Local versus long-range neurotrophin receptor signalling: endosomes are not just carriers for axonal transport.

Neurotrophins play a critical role in neuronal development and survival, as well as maintenance of the adult nervous system. Neurotrophins can mediate their effects by signalling locally at the nerve terminal, or signalling retrogradely from the axonal terminal to the cell soma to regulate gene expression. Given that the axon terminals of many nerve cells can be up to a metre away from their soma, neurons have evolved specialized long-range signalling platforms that depend on a highly regulated network of intracellular membrane compartments termed "signalling endosomes". Endosomal trafficking of activated receptors controls not only the axonal retrograde signals but also local receptor recycling and degradation. Endosomal trafficking involving the sorting and compartmentalizing of different signals, which are subsequently distributed to the appropriate cellular destination, can at least partially explain how neurotrophins generate a diverse array of signalling outcomes. Although signalling endosomes provide a useful model for understanding how different cell surface receptor-mediated signals are generated and transported, the precise role, identity and functional definition of a signalling endosome remains unclear. In this review we will discuss the regulation of local versus long-range neurotrophin signalling, with a specific focus on recent developments in the role of endosomes in regulating the fate of Trk receptors.

An intracellular domain fragment of the p75 neurotrophin receptor (p75(NTR)) enhances tropomyosin receptor kinase A (TrkA) receptor function.

Facilitation of nerve growth factor (NGF) signaling by the p75 neurotrophin receptor (p75(NTR)) is critical for neuronal survival and differentiation. However, the interaction between p75(NTR) and TrkA receptors required for this activity is not understood. Here, we report that a specific 29-amino acid peptide derived from the intracellular domain fragment of p75(NTR) interacts with and potentiates binding of NGF to TrkA-expressing cells, leading to increased neurite outgrowth in sympathetic neurons as a result of enhanced Erk1/2 and Akt signaling. An endogenous intracellular domain fragment of p75(NTR) (p75(ICD)) containing these 29 amino acids is produced by regulated proteolysis of the full-length receptor. We demonstrate that generation of this fragment is a requirement for p75(NTR) to facilitate TrkA signaling in neurons and propose that the juxtamembrane region of p75(ICD) acts to cause a conformational change within the extracellular domain of TrkA. This finding provides new insight into the mechanism by which p75(NTR) and TrkA interact to enhance neurotrophic signaling.

The effects of transmembrane sequence and dimerization on cleavage of the p75 neurotrophin receptor by γ-secretase.

Cleavage of transmembrane receptors by γ-secretase is the final step in the process of regulated intramembrane proteolysis (RIP) and has a significant impact on receptor function. Although relatively little is known about the molecular mechanism of γ-secretase enzymatic activity, it is becoming clear that substrate dimerization and/or the α-helical structure of the substrate can regulate the site and rate of γ-secretase activity. Here we show that the transmembrane domain of the pan-neurotrophin receptor p75(NTR), best known for regulating neuronal death, is sufficient for its homodimerization. Although the p75(NTR) ligands NGF and pro-NGF do not induce homerdimerization or RIP, homodimers of p75(NTR) are γ-secretase substrates. However, dimerization is not a requirement for p75(NTR) cleavage, suggesting that γ-secretase has the ability to recognize and cleave each receptor molecule independently. The transmembrane cysteine 257, which mediates covalent p75(NTR) interactions, is not crucial for homodimerization, but this residue is required for normal rates of γ-secretase cleavage. Similarly, mutation of the residues alanine 262 and glycine 266 of an AXXXG dimerization motif flanking the γ-secretase cleavage site within the p75(NTR) transmembrane domain alters the orientation of the domain and inhibits γ-secretase cleavage of p75(NTR). Nonetheless, heteromer interactions of p75(NTR) with TrkA increase full-length p75(NTR) homodimerization, which in turn potentiates the rate of γ-cleavage following TrkA activation independently of rates of α-cleavage. These results provide support for the idea that the helical structure of the p75(NTR) transmembrane domain, which may be affected by co-receptor interactions, is a key element in γ-secretase-catalyzed cleavage.

Mapping of the interaction site between sortilin and the p75 neurotrophin receptor reveals a regulatory role for the sortilin intracellular domain in p75 neurotrophin receptor shedding and apoptosis.

Neurotrophins comprise a group of neuronal growth factors that are essential for the development and maintenance of the nervous system. However, the immature pro-neurotrophins promote apoptosis by engaging in a complex with sortilin and the p75 neurotrophin receptor (p75(NTR)). To identify the interaction site between sortilin and p75(NTR), we analyzed binding between chimeric receptor constructs and truncated p75(NTR) variants by co-immunoprecipitation experiments, surface plasmon resonance analysis, and FRET. We found that complex formation between sortilin and p75(NTR) relies on contact points in the extracellular domains of the receptors. We also determined that the interaction critically depends on an extracellular juxtamembrane 23-amino acid sequence of p75(NTR). Functional studies further revealed an important regulatory function of the sortilin intracellular domain in p75(NTR)-regulated intramembrane proteolysis and apoptosis. Thus, although the intracellular domain of sortilin does not contribute to p75(NTR) binding, it does regulate the rates of p75(NTR) cleavage, which is required to mediate pro-neurotrophin-stimulated cell death.

Proteolytic processing of the p75 neurotrophin receptor: A prerequisite for signalling?: Neuronal life, growth and death signalling are crucially regulated by intra-membrane proteolysis and trafficking of p75(NTR).

The common neurotrophin receptor (p75(NTR) ) regulates various functions in the developing and adult nervous system. Cell survival, cell death, axonal and growth cone retraction, and regulation of the cell cycle can be regulated by p75(NTR) -mediated signals following activation by either mature or pro-neurotrophins and in combination with various co-receptors, including Trk receptors and sortilin. Here, we review the known functions of p75(NTR) by cell type, receptor-ligand combination, and whether regulated intra-membrane proteolysis of p75(NTR) is required for signalling. We highlight that the generation of the intracellular domain fragment of p75(NTR) is associated with many of the receptor functions, regardless of its ligand and co-receptor interactions.

Dynamin regulates L cell secretion in human gut.

BACKGROUND: The mechanochemical enzyme dynamin mediates endocytosis and regulates neuroendocrine cell exocytosis. Enteroendocrine L cells co-secrete the anorectic gut hormones glucagon-like peptide 1 (GLP-1) and peptide YY (PYY) postprandially and is a potential therapeutic target for metabolic diseases. In the present study, we aimed to determine if dynamin is implicated in human L cell secretion. METHODS: Western blot was performed on the murine L cell line GLUTag. Static incubation of human colonic mucosae with activators and inhibitors of dynamin was carried out. GLP-1 and PYY contents of the secretion supernatants were assayed using ELISA. RESULTS AND CONCLUSION: s: Both dynamin I and II are expressed in GLUTag cells. The dynamin activator Ryngo 1-23 evoked significant GLP-1 and PYY release from human colonic mucosae while the dynamin inhibitor Dynole 3-42 significantly inhibited release triggered by known L cell secretagogues. Thus, the cell signaling regulator dynamin is able to bi-directionally regulate L cell hormone secretion in the human gut and may represent a novel target for gastrointestinal-targeted metabolic drug development.

Clodronate Treatment Prevents Vaginal Hypersensitivity in a Mouse Model of Vestibulodynia.

INTRODUCTION: Improved understanding of vestibulodynia pathophysiology is required to develop appropriately targeted treatments. Established features include vulvovaginal hyperinnervation, increased nociceptive signalling and hypersensitivity. Emerging evidence indicates macrophage-neuron signalling contributes to chronic pain pathophysiology. Macrophages are broadly classified as M1 or M2, demonstrating pro-nociceptive or anti-nociceptive effects respectively. This study investigates the impact of clodronate liposomes, a macrophage depleting agent, on nociceptive signalling in a mouse model of vestibulodynia. METHODS: Microinjection of complete Freund's adjuvant (CFA) at the vaginal introitus induced mild chronic inflammation in C57Bl/6J mice. A subgroup was treated with the macrophage depleting agent clodronate. Control mice received saline. After 7 days, immunolabelling for PGP9.5, F4/80+CD11c+ and F4/80+CD206+ was used to compare innervation density and presence of M1 and M2 macrophages respectively in experimental groups. Nociceptive signalling evoked by vaginal distension was assessed using immunolabelling for phosphorylated MAP extracellular signal-related kinase (pERK) in spinal cord sections. Hyperalgesia was assessed by visceromotor response to graded vaginal distension. RESULTS: CFA led to increased vaginal innervation (p < 0.05), increased pERK-immunoreactive spinal cord dorsal horn neurons evoked by vaginal-distension (p < 0.01) and enhanced visceromotor responses compared control mice (p < 0.01). Clodronate did not reduce vaginal hyperinnervation but significantly reduced the abundance of M1 and M2 vaginal macrophages and restored vaginal nociceptive signalling and vaginal sensitivity to that of healthy control animals. CONCLUSIONS: We have developed a robust mouse model of vestibulodynia that demonstrates vaginal hyperinnervation, enhanced nociceptive signalling, hyperalgesia and allodynia. Macrophages contribute to hypersensitivity in this model. Macrophage-sensory neuron signalling pathways may present useful pathophysiological targets.

Immortalized Dorsal Root Ganglion Neuron Cell Lines.

Pain is one of the most significant causes of suffering and disability world-wide, and arguably the most burdensome global health challenge. The growing number of patients suffering from chronic pain conditions such as fibromyalgia, complex regional pain syndrome, migraine and irritable bowel syndrome, not only reflect the complexity and heterogeneity of pain types, but also our lack of understanding of the underlying mechanisms. Sensory neurons within the dorsal root ganglia (DRG) have emerged as viable targets for effective chronic pain therapy. However, DRG's contain different classes of primary sensory neurons including pain-associated nociceptive neurons, non-nociceptive temperature sensing, mechanosensory and chemoreceptive neurons, as well as multiple types of immune and endothelial cells. This cell-population heterogeneity makes investigations of individual subgroups of DRG neurons, such as nociceptors, difficult. In attempts to overcome some of these difficulties, a limited number of immortalized DRG-derived cell lines have been generated over the past few decades. In vitro experiments using DRG-derived cell lines have been useful in understanding sensory neuron function. In addition to retaining phenotypic similarities to primary cultured DRG neurons, these cells offer greater suitability for high throughput assays due to ease of culture, maintenance, growth efficiency and cost-effectiveness. For accurate interpretation and translation of results it is critical, however, that phenotypic similarities and differences of DRG-derived cells lines are methodically compared to native neurons. Published reports to date show notable variability in how these DRG-derived cells are maintained and differentiated. Understanding the cellular and molecular differences stemming from different culture methods, is essential to validate past and future experiments, and enable these cells to be used to their full potential. This review describes currently available DRG-derived cell lines, their known sensory and nociceptor specific molecular profiles, and summarize their morphological features related to differentiation and neurite outgrowth.

Emerging Evidence of Macrophage Contribution to Hyperinnervation and Nociceptor Sensitization in Vulvodynia.

Vulvodynia is an idiopathic chronic pain disorder and a leading cause of dyspareunia, or pain associated with sexual intercourse, for women. The key pathophysiological features of vulvodynia are vaginal hyperinnervation and nociceptor sensitization. These features have been described consistently by research groups over the past 30 years, but currently there is no first-line recommended treatment that targets this pathophysiology. Instead, psychological interventions, pelvic floor physiotherapy and surgery to remove painful tissue are recommended, as these are the few interventions that have shown some benefit in clinical trials. Recurrence of vulvodynia is frequent, even after vestibulectomy and questions regarding etiology remain. Vestibular biopsies from women with vulvodynia contain increased abundance of immune cells including macrophages as well as increased numbers of nerve fibers. Macrophages have multiple roles in the induction and resolution of inflammation and their function can be broadly described as pro-inflammatory or anti-inflammatory depending on their polarization state. This state is not fixed and can alter rapidly in response to the microenvironment. Essentially, M1, or classically activated macrophages, produce pro-inflammatory cytokines and promote nociceptor sensitization and mechanical allodynia, whereas M2, or alternatively activated macrophages produce anti-inflammatory cytokines and promote functions such as wound healing. Signaling between macrophages and neurons has been shown to promote axonal sprouting and nociceptor sensitization. This mini review considers emerging evidence that macrophages may play a role in nociceptor sensitization and hyperinnervation relevant to vulvodynia and considers the implications for development of new therapeutic strategies.

Human Dorsal Root Ganglia.

Sensory neurons with cell bodies situated in dorsal root ganglia convey information from external or internal sites of the body such as actual or potential harm, temperature or muscle length to the central nervous system. In recent years, large investigative efforts have worked toward an understanding of different types of DRG neurons at transcriptional, translational, and functional levels. These studies most commonly rely on data obtained from laboratory animals. Human DRG, however, have received far less investigative focus over the last 30 years. Nevertheless, knowledge about human sensory neurons is critical for a translational research approach and future therapeutic development. This review aims to summarize both historical and emerging information about the size and location of human DRG, and highlight advances in the understanding of the neurochemical characteristics of human DRG neurons, in particular nociceptive neurons.

Characterization and use of the NSC-34 cell line for study of neurotrophin receptor trafficking.

This study addressed the suitability of the NSC-34 cell line as a motor neuron-like model for investigating neurotrophin receptor trafficking and associated subcellular processes. Initially, culture conditions were optimized for the use of NSC-34 cells in confocal microscopy. Cell surface markers, as well as markers associated with the regulated endosomal pathway thought to be associated with neurotrophin receptor transport, were identified. The study revealed the presence of a number of molecules previously not described in the literature, including the tropomyosin-like receptor kinase C (TrkC), sortilin, the vesicular acetylcholine transporter (VAChT), and the lipid raft-associated ganglioside GT1b. The presence of both sortilin and Gt1b was of special interest, insofar as these markers have been implicated in direct relationships with the p75NTR receptor. Evidence is provided for neurotrophin-dependent internalization of p75NTR and TrkB. Both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) increased the rate of internalization of p75NTR, with internalization dynamics comparable to those described for other cell lines. Thus, these studies not only describe components of the regulatory process governing the trafficking of this important receptor but also clearly demonstrate the value of NSC-34 cells as a suitable motor neuron model for the study of internalization and trafficking of cell surface molecules.

Sphingosine-1-Phosphate and the S1P3 Receptor Initiate Neuronal Retraction via RhoA/ROCK Associated with CRMP2 Phosphorylation.

The bioactive lipid sphingosine-1-phosphate (S1P) is an important regulator in the nervous system. Here, we explored the role of S1P and its receptors in vitro and in preclinical models of peripheral nerve regeneration. Adult sensory neurons and motor neuron-like cells were exposed to S1P in an in vitro assay, and virtually all neurons responded with a rapid retraction of neurites and growth cone collapse which were associated with RhoA and ROCK activation. The S1P1 receptor agonist SEW2871 neither activated RhoA or neurite retraction, nor was S1P-induced neurite retraction mitigated in S1P1-deficient neurons. Depletion of S1P3 receptors however resulted in a dramatic inhibition of S1P-induced neurite retraction and was on the contrary associated with a significant elongation of neuronal processes in response to S1P. Opposing responses to S1P could be observed in the same neuron population, where S1P could activate S1P1 receptors to stimulate elongation or S1P3 receptors and retraction. S1P was, for the first time in sensory neurons, linked to the phosphorylation of collapsin response-mediated protein-2 (CRMP2), which was inhibited by ROCK inhibition. The improved sensory recovery after crush injury further supported the relevance of a critical role for S1P and receptors in fine-tuning axonal outgrowth in peripheral neurons.

G-Protein-Coupled Inwardly Rectifying Potassium (GIRK) Channel Activation by the p75 Neurotrophin Receptor Is Required for Amyloid ß Toxicity.

Alzheimer's disease is characterized by cognitive decline, neuronal degeneration, and the accumulation of amyloid-beta (Aß). Although, the neurotoxic Aß peptide is widely believed to trigger neuronal dysfunction and degeneration in Alzheimer's disease, the mechanism by which this occurs is poorly defined. Here we describe a novel, Aß-triggered apoptotic pathway in which Aß treatment leads to the upregulation of G-protein activated inwardly rectifying potassium (GIRK/Kir3) channels, causing potassium efflux from neurons and Aß-mediated apoptosis. Although, GIRK channel activity is required for Aß-induced neuronal degeneration, we show that it is not sufficient, with coincident signaling by the p75 neurotrophin receptor (p75NTR) also required for potassium efflux and cell death. Our results identify a novel role for GIRK channels in mediating apoptosis, and provide a previously missing mechanistic link between the excitotoxicity of Aß and its ability to trigger cell death pathways, such as that mediated by p75NTR. We propose that this death-signaling pathway contributes to the dysfunction of neurons in Alzheimer's disease and is responsible for their eventual degeneration.

Morphological and neurochemical differences in peptidergic nerve fibers of the mouse vagina.

The vagina is innervated by a complex arrangement of sensory, sympathetic, and parasympathetic nerve fibers that contain classical transmitters plus an array of neuropeptides and enzymes known to regulate diverse processes including blood flow and nociception. The neurochemical characteristics and distributions of peptide-containing nerves in the mouse vagina are unknown. This study used multiple labeling immunohistochemistry, confocal maging and analysis to investigate the presence and colocalization of the peptides vasoactive intestinal polypeptide (VIP), calcitonin-gene related peptide (CGRP), substance P (SP), neuropeptide tyrosine (NPY), and the nitric oxide synthesizing enzyme neuronal nitric oxide synthase (nNOS) in nerve fibers of the murine vaginal wall. We compared cervical and vulvar areas of the vagina in young nullipara and older multipara C57Bl/6 mice, and identified differences including that small ganglia were restricted to cervical segments, epithelial fibers were mainly present in vulvar segments and most nerve fibers were found in the lamina propria of the cervical region of the vagina, where a higher number of fibers containing immunoreactivity for VIP, CGRP, SP, or nNOS were found. Two populations of VIP-containing fibers were identified: fibers containing CGRP and fibers containing VIP but not CGRP. Differences between young and older mice were present in multiple layers of the vaginal wall, with older mice showing overall loss of innervation of epithelium of the proximal vagina and reduced proportions of VIP, CGRP, and SP containing nerve fibers in the distal epithelium. The distal vagina also showed increased vascularization and perivascular fibers containing NPY. Immunolabeling of ganglia associated with the vagina indicated the likely origin of some peptidergic fibers. Our results reveal regional differences and age- or parity-related changes in innervation of the mouse vagina, effecting the distribution of neuropeptides with diverse roles in function of the female genital tract.

Functional monoclonal antibodies to p75 neurotrophin receptor raised in knockout mice.

In this study, p75NTREXONIII knockout mice were used as immune-naive hosts to produce functional antibodies to human p75NTR. Three monoclonal antibodies were produced and named MLR1, MLR2 and MLR3, and isotyped as IgG1, IgG2a and IgG2a, respectively. MLR1 and MLR2 bound to human p75NTR with higher affinity than the well-characterized ME20.4 in ELISA and also recognized p75NTR present on neurons in both rat and mouse. MLR1 and MLR2 bound to nerves known to express p75NTR following injection into Balb/C mice but not p75NTREXONIII knockout mice, indicating the antibodies are directed against the ligand binding extracellular region absent in knockout mice. Both MLR1 and MLR2 partially blocked NGF induced cell death in a mouse cell-line that expresses p75NTR but not TrKA. Importantly, intracerebroventricular injections indicated MLR2 was internalized within the cell bodies of mouse basal forebrain neurons, further demonstrating that this antibody is biologically active.

Sphingosine kinase 2-deficiency mediated changes in spinal pain processing.

Chronic pain is one of the most burdensome health issues facing the planet (as costly as diabetes and cancer combined), and in desperate need for new diagnostic targets leading to better therapies. The bioactive lipid sphingosine 1-phosphate (S1P) and its receptors have recently been shown to modulate nociceptive signaling at the level of peripheral nociceptors and central neurons. However, the exact role of S1P generating enzymes, in particular sphingosine kinase 2 (Sphk2), in nociception remains unknown. We found that both sphingosine kinases, Sphk1 and Sphk2, were expressed in spinal cord (SC) with higher levels of Sphk2 mRNA compared to Sphk1. All three Sphk2 mRNA-isoforms were present with the Sphk2.1 mRNA showing the highest relative expression. Mice deficient in Sphk2 (Sphk2(-/-)) showed in contrast to mice deficient in Sphk1 (Sphk1(-/-)) substantially lower spinal S1P levels compared to wild-type C57BL/6 mice. In the formalin model of acute peripheral inflammatory pain, Sphk2(-/-) mice showed facilitation of nociceptive transmission during the late response, whereas responses to early acute pain, and the number of c-Fos immunoreactive dorsal horn neurons were not different between Sphk2(-/-) and wild-type mice. Chronic peripheral inflammation (CPI) caused a bilateral increase in mechanical sensitivity in Sphk2(-/-) mice. Additionally, CPI increased the relative mRNA expression of P2X4 receptor, brain-derived neurotrophic factor and inducible nitric oxide synthase in the ipsilateral SC of wild-type but not Sphk2(-/-) mice. Similarly, Sphk2(-/-) mice showed in contrast to wild-type no CPI-dependent increase in areas of the dorsal horn immunoreactive for the microglia marker Iba-1 and the astrocyte marker Glial fibrillary acidic protein (GFAP). Our results suggest that the tightly regulated cell signaling enzyme Sphk2 may be a key component for facilitation of nociceptive circuits in the CNS leading to central sensitization and pain memory formation.