RESUMEN
The relationship between transcription and protein expression is complex. We identified polysome-associated RNA transcripts in the somata and central terminals of mouse sensory neurons in control, painful (plus nerve growth factor), and pain-free conditions (Nav1.7-null mice). The majority (98%) of translated transcripts are shared between male and female mice in both the somata and terminals. Some transcripts are highly enriched in the somata or terminals. Changes in the translatome in painful and pain-free conditions include novel and known regulators of pain pathways. Antisense knockdown of selected somatic and terminal polysome-associated transcripts that correlate with pain states diminished pain behavior. Terminal-enriched transcripts included those encoding synaptic proteins (e.g., synaptotagmin), non-coding RNAs, transcription factors (e.g., Znf431), proteins associated with transsynaptic trafficking (HoxC9), GABA-generating enzymes (Gad1 and Gad2), and neuropeptides (Penk). Thus, central terminal translation may well be a significant regulatory locus for peripheral input from sensory neurons.
Asunto(s)
Dolor , Células Receptoras Sensoriales , Animales , Células Receptoras Sensoriales/metabolismo , Ratones , Masculino , Femenino , Dolor/metabolismo , Biosíntesis de Proteínas , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/genética , Glutamato Descarboxilasa/metabolismo , Glutamato Descarboxilasa/genética , Polirribosomas/metabolismo , Ratones Endogámicos C57BL , Ganglios Espinales/metabolismoRESUMEN
Drive from peripheral neurons is essential in almost all pain states, but pharmacological silencing of these neurons to effect analgesia has proved problematic. Reversible gene therapy using long-lived chemogenetic approaches is an appealing option. We used the genetically activated chloride channel PSAM4-GlyR to examine pain pathways in mice. Using recombinant AAV9-based delivery to sensory neurons, we found a reversal of acute pain behavior and diminished neuronal activity using in vitro and in vivo GCaMP imaging on activation of PSAM4-GlyR with varenicline. A significant reduction in inflammatory heat hyperalgesia and oxaliplatin-induced cold allodynia was also observed. Importantly, there was no impairment of motor coordination, but innocuous von Frey sensation was inhibited. We generated a transgenic mouse that expresses a CAG-driven FLExed PSAM4-GlyR downstream of the Rosa26 locus that requires Cre recombinase to enable the expression of PSAM4-GlyR and tdTomato. We used NaV1.8 Cre to examine the role of predominantly nociceptive NaV1.8+ neurons in cancer-induced bone pain (CIBP) and neuropathic pain caused by chronic constriction injury (CCI). Varenicline activation of PSAM4-GlyR in NaV1.8-positive neurons reversed CCI-driven mechanical, thermal, and cold sensitivity. Additionally, varenicline treatment of mice with CIBP expressing PSAM4-GlyR in NaV1.8+ sensory neurons reversed cancer pain as assessed by weight-bearing. Moreover, when these mice were subjected to acute pain assays, an elevation in withdrawal thresholds to noxious mechanical and thermal stimuli was detected, but innocuous mechanical sensations remained unaffected. These studies confirm the utility of PSAM4-GlyR chemogenetic silencing in chronic pain states for mechanistic analysis and potential future therapeutic use.
Asunto(s)
Dolor Agudo , Dolor en Cáncer , Neoplasias , Ratones , Animales , Dolor en Cáncer/terapia , Dolor en Cáncer/metabolismo , Dolor Agudo/metabolismo , Vareniclina , Células Receptoras Sensoriales/fisiología , Hiperalgesia/metabolismo , Ratones Transgénicos , Neoplasias/metabolismo , Ganglios Espinales/metabolismoRESUMEN
Background: Somatosensation depends on primary sensory neurons of the trigeminal and dorsal root ganglia (DRG). Transcriptional profiling of mouse DRG sensory neurons has defined at least 18 distinct neuronal cell types. Using an advillin promoter, we have generated a transgenic mouse line that only expresses diphtheria toxin A (DTA) in sensory neurons in the presence of Cre recombinase. This has allowed us to ablate specific neuronal subsets within the DRG using a range of established and novel Cre lines that encompass all sets of sensory neurons. Methods: A floxed-tdTomato-stop-DTA bacterial artificial chromosome (BAC) transgenic reporter line (AdvDTA) under the control of the mouse advillin DRG promoter was generated. The line was first validated using a Na v1.8 Cre and then crossed to CGRP CreER (Calca), Th CreERT2, Tmem45b Cre, Tmem233 Cre, Ntng1 Cre and TrkB CreER (Ntrk2) lines. Pain behavioural assays included Hargreaves', hot plate, Randall-Selitto, cold plantar, partial sciatic nerve ligation and formalin tests. Results: Motor activity, as assessed by the rotarod test, was normal for all lines tested. Noxious mechanosensation was significantly reduced when either Na v1.8 positive neurons or Tmem45b positive neurons were ablated whilst acute heat pain was unaffected. In contrast, noxious mechanosensation was normal following ablation of CGRP-positive neurons but acute heat pain thresholds were significantly elevated and a reduction in nocifensive responses was observed in the second phase of the formalin test. Ablation of TrkB-positive neurons led to significant deficits in mechanical hypersensitivity in the partial sciatic nerve ligation neuropathic pain model. Conclusions: Ablation of specific DRG neuronal subsets using the AdvDTA line will be a useful resource for further functional characterization of somatosensory processing, neuro-immune interactions and chronic pain disorders.
RESUMEN
The study of rare families with inherited pain insensitivity can identify new human-validated analgesic drug targets. Here, a 66-yr-old female presented with nil requirement for postoperative analgesia after a normally painful orthopaedic hand surgery (trapeziectomy). Further investigations revealed a lifelong history of painless injuries, such as frequent cuts and burns, which were observed to heal quickly. We report the causative mutations for this new pain insensitivity disorder: the co-inheritance of (i) a microdeletion in dorsal root ganglia and brain-expressed pseudogene, FAAH-OUT, which we cloned from the fatty-acid amide hydrolase (FAAH) chromosomal region; and (ii) a common functional single-nucleotide polymorphism in FAAH conferring reduced expression and activity. Circulating concentrations of anandamide and related fatty-acid amides (palmitoylethanolamide and oleoylethanolamine) that are all normally degraded by FAAH were significantly elevated in peripheral blood compared with normal control carriers of the hypomorphic single-nucleotide polymorphism. The genetic findings and elevated circulating fatty-acid amides are consistent with a phenotype resulting from enhanced endocannabinoid signalling and a loss of function of FAAH. Our results highlight previously unknown complexity at the FAAH genomic locus involving the expression of FAAH-OUT, a novel pseudogene and long non-coding RNA. These data suggest new routes to develop FAAH-based analgesia by targeting of FAAH-OUT, which could significantly improve the treatment of postoperative pain and potentially chronic pain and anxiety disorders.
Asunto(s)
Amidohidrolasas/genética , Ácidos Araquidónicos/sangre , Endocannabinoides/sangre , Insensibilidad Congénita al Dolor/sangre , Insensibilidad Congénita al Dolor/genética , Alcamidas Poliinsaturadas/sangre , Seudogenes/genética , Anciano , Amidohidrolasas/sangre , Femenino , Humanos , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Background: Sensory neurons play an essential role in almost all pain conditions, and have recently been classified into distinct subsets on the basis of their transcriptomes. Here we have analysed alterations in dorsal root ganglia (DRG) gene expression using microarrays in mouse models related to human chronic pain. Methods: Six different pain models were studied in male C57BL/6J mice: (1) bone cancer pain using cancer cell injection in the intramedullary space of the femur; (2) neuropathic pain using partial sciatic nerve ligation; (3) osteoarthritis pain using mechanical joint loading; (4) chemotherapy-induced pain with oxaliplatin; (5) chronic muscle pain using hyperalgesic priming; and (6) inflammatory pain using intraplantar complete Freund's adjuvant. Microarray analyses were performed using RNA isolated from dorsal root ganglia and compared to sham/vehicle treated controls. Results: Differentially expressed genes (DEGs) were identified. Known and previously unreported genes were found to be dysregulated in each pain model. The transcriptomic profiles for each model were compared and expression profiles of DEGs within subsets of DRG neuronal populations were analysed to determine whether specific neuronal subsets could be linked to each of the pain models. Conclusions: Each pain model exhibits a unique set of altered transcripts implying distinct cellular responses to different painful stimuli. No simple direct link between genetically distinct sets of neurons and particular pain models could be discerned.
RESUMEN
The voltage-gated sodium channel NaV1.7 plays a critical role in pain pathways. We generated an epitope-tagged NaV1.7 mouse that showed normal pain behaviours to identify channel-interacting proteins. Analysis of NaV1.7 complexes affinity-purified under native conditions by mass spectrometry revealed 267 proteins associated with Nav1.7 in vivo The sodium channel ß3 (Scn3b), rather than the ß1 subunit, complexes with Nav1.7, and we demonstrate an interaction between collapsing-response mediator protein (Crmp2) and Nav1.7, through which the analgesic drug lacosamide regulates Nav1.7 current density. Novel NaV1.7 protein interactors including membrane-trafficking protein synaptotagmin-2 (Syt2), L-type amino acid transporter 1 (Lat1) and transmembrane P24-trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated by co-immunoprecipitation (Co-IP) from sensory neuron extract. Nav1.7, known to regulate opioid receptor efficacy, interacts with the G protein-regulated inducer of neurite outgrowth (Gprin1), an opioid receptor-binding protein, demonstrating a physical and functional link between Nav1.7 and opioid signalling. Further information on physiological interactions provided with this normal epitope-tagged mouse should provide useful insights into the many functions now associated with the NaV1.7 channel.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Dolor/fisiopatología , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Opioides/metabolismo , Células Receptoras Sensoriales/metabolismo , Acetamidas/farmacología , Analgésicos/farmacología , Animales , Línea Celular , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lacosamida , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Canal de Sodio Activado por Voltaje NAV1.7/genética , Unión Proteica , Mapeo de Interacción de Proteínas , Transporte de Proteínas/fisiología , Sinaptotagmina II/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Subunidad beta-3 de Canal de Sodio Activado por Voltaje/metabolismoRESUMEN
Loss of function of the gene SCN9A, encoding the voltage-gated sodium channel Na(v)1.7, causes a congenital inability to experience pain in humans. Here we show that Na(v)1.7 is not only necessary for pain sensation but is also an essential requirement for odour perception in both mice and humans. We examined human patients with loss-of-function mutations in SCN9A and show that they are unable to sense odours. To establish the essential role of Na(v)1.7 in odour perception, we generated conditional null mice in which Na(v)1.7 was removed from all olfactory sensory neurons. In the absence of Na(v)1.7, these neurons still produce odour-evoked action potentials but fail to initiate synaptic signalling from their axon terminals at the first synapse in the olfactory system. The mutant mice no longer display vital, odour-guided behaviours such as innate odour recognition and avoidance, short-term odour learning, and maternal pup retrieval. Our study creates a mouse model of congenital general anosmia and provides new strategies to explore the genetic basis of the human sense of smell.
Asunto(s)
Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación/genética , Trastornos del Olfato/genética , Trastornos del Olfato/fisiopatología , Canales de Sodio/genética , Potenciales de Acción , Animales , Conducta Animal , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Canal de Sodio Activado por Voltaje NAV1.7 , Odorantes/análisis , Trastornos del Olfato/congénito , Trastornos del Olfato/patología , Mucosa Olfatoria/citología , Mucosa Olfatoria/patología , Vías Olfatorias/metabolismo , Vías Olfatorias/patología , Vías Olfatorias/fisiopatología , Percepción Olfatoria/genética , Percepción Olfatoria/fisiología , Neuronas Receptoras Olfatorias/metabolismo , Neuronas Receptoras Olfatorias/patología , Dolor/genética , Dolor/fisiopatología , Fenotipo , Olfato/genética , Olfato/fisiología , Canales de Sodio/deficiencia , Canales de Sodio/metabolismo , Sinapsis/metabolismo , Sinapsis/patología , Orina/químicaRESUMEN
Several members of the ADAM (a disintegrin and metalloprotease) family of proteins have been implicated in biological processes ranging from fertilization to myoblast fusion and neural cell fate determination. These proteins have so far been studied mostly in terms of their protease activity, but a considerable amount of evidence suggests that many ADAMs are also important as receptors for cell-surface integrins. We have shown that, for one such member of the family, ADAM23, mRNA transcripts are expressed in neuronal cells throughout the rat brain, at all stages of postnatal development, and that particularly high transcript concentrations are found in the hippocampus and cerebellum. Using an antibody that we raised against the rat ADAM23 disintegrin domain, we found that ADAM23 is present at detectable levels only in nervous system tissue. Our analysis of ADAM23 expression in cultured cerebellar granule cells (CGCs) furthermore suggested that this protein is synthesized as a glycosylated precursor of about 100 kD whose maturation depends on cleavage by furin or a related enzyme. We have also shown ADAM23 to be expressed primarily as a cell-surface protein that appears to be localized to sites of intercellular contact. Taken together, these data are consistent with a model wherein ADAM23 serves to mediate cell-cell interactions within the mammalian CNS.