RESUMEN
Ubiquitin (Ub)-mediated regulation of plasmalemmal ion channel activity canonically occurs via stimulation of endocytosis. Whether ubiquitination can modulate channel activity by alternative mechanisms remains unknown. Here, we show that the transient receptor potential vanilloid 4 (TRPV4) cation channel is multiubiquitinated within its cytosolic N-terminal and C-terminal intrinsically disordered regions (IDRs). Mutagenizing select lysine residues to block ubiquitination of the N-terminal but not C-terminal IDR resulted in a marked elevation of TRPV4-mediated intracellular calcium influx, without increasing cell surface expression levels. Conversely, enhancing TRPV4 ubiquitination via expression of an E3 Ub ligase reduced TRPV4 channel activity but did not decrease plasma membrane abundance. These results demonstrate Ub-dependent regulation of TRPV4 channel function independent of effects on plasma membrane localization. Consistent with ubiquitination playing a key negative modulatory role of the channel, gain-of-function neuropathy-causing mutations in the TRPV4 gene led to reduced channel ubiquitination in both cellular and Drosophila models of TRPV4 neuropathy, whereas increasing mutant TRPV4 ubiquitination partially suppressed channel overactivity. Together, these data reveal a novel mechanism via which ubiquitination of an intracellular flexible IDR domain modulates ion channel function independently of endocytic trafficking and identify a contributory role for this pathway in the dysregulation of TRPV4 channel activity by neuropathy-causing mutations.
Asunto(s)
Canales Catiónicos TRPV , Ubiquitinación , Animales , Calcio/metabolismo , Membrana Celular/metabolismo , Drosophila/genética , Drosophila/metabolismo , Humanos , Ratones , Mutación , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Ubiquitina/metabolismoRESUMEN
OBJECTIVE: Myotonia is caused by involuntary firing of skeletal muscle action potentials and causes debilitating stiffness. Current treatments are insufficiently efficacious and associated with side effects. Myotonia can be triggered by voluntary movement (electrically induced myotonia) or percussion (mechanically induced myotonia). Whether distinct molecular mechanisms underlie these triggers is unknown. Our goal was to identify ion channels involved in mechanically induced myotonia and to evaluate block of the channels involved as a novel approach to therapy. METHODS: We developed a novel system to enable study of mechanically induced myotonia using both genetic and pharmacologic mouse models of myotonia congenita. We extended ex vivo studies of excitability to in vivo studies of muscle stiffness. RESULTS: As previous work suggests activation of transient receptor potential vanilloid 4 (TRPV4) channels by mechanical stimuli in muscle, we examined the role of this cation channel. Mechanically induced myotonia was markedly suppressed in TRPV4-null muscles and in muscles treated with TRPV4 small molecule antagonists. The suppression of mechanically induced myotonia occurred without altering intrinsic muscle excitability, such that myotonia triggered by firing of action potentials (electrically induced myotonia) was unaffected. When injected intraperitoneally, TRPV4 antagonists lessened the severity of myotonia in vivo by approximately 80%. INTERPRETATION: These data demonstrate that there are distinct molecular mechanisms triggering electrically induced and mechanically induced myotonia. Our data indicates that activation of TRPV4 during muscle contraction plays an important role in triggering myotonia in vivo. Elimination of mechanically induced myotonia by TRPV4 inhibition offers a new approach to treating myotonia. ANN NEUROL 2020;88:297-308.
Asunto(s)
Contracción Isométrica/fisiología , Morfolinas/farmacología , Miotonía Congénita/genética , Miotonía Congénita/metabolismo , Pirroles/farmacología , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/deficiencia , Animales , Antracenos/farmacología , Contracción Isométrica/efectos de los fármacos , Ratones , Ratones Noqueados , Morfolinas/uso terapéutico , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología , Miotonía Congénita/prevención & control , Pirroles/uso terapéuticoRESUMEN
Blood-CNS barrier disruption is a hallmark of numerous neurological disorders, yet whether barrier breakdown is sufficient to trigger neurodegenerative disease remains unresolved. Therapeutic strategies to mitigate barrier hyperpermeability are also limited. Dominant missense mutations of the cation channel transient receptor potential vanilloid 4 (TRPV4) cause forms of hereditary motor neuron disease. To gain insights into the cellular basis of these disorders, we generated knock-in mouse models of TRPV4 channelopathy by introducing two disease-causing mutations (R269C and R232C) into the endogenous mouse Trpv4 gene. TRPV4 mutant mice exhibited weakness, early lethality, and regional motor neuron loss. Genetic deletion of the mutant Trpv4 allele from endothelial cells (but not neurons, glia, or muscle) rescued these phenotypes. Symptomatic mutant mice exhibited focal disruptions of blood-spinal cord barrier (BSCB) integrity, associated with a gain of function of mutant TRPV4 channel activity in neural vascular endothelial cells (NVECs) and alterations of NVEC tight junction structure. Systemic administration of a TRPV4-specific antagonist abrogated channel-mediated BSCB impairments and provided a marked phenotypic rescue of symptomatic mutant mice. Together, our findings show that mutant TRPV4 channels can drive motor neuron degeneration in a non-cell autonomous manner by precipitating focal breakdown of the BSCB. Further, these data highlight the reversibility of TRPV4-mediated BSCB impairments and identify a potential therapeutic strategy for patients with TRPV4 mutations.
Asunto(s)
Barrera Hematoencefálica , Células Endoteliales , Mutación con Ganancia de Función , Neuronas Motoras , Canales Catiónicos TRPV , Animales , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genética , Neuronas Motoras/patología , Neuronas Motoras/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Ratones , Degeneración Nerviosa/patología , Degeneración Nerviosa/genética , Fenotipo , Médula Espinal/patología , Médula Espinal/metabolismoRESUMEN
Crosstalk between ion channels and small GTPases is critical during homeostasis and disease, but little is known about the structural underpinnings of these interactions. TRPV4 is a polymodal, calcium-permeable cation channel that has emerged as a potential therapeutic target in multiple conditions. Gain-of-function mutations also cause hereditary neuromuscular disease. Here, we present cryo-EM structures of human TRPV4 in complex with RhoA in the ligand-free, antagonist-bound closed, and agonist-bound open states. These structures reveal the mechanism of ligand-dependent TRPV4 gating. Channel activation is associated with rigid-body rotation of the intracellular ankyrin repeat domain, but state-dependent interaction with membrane-anchored RhoA constrains this movement. Notably, many residues at the TRPV4-RhoA interface are mutated in disease and perturbing this interface by introducing mutations into either TRPV4 or RhoA increases TRPV4 channel activity. Together, these results suggest that RhoA serves as an auxiliary subunit for TRPV4, regulating TRPV4-mediated calcium homeostasis and disruption of TRPV4-RhoA interactions can lead to TRPV4-related neuromuscular disease. These insights will help facilitate TRPV4 therapeutics development.
Asunto(s)
Canales Catiónicos TRPV , Proteína de Unión al GTP rhoA , Humanos , Repetición de Anquirina , Calcio/metabolismo , Mutación , Canales Catiónicos TRPV/química , Proteína de Unión al GTP rhoA/químicaRESUMEN
Crosstalk between ion channels and small GTPases is critical during homeostasis and disease 1 , but little is known about the structural underpinnings of these interactions. TRPV4 is a polymodal, calcium-permeable cation channel that has emerged as a potential therapeutic target in multiple conditions 2-5 . Gain-of-function mutations also cause hereditary neuromuscular disease 6-11 . Here, we present cryo-EM structures of human TRPV4 in complex with RhoA in the apo, antagonist-bound closed, and agonist-bound open states. These structures reveal the mechanism of ligand-dependent TRPV4 gating. Channel activation is associated with rigid-body rotation of the intracellular ankyrin repeat domain, but state-dependent interaction with membrane-anchored RhoA constrains this movement. Notably, many residues at the TRPV4-RhoA interface are mutated in disease and perturbing this interface by introducing mutations into either TRPV4 or RhoA increases TRPV4 channel activity. Together, these results suggest that the interaction strength between TRPV4 and RhoA tunes TRPV4-mediated calcium homeostasis and actin remodeling, and that disruption of TRPV4-RhoA interactions leads to TRPV4-related neuromuscular disease, findings that will guide TRPV4 therapeutics development.
RESUMEN
Transplantation of exogenous stem cells has been proposed as a treatment to prevent or reverse sensorineural hearing loss. Here, we investigate the effects of transplantation of adult human olfactory mucosa-derived stem cells on auditory function in A/J mice, a strain exhibiting early-onset progressive sensorineural hearing loss. Recent evidence indicates that these stem cells exhibit multipotency in transplantation settings and may represent a subtype of mesenchymal stem cell. Olfactory stem cells were injected into the cochleae of A/J mice via a lateral wall cochleostomy during the time period in which hearing loss first becomes apparent. Changes in auditory function were assessed 1 month after transplantation and compared against animals that received sham injections. Hearing threshold levels in stem cell-transplanted mice were found to be significantly lower than those of sham-injected mice (p < .05) for both click and pure tone stimuli. Transplanted cells survived within the perilymphatic compartments but did not integrate into cochlear tissues. These results indicate that transplantation of adult human olfactory mucosa-derived stem cells can help preserve auditory function during early-onset progressive sensorineural hearing loss.
Asunto(s)
Células Madre Adultas/trasplante , Pérdida Auditiva Sensorineural/fisiopatología , Pérdida Auditiva Sensorineural/terapia , Vías Olfatorias , Animales , Umbral Auditivo/fisiología , Células Cultivadas , Cóclea/citología , Cóclea/fisiopatología , Células Ciliadas Auditivas , Audición/fisiología , Pérdida Auditiva Sensorineural/patología , Pruebas Auditivas , Humanos , Ratones , Ratones Endogámicos ARESUMEN
Noise trauma in mammals can result in damage to multiple epithelial cochlear cell types, producing permanent hearing loss. Here we investigate whether epithelial stem cell transplantation can ameliorate noise-induced hearing loss in mice. Epithelial stem/progenitor cells isolated from adult mouse tongue displayed extensive proliferation in vitro as well as positive immunolabelling for the epithelial stem cell marker p63. To examine the functional effects of cochlear transplantation of these cells, mice were exposed to noise trauma and the cells were transplanted via a lateral wall cochleostomy 2 days post-trauma. Changes in auditory function were assessed by determining auditory brainstem response (ABR) threshold shifts 4 weeks after stem cell transplantation or sham surgery. Stem/progenitor cell transplantation resulted in a significantly reduced permanent ABR threshold shift for click stimuli compared to sham-injected mice, as corroborated using two distinct analyses. Cell fate analyses revealed stem/progenitor cell survival and integration into suprastrial regions of the spiral ligament. These results suggest that transplantation of adult epithelial stem/progenitor cells can attenuate the ototoxic effects of noise trauma in a mammalian model of noise-induced hearing loss.
Asunto(s)
Cóclea/cirugía , Modelos Animales de Enfermedad , Células Epiteliales/trasplante , Pérdida Auditiva Provocada por Ruido/cirugía , Trasplante de Células Madre/métodos , Células Madre/fisiología , Animales , Proliferación Celular , Células Cultivadas , Cóclea/citología , Cóclea/patología , Células Epiteliales/citología , Células Epiteliales/patología , Femenino , Pérdida Auditiva Provocada por Ruido/patología , Pérdida Auditiva Provocada por Ruido/fisiopatología , Masculino , Ratones , Ratones Endogámicos CBA , Regeneración Nerviosa/fisiología , Fenotipo , Recuperación de la Función/fisiología , Células Madre/citología , Lengua/citología , Lengua/patología , Lengua/fisiologíaRESUMEN
TRPV4 is a cell surface-expressed calcium-permeable cation channel that mediates cell-specific effects on cellular morphology and function. Dominant missense mutations of TRPV4 cause distinct, tissue-specific diseases, but the pathogenic mechanisms are unknown. Mutations causing peripheral neuropathy localize to the intracellular N-terminal domain whereas skeletal dysplasia mutations are in multiple domains. Using an unbiased screen, we identified the cytoskeletal remodeling GTPase RhoA as a TRPV4 interactor. TRPV4-RhoA binding occurs via the TRPV4 N-terminal domain, resulting in suppression of TRPV4 channel activity, inhibition of RhoA activation, and extension of neurites in vitro. Neuropathy but not skeletal dysplasia mutations disrupt TRPV4-RhoA binding and cytoskeletal outgrowth. However, inhibition of RhoA restores neurite length in vitro and in a fly model of TRPV4 neuropathy. Together these results identify RhoA as a critical mediator of TRPV4-induced cell structure changes and suggest that disruption of TRPV4-RhoA binding may contribute to tissue-specific toxicity of TRPV4 neuropathy mutations.
Asunto(s)
Neuritas/metabolismo , Enfermedades del Sistema Nervioso Periférico/genética , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Células COS , Calcio/metabolismo , Línea Celular , Chlorocebus aethiops , Drosophila , Células HEK293 , HumanosRESUMEN
Adult taste buds are maintained by the lifelong proliferation of epithelial stem and progenitor cells, the identities of which have remained elusive. It has been proposed that these cells reside either within the taste bud (intragemmal) or in the surrounding epithelium (perigemmal). Here, we apply three different in vivo approaches enabling single-cell resolution of proliferative history to identify putative stem and progenitor cells associated with adult mouse taste buds. Experiments were performed across the circadian peak in oral epithelial proliferation (04:00 h), a time period in which mitotic activity in taste buds has not yet been detailed. Using double label pulse-chase experiments, we show that defined intragemmal cells (taste and basal) and perigemmal cells undergo rapid, sequential cell divisions and thus represent potential progenitor cells. Strikingly, mitotic activity was observed in taste cells previously thought to be postmitotic (labelled cells occur in 30% of palatal taste buds after 1 h of BrdU exposure). Basal cells showed expression of the transcription factor p63, required for maintaining the self-renewal potential of various epithelial stem cell types. Candidate taste stem cells were identified almost exclusively as basal cells using the label-retaining cell approach to localize slow-cycling cells (0.06 +/- 0.01 cells per taste bud; n = 436 taste buds). Together, these results indicate that both stem- and progenitor-like cells reside within the mammalian taste bud.
Asunto(s)
Células Madre Adultas/fisiología , Epitelio/fisiología , Nicho de Células Madre/fisiología , Papilas Gustativas/fisiología , Envejecimiento , Animales , División Celular , Proliferación Celular , Ritmo Circadiano/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos CBA , Mitosis , Fosfoproteínas/metabolismo , Transactivadores/metabolismoRESUMEN
The cation channel transient receptor potential vanilloid 4 (TRPV4) is one of the few identified ion channels that can directly cause inherited neurodegeneration syndromes, but the molecular mechanisms are unknown. Here, we show that in vivo expression of a neuropathy-causing TRPV4 mutant (TRPV4R269C) causes dose-dependent neuronal dysfunction and axonal degeneration, which are rescued by genetic or pharmacological blockade of TRPV4 channel activity. TRPV4R269C triggers increased intracellular Ca2+ through a Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated mechanism, and CaMKII inhibition prevents both increased intracellular Ca2+ and neurotoxicity in Drosophila and cultured primary mouse neurons. Importantly, TRPV4 activity impairs axonal mitochondrial transport, and TRPV4-mediated neurotoxicity is modulated by the Ca2+-binding mitochondrial GTPase Miro. Our data highlight an integral role for CaMKII in neuronal TRPV4-associated Ca2+ responses, the importance of tightly regulated Ca2+ dynamics for mitochondrial axonal transport, and the therapeutic promise of TRPV4 antagonists for patients with TRPV4-related neurodegenerative diseases.
Asunto(s)
Señalización del Calcio/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Drosophila melanogaster/metabolismo , Enfermedades Neurodegenerativas/genética , Canales Catiónicos TRPV/genética , Animales , Animales Modificados Genéticamente , Axones/patología , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Enfermedades Neurodegenerativas/patología , Alas de Animales/crecimiento & desarrolloRESUMEN
Notch signaling is a highly conserved intercellular pathway with tightly regulated and pleiotropic roles in normal tissue development and homeostasis. Dysregulated Notch signaling has also been implicated in human disease, including multiple forms of cancer, and represents an emerging therapeutic target. Successful development of such therapeutics requires a detailed understanding of potential on-target toxicities. Here, we identify autosomal dominant mutations of the canonical Notch ligand Jagged1 (or JAG1) as a cause of peripheral nerve disease in 2 unrelated families with the hereditary axonal neuropathy Charcot-Marie-Tooth disease type 2 (CMT2). Affected individuals in both families exhibited severe vocal fold paresis, a rare feature of peripheral nerve disease that can be life-threatening. Our studies of mutant protein posttranslational modification and localization indicated that the mutations (p.Ser577Arg, p.Ser650Pro) impair protein glycosylation and reduce JAG1 cell surface expression. Mice harboring heterozygous CMT2-associated mutations exhibited mild peripheral neuropathy, and homozygous expression resulted in embryonic lethality by midgestation. Together, our findings highlight a critical role for JAG1 in maintaining peripheral nerve integrity, particularly in the recurrent laryngeal nerve, and provide a basis for the evaluation of peripheral neuropathy as part of the clinical development of Notch pathway-modulating therapeutics.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Genes Dominantes , Proteína Jagged-1 , Mutación Missense , Transducción de Señal/genética , Sustitución de Aminoácidos , Animales , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/metabolismo , Femenino , Glicosilación , Humanos , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Masculino , Ratones , Receptores Notch/genética , Receptores Notch/metabolismoRESUMEN
BACKGROUND: Auditory brainstem responses (ABRs) are used to study auditory acuity in animal-based medical research. ABRs are evoked by acoustic stimuli, and consist of an electrical signal resulting from summated activity in the auditory nerve and brainstem nuclei. ABR analysis determines the sound intensity at which a neural response first appears (hearing threshold). Traditionally, threshold has been assessed by visual estimation of a series of ABRs evoked by different sound intensities. Here we develop an automated threshold detection method that eliminates the variability and subjectivity associated with visual estimation. RESULTS: The automated method is a robust computational procedure that detects the sound level at which the peak amplitude of the evoked ABR signal first exceeds four times the standard deviation of the baseline noise. Implementation of the procedure was achieved by evoking ABRs in response to click and tone stimuli, under normal and experimental conditions (adult stem cell transplantation into cochlea). Automated detection revealed that the threshold shift from pre- to post-surgery hearing levels was similar in mice receiving stem cell transplantation or sham injection for click and tone stimuli. Visual estimation by independent observers corroborated these results but revealed variability in ABR threshold shifts and significance levels for stem cell-transplanted and sham-injected animals. CONCLUSION: In summary, the automated detection method avoids the subjectivity of visual analysis and offers a rapid, easily accessible http://axograph.com/source/abr.html approach to measure hearing threshold levels in auditory brainstem response.
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Umbral Diferencial , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Trasplante de Células Madre , Estimulación Acústica , Animales , Umbral Auditivo/fisiología , Células Cultivadas , Sordera/fisiopatología , Oído Interno/citología , Oído Interno/trasplante , Femenino , Audición/fisiología , Masculino , RatonesRESUMEN
Adult neurogenesis, the generation of new neurons from adult precursor cells, occurs in the brains of a phylogenetically diverse array of animals. In the higher (amniotic) vertebrates, these precursor cells are glial cells that reside within specialized regions, known as neurogenic niches, the elements of which both support and regulate neurogenesis. The in vivo identity and location of the precursor cells responsible for adult neurogenesis in nonvertebrate taxa, however, remain largely unknown. Among the invertebrates, adult neurogenesis has been particularly well characterized in freshwater crayfish (Arthropoda, Crustacea), although the identity of the precursor cells sustaining continuous neuronal proliferation in these animals has yet to be established. Here we provide evidence suggesting that, as in the higher vertebrates, the precursor cells maintaining adult neurogenesis in the crayfish Procambarus clarkii are glial cells. These precursor cells reside within a specialized region, or niche, on the ventral surface of the brain, and their progeny migrate from this niche along glial fibers and then proliferate to form new neurons in the central olfactory pathway. The niche in which these precursor cells reside has many features in common with the neurogenic niches of higher vertebrates. These commonalities include: glial cells functioning as both precursor and support cells, directed migration, close association with the brain vasculature, and specialized basal laminae. The cellular machinery maintaining adult neurogenesis appears, therefore, to be shared by widely disparate taxa. These extensive structural and functional parallels suggest a common strategy for the generation of new neurons in adult brains.
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Astacoidea/fisiología , Encéfalo/fisiología , Diferenciación Celular/fisiología , Neuroglía/fisiología , Neuronas/fisiología , Células Madre/fisiología , Animales , Astacoidea/citología , Encéfalo/citología , Movimiento Celular/fisiología , Femenino , Colorantes Fluorescentes , Masculino , Neuroglía/citología , Neuronas/citología , Vías Olfatorias/citología , Vías Olfatorias/fisiología , Filogenia , Especificidad de la Especie , Células Madre/citologíaRESUMEN
Adult neurogenesis is a characteristic feature of the olfactory pathways of decapod crustaceans. In crayfish and clawed lobsters, adult-born neurons are the progeny of precursor cells with glial characteristics located in a neurogenic niche on the ventral surface of the brain. The daughters of these precursor cells migrate during S and G(2 )stages of the cell cycle along glial fibers to lateral (cluster 10) and medial (cluster 9) proliferation zones. Here, they divide (M phase) producing offspring that differentiate into olfactory interneurons. The complete lineage of cells producing neurons in these animals, therefore, is arranged along the migratory stream according to cell cycle stage. We have exploited this model to examine the influence of environmental and endogenous factors on adult neurogenesis. We find that increased levels of serotonin upregulate neuronal production, as does maintaining animals in an enriched (versus deprived) environment or augmenting their diet with omega-3 fatty acids; increased levels of nitric oxide, on the other hand, decrease the rate of neurogenesis. The features of the neurogenic niche and migratory streams, and the fact that these continue to function in vitro, provide opportunities unavailable in other organisms to explore the sequence of cellular and molecular events leading to the production of new neurons in adult brains.
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Crustáceos/fisiología , Interneuronas/fisiología , Neuronas/citología , Bulbo Olfatorio/citología , Vías Olfatorias/citología , Envejecimiento , Animales , Ciclo Celular/fisiología , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Crustáceos/citología , Ácidos Grasos Omega-3/metabolismo , Neuronas/fisiología , Óxido Nítrico/metabolismo , Bulbo Olfatorio/fisiología , Vías Olfatorias/fisiología , Serotonina/metabolismo , Células Madre/fisiologíaRESUMEN
Information about the input and output pathways of higher-order brain neuropils is essential for gaining an understanding of their functions. The present study examines the connectivity of two higher-order neuropils in the central olfactory pathway of the crayfish: the accessory lobe and its target neuropil, the hemiellipsoid body. It is known that the two subregions of the accessory lobe, the cortex and medulla, receive different inputs; the medulla receives visual and tactile inputs, whereas the cortex receives neither (Sandeman et al. [1995] J Comp Neurol 352:263-279). By using dye injections into the olfactory lobe, we demonstrate that the accessory lobe cortex and medulla also have differing connections with the olfactory lobe. These injections show that local interneurons joining the olfactory and accessory lobes branch primarily within the cortex with only limited branching within the medulla. Injections of different dyes into the two subregions of the hemiellipsoid body, HBI and HBII, show that the accessory lobe cortex and medulla also have separate output pathways. HBI is innervated by the output pathway from the cortex while HBII is innervated by the output pathway from the medulla. These injections also show that HBI and HBII are innervated by separate populations of local interneurons with differing connections to higher-order neuropils in the olfactory and visual pathways. These results suggest a segregation of olfactory and multimodal (including olfactory) inputs within both the accessory lobe and the hemiellipsoid body and provide evidence of important functional subdivisions within both neuropils.
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Astacoidea/fisiología , Encéfalo/fisiología , Interneuronas/fisiología , Neuronas/fisiología , Neurópilo/fisiología , Vías Olfatorias/fisiología , Animales , Astacoidea/anatomía & histología , Encéfalo/citología , Femenino , Ganglios de Invertebrados/citología , Ganglios de Invertebrados/fisiología , Masculino , Neurópilo/citología , Vías Olfatorias/citología , Percepción/fisiología , Vías Visuales/citología , Vías Visuales/fisiologíaRESUMEN
OBJECTIVE: To characterize 2 novel TRPV4 mutations in 2 unrelated families exhibiting the Charcot-Marie-Tooth disease type 2C (CMT2C) phenotype. METHODS: Direct CMT gene testing was performed on 2 unrelated families with CMT2C. A 4-fold symmetric tetramer model of human TRPV4 was generated to map the locations of novel TRPV4 mutations in these families relative to previously identified disease-causing mutations (neuropathy, skeletal dysplasia, and osteoarthropathy). Effects of the mutations on TRPV4 expression, localization, and channel activity were determined by immunocytochemical, immunoblotting, Ca(2+) imaging, and cytotoxicity assays. RESULTS: Previous studies suggest that neuropathy-causing mutations occur primarily at arginine residues on the convex face of the TRPV4 ankyrin repeat domain (ARD). Further highlighting the key role of this domain in TRPV4-mediated hereditary neuropathy, we report 2 novel heterozygous missense mutations in the TRPV4-ARD convex face (p.Arg237Gly and p.Arg237Leu). Generation of a model of the TRPV4 homotetramer revealed that while ARD residues mutated in neuropathy (including Arg237) are likely accessible for intermolecular interactions, skeletal dysplasia-causing TRPV4 mutations occur at sites suggesting disruption of intramolecular and/or intersubunit interactions. Like previously described neuropathy-causing mutations, the p.Arg237Gly and p.Arg237Leu substitutions do not alter TRPV4 subcellular localization in transfected cells but cause elevations of cytosolic Ca(2+) levels and marked cytotoxicity. CONCLUSIONS: These findings expand the number of ARD residues mutated in TRPV4-mediated neuropathy, providing further evidence of the central importance of this domain to TRPV4 function in peripheral nerve.
RESUMEN
Output from the olfactory lobe (primary olfactory center) of eumalacostracan crustaceans is transmitted to the medulla terminalis (MT) and hemiellipsoid body (HB) in the lateral protocerebrum (higher order center) by a large population of projection neurons. In eureptantian crustaceans (lobsters, crayfish, and crabs), these projection neurons also form the output pathway from an additional neuropil, the accessory lobe (higher order center), which appears to have arisen de novo in these animals. In a previous study of lobsters and crayfish we showed that whereas projection neurons innervating the olfactory lobe project primarily to the MT, those innervating the accessory lobe project exclusively to the HB (Sullivan and Beltz [ 2001a] J. Comp. Neurol. 441:9-22). In the present study, we used focal dye injections to examine the olfactory projection neuron pathways of representatives of four eumalacostracan taxa (Stomatopoda, Dendrobranchiata, Caridea, and Stenopodidea) that diverged from the eureptantian line prior to the appearance of the accessory lobe. These experiments were undertaken both to examine the evolution of the olfactory pathway in the Eumalacostraca and to provide insights into the changes in this pathway that accompanied the appearance of the accessory lobe. The innervation patterns of the olfactory projection neurons of the species examined were found to differ markedly, varying from that observed in the most basal taxon examined (Stomatopoda), in which the neurons primarily project to the MT, to those observed in the two highest taxa examined (Caridea and Stenopodidea), in which they primarily target the HB. These results suggest that substantial changes in the relative importance of the MT and HB within the olfactory pathway have occurred during the evolution of the Eumalacostraca.
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Evolución Biológica , Neuronas/fisiología , Vías Olfatorias/metabolismo , Animales , Crustáceos , Drosophila , Técnica del Anticuerpo Fluorescente/métodos , Lateralidad Funcional , Ganglios de Invertebrados/citología , Bulbo Raquídeo/citología , Bulbo Raquídeo/metabolismo , Microscopía Confocal/métodos , Modelos Neurológicos , Neurópilo/citología , Neurópilo/metabolismo , Vías Olfatorias/citología , Compuestos de Piridinio/metabolismo , Sinapsinas/metabolismoRESUMEN
Presynaptic resting Ca(2+) influences synaptic vesicle (SV) release probability. Here, we report that a TRPV channel, Inactive (Iav), maintains presynaptic resting [Ca(2+)] by promoting Ca(2+) release from the endoplasmic reticulum in Drosophila motor neurons, and is required for both synapse development and neurotransmission. We find that Iav activates the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin, which is essential for presynaptic microtubule stabilization at the neuromuscular junction. Thus, loss of Iav induces destabilization of presynaptic microtubules, resulting in diminished synaptic growth. Interestingly, expression of human TRPV1 in Iav-deficient motor neurons rescues these defects. We also show that the absence of Iav causes lower SV release probability and diminished synaptic transmission, whereas Iav overexpression elevates these synaptic parameters. Together, our findings indicate that Iav acts as a key regulator of synaptic development and function by influencing presynaptic resting [Ca(2+)].