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1.
J Cell Sci ; 135(5)2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33771931

RESUMO

The lipid composition of the primary cilia membrane is emerging as a critical regulator of cilia formation, maintenance and function. Here, we show that conditional deletion of the phosphoinositide 5'-phosphatase gene Inpp5e, mutation of which is causative of Joubert syndrome, in terminally developed mouse olfactory sensory neurons (OSNs), leads to a dramatic remodeling of ciliary phospholipids that is accompanied by marked elongation of cilia. Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2], which is normally restricted to the proximal segment redistributed to the entire length of cilia in Inpp5e knockout mice with a reduction in phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2] and elevation of phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] in the dendritic knob. The redistribution of phosphoinositides impaired odor adaptation, resulting in less efficient recovery and altered inactivation kinetics of the odor-evoked electrical response and the odor-induced elevation of cytoplasmic Ca2+. Gene replacement of Inpp5e through adenoviral expression restored the ciliary localization of PI(4,5)P2 and odor response kinetics in OSNs. Our findings support the role of phosphoinositides as a modulator of the odor response and in ciliary biology of native multi-ciliated OSNs.

2.
Chem Senses ; 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33690843

RESUMO

Olfactory dysfunction is a common disorder in the general population. There are multiple causes, one of which being ciliopathies, an emerging class of human hereditary genetic disorders characterized by multiple symptoms due to defects in ciliary biogenesis, maintenance, and/or function. Mutations/deletions in a wide spectrum of ciliary genes have been identified to cause ciliopathies. Currently, besides symptomatic therapy, there is no available therapeutic treatment option for olfactory dysfunction caused by ciliopathies. Multiple studies have demonstrated that targeted gene replacement can restore the morphology and function of olfactory cilia in olfactory sensory neurons and further re-establish the odor-guided behaviors in animals. Therefore, targeted gene replacement could be potentially used to treat olfactory dysfunction in ciliopathies. However, due to the potential limitations of single gene therapy for polygenic mutation-induced diseases, alternative therapeutic targets for broader curative measures need to be developed for olfactory dysfunction, and also for other symptoms in ciliopathies. Here we review the current understanding of ciliogenesis and maintenance of olfactory cilia. Furthermore, we emphasize signaling mechanisms that may be involved in the regulation of olfactory ciliary length and highlight potential alternative therapeutic targets for the treatment of ciliopathy induced dysfunction in the olfactory system and even in other ciliated organ systems.

3.
Mol Cell Neurosci ; 110: 103585, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33358996

RESUMO

Olfactory GPCRs (ORs) in mammalian olfactory receptor neurons (ORNs) mediate excitation through the Gαs family member Gαolf. Here we tentatively associate a second G protein, Gαo, with inhibitory signaling in mammalian olfactory transduction by first showing that odor evoked phosphoinositide 3-kinase (PI3K)-dependent inhibition of signal transduction is absent in the native ORNs of mice carrying a conditional OMP-Cre based knockout of Gαo. We then identify an OR from native rat ORNs that are activated by octanol through cyclic nucleotide signaling and inhibited by citral in a PI3K-dependent manner. We show that the OR activates cyclic nucleotide signaling and PI3K signaling in a manner that reflects its functionality in native ORNs. Our findings lay the groundwork to explore the interesting possibility that ORs can interact with two different G proteins in a functionally identified, ligand-dependent manner to mediate opponent signaling in mature mammalian ORNs.

4.
Chem Senses ; 45(9): 805-822, 2020 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-33075817

RESUMO

Olfactory sensory neurons (OSNs) are bipolar neurons, unusual because they turn over continuously and have a multiciliated dendrite. The extensive changes in gene expression accompanying OSN differentiation in mice are largely known, especially the transcriptional regulators responsible for altering gene expression, revealing much about how differentiation proceeds. Basal progenitor cells of the olfactory epithelium transition into nascent OSNs marked by Cxcr4 expression and the initial extension of basal and apical neurites. Nascent OSNs become immature OSNs within 24-48 h. Immature OSN differentiation requires about a week and at least 2 stages. Early-stage immature OSNs initiate expression of genes encoding key transcriptional regulators and structural proteins necessary for further neuritogenesis. Late-stage immature OSNs begin expressing genes encoding proteins important for energy production and neuronal homeostasis that carry over into mature OSNs. The transition to maturity depends on massive expression of one allele of one odorant receptor gene, and this results in expression of the last 8% of genes expressed by mature OSNs. Many of these genes encode proteins necessary for mature function of axons and synapses or for completing the elaboration of non-motile cilia, which began extending from the newly formed dendritic knobs of immature OSNs. The cilia from adjoining OSNs form a meshwork in the olfactory mucus and are the site of olfactory transduction. Immature OSNs also have a primary cilium, but its role is unknown, unlike the critical role in proliferation and differentiation played by the primary cilium of the olfactory epithelium's horizontal basal cell.

5.
ACS Chem Biol ; 15(9): 2516-2528, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32865973

RESUMO

The chemosensory system of any animal relies on a vast array of detectors tuned to distinct chemical cues. Odorant receptors and the ion channels of the TRP family are all uniquely expressed in olfactory tissues in a species-specific manner. Great effort has been made to characterize the molecular and pharmacological properties of these proteins. Nevertheless, most of the natural ligands are highly hydrophobic molecules that are not amenable to controlled delivery. We sought to develop photoreleasable, biologically inactive odorants that could be delivered to the target receptor or ion channel and effectively activated by a short light pulse. Chemically distinct ligands eugenol, benzaldehyde, 2-phenethylamine, ethanethiol, butane-1-thiol, and 2,2-dimethylethane-1-thiol were modified by covalently attaching the photoremovable protecting group (8-cyano-7-hydroxyquinolin-2-yl)methyl (CyHQ). The CyHQ derivatives were shown to release the active odorant upon illumination with 365 and 405 nm light. We characterized their bioactivity by measuring activation of recombinant TRPV1 and TRPA1 ion channels expressed in HEK 293 cells and the electroolfactogram (EOG) response from intact mouse olfactory epithelium (OE). Illumination with 405 nm light was sufficient to robustly activate TRP channels within milliseconds of the light pulse. Photoactivation of channels was superior to activation by conventional bath application of the ligands. Photolysis of the CyHQ-protected odorants efficiently activated an EOG response in a dose-dependent manner with kinetics similar to that evoked by the vaporized odorant amyl acetate (AAc). We conclude that CyHQ-based, photoreleasable odorants can be successfully implemented in chemosensory research.

6.
Chem Senses ; 45(7): 493-502, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32556127

RESUMO

The chemical senses of taste and smell play a vital role in conveying information about ourselves and our environment. Tastes and smells can warn against danger and also contribute to the daily enjoyment of food, friends and family, and our surroundings. Over 12% of the US population is estimated to experience taste and smell (chemosensory) dysfunction. Yet, despite this high prevalence, long-term, effective treatments for these disorders have been largely elusive. Clinical successes in other sensory systems, including hearing and vision, have led to new hope for developments in the treatment of chemosensory disorders. To accelerate cures, we convened the "Identifying Treatments for Taste and Smell Disorders" conference, bringing together basic and translational sensory scientists, health care professionals, and patients to identify gaps in our current understanding of chemosensory dysfunction and next steps in a broad-based research strategy. Their suggestions for high-yield next steps were focused in 3 areas: increasing awareness and research capacity (e.g., patient advocacy), developing and enhancing clinical measures of taste and smell, and supporting new avenues of research into cellular and therapeutic approaches (e.g., developing human chemosensory cell lines, stem cells, and gene therapy approaches). These long-term strategies led to specific suggestions for immediate research priorities that focus on expanding our understanding of specific responses of chemosensory cells and developing valuable assays to identify and document cell development, regeneration, and function. Addressing these high-priority areas should accelerate the development of novel and effective treatments for taste and smell disorders.

7.
Chem Senses ; 44(8): 583-592, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31420672

RESUMO

Published evidence suggests that inherent rhythmically active or "bursting" primary olfactory receptor neurons (bORNs) in crustaceans have the previously undescribed functional property of encoding olfactory information by having their rhythmicity entrained by the odor stimulus. In order to determine whether such bORN-based encoding is a fundamental feature of olfaction that extends beyond crustaceans, we patch-clamped bORN-like ORNs in mice, characterized their dynamic properties, and show they align with the dynamic properties of lobster bORNs. We then characterized bORN-like activity by imaging the olfactory epithelium of OMP-GCaMP6f mice. Next, we showed rhythmic activity is not dependent upon the endogenous OR by patching ORNs in OR/GFP mice. Lastly, we showed the properties of bORN-like ORNs characterized in mice generalize to rats. Our findings suggest encoding odor time should be viewed as a fundamental feature of olfaction with the potential to be used to navigate odor plumes in animals as diverse as crustaceans and mammals.


Assuntos
Cálcio/fisiologia , Potenciais Somatossensoriais Evocados/fisiologia , Odorantes/análise , Neurônios Receptores Olfatórios/fisiologia , Olfato/fisiologia , Animais , Cálcio/análise , Potenciais Somatossensoriais Evocados/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Imagem Molecular , Nephropidae , Mucosa Olfatória/citologia , Mucosa Olfatória/fisiologia , Neurônios Receptores Olfatórios/citologia , Técnicas de Patch-Clamp , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley
8.
Methods Mol Biol ; 1950: 283-297, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30783981

RESUMO

Intranasal delivery of solutions is a straightforward methodology for viral vector transduction and gene transfer to the epithelia within the nasal cavity. Beyond the simplicity of the technique, intranasal delivery has demonstrated restricted transduction of the olfactory and respiratory epithelial tissues. Here we outline the procedure of viral vector intranasal delivery in early postnatal and adult mice, as well as adult rats. The procedure allows for robust transduction and ectopic gene delivery that can be used for the visualization of cellular structures, protein distribution, and assessment of viral vector-mediated therapies.


Assuntos
Adenoviridae/genética , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Mucosa Olfatória/metabolismo , Transdução Genética , Administração Intranasal , Animais , Injeções , Mamíferos , Camundongos , Ratos
9.
J Cell Sci ; 132(5)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30665891

RESUMO

Bardet-Beidl syndrome (BBS) manifests from genetic mutations encoding for one or more BBS proteins. BBS4 loss impacts olfactory ciliation and odor detection, yet the cellular mechanisms remain unclear. Here, we report that Bbs4-/- mice exhibit shorter and fewer olfactory sensory neuron (OSN) cilia despite retaining odorant receptor localization. Within Bbs4-/- OSN cilia, we observed asynchronous rates of IFT-A/B particle movements, indicating miscoordination in IFT complex trafficking. Within the OSN dendritic knob, the basal bodies are dynamic, with incorporation of ectopically expressed centrin-2 and γ-tubulin occurring after nascent ciliogenesis. Importantly, BBS4 loss results in the reduction of basal body numbers separate from cilia loss. Adenoviral expression of BBS4 restored OSN cilia lengths and was sufficient to re-establish odor detection, but failed to rescue ciliary and basal body numbers. Our results yield a model for the plurality of BBS4 functions in OSNs that includes intraciliary and periciliary roles that can explain the loss of cilia and penetrance of ciliopathy phenotypes in olfactory neurons.


Assuntos
Síndrome de Bardet-Biedl/metabolismo , Cílios/fisiologia , Flagelos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios Receptores Olfatórios/fisiologia , Animais , Corpos Basais/patologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Fenótipo , Transporte Proteico , Olfato , Combinação Trimetoprima e Sulfametoxazol/metabolismo , Tubulina (Proteína)/metabolismo
10.
Curr Med Chem ; 26(17): 3103-3119, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-29303074

RESUMO

BACKGROUND: Ciliopathies are a class of inherited pleiotropic genetic disorders in which alterations in cilia assembly, maintenance, and/or function exhibit penetrance in the multiple organ systems. Olfactory dysfunction is one such clinical manifestation that has been shown in both patients and model organisms. Existing therapies for ciliopathies are limited to the treatment or management of symptoms. The last decade has seen an increase in potential curative therapeutic options including small molecules and biologics. Recent work in multiciliated olfactory sensory neurons has demonstrated the capacity of targeted gene therapy to restore ciliation in terminally differentiated cells and rescue olfactory function. This review will discuss the current understanding of the penetrance of ciliopathies in the olfactory system. Importantly, it will highlight both pharmacological and biological approaches, and their potential therapeutic value in the olfactory system and other ciliated tissues. METHODS: We undertook a structured and comprehensive search of peer-reviewed research literature encompassing in vitro, in vivo, model organism, and clinical studies. From these publications, we describe the olfactory system, and discuss the penetrance of ciliopathies and impact of cilia loss on olfactory function. In addition, we outlined the developing therapies for ciliopathies across different organ and cell culture systems, and discussed their potential therapeutic application to the mammalian olfactory system. RESULTS: One-hundred sixty-one manuscripts were included in the review, centering on the understanding of olfactory penetrance of ciliopathies, and discussing the potential therapeutic options for ciliopathies in the context of the mammalian olfactory system. Forty-four manuscripts were used to generate a table listing the known congenital causes of olfactory dysfunction, with the first ten listed are linked to ciliopathies. Twenty-three manuscripts were used to outline the potential of small molecules for the olfactory system. Emphasis was placed on HDAC6 inhibitors and lithium, both of which were shown to stabilize microtubule structures, contributing to ciliogenesis and cilia lengthening. Seventy-five manuscripts were used to describe gene therapy and gene therapeutic strategies. Included were the implementation of adenoviral, adeno-associated virus (AAV), and lentiviral vectors to treat ciliopathies across different organ systems and application toward the olfactory system. Thus far, adenoviral and AAVmeditated ciliary restoration demonstrated successful proof-of-principle preclinical studies. In addition, gene editing, ex vivo gene therapy, and transplantation could serve as alternative therapeutic and long-term approaches. But for all approaches, additional assessment of vector immunogenicity, specificity, and efficacy need further investigation. Currently, ciliopathy treatments are limited to symptomatic management with no curative options. However, the accessibility and amenability of the olfactory system to treatment would facilitate development and advancement of a viable therapy. CONCLUSION: The findings of this review highlight the contribution of ciliopathies to a growing list of congenial olfactory dysfunctions. Promising results from other organ systems imply the feasibility of biologics, with results from gene therapies proving to be a viable therapeutic option for ciliopathies and olfactory dysfunction.


Assuntos
Cílios/efeitos dos fármacos , Ciliopatias/terapia , Terapia Genética , Transtornos do Olfato/terapia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Cílios/metabolismo , Ciliopatias/metabolismo , Humanos , Transtornos do Olfato/metabolismo
11.
FASEB J ; 33(1): 1440-1455, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30133325

RESUMO

The transition zone (TZ) is a domain at the base of the cilium that is involved in maintaining ciliary compartment-specific sensory and signaling activity by regulating cilia protein composition. Mutations in TZ proteins result in cilia dysfunction, often causing pleiotropic effects observed in a group of human diseases classified as ciliopathies. The purpose of this study is to describe the importance of the TZ component Meckel-Grüber syndrome 6 ( Mks6) in several organ systems and tissues regarding ciliogenesis and cilia maintenance using congenital and conditional mutant mouse models. Similar to MKS, congenital loss of Mks6 is embryonic lethal, displaying cilia loss and altered cytoskeletal microtubule modifications but only in specific cell types. Conditional Mks6 mutants have a variable cystic kidney phenotype along with severe retinal degeneration with mislocalization of phototransduction cascade proteins. However, other phenotypes, such as anosmia and obesity, which are typically associated with cilia and TZ dysfunction, were not evident. These data indicate that despite Mks6 being a core TZ component, it has tissue- or cell type-specific functions important for cilia formation and cilia sensory and signaling activities. Lewis, W. R., Bales, K. L., Revell, D. Z., Croyle, M. J., Engle, S. E., Song, C. J., Malarkey, E. B., Uytingco, C. R., Shan, D., Antonellis, P. J., Nagy, T. R., Kesterson, R. A., Mrug, M. M., Martens, J. R., Berbari, N. F., Gross, A. K., Yoder, B. K. Mks6 mutations reveal tissue- and cell type-specific roles for the cilia transition zone.


Assuntos
Cílios/metabolismo , Proteínas do Citoesqueleto/genética , Mutação , Acetilação , Animais , Transtornos da Motilidade Ciliar/genética , Citoplasma/metabolismo , Encefalocele/genética , Feminino , Genes Letais , Doenças Renais Císticas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Transtornos do Olfato/genética , Fenótipo , Doenças Renais Policísticas/genética , Degeneração Retiniana/genética , Retinite Pigmentosa/genética , Tubulina (Proteína)/metabolismo , Ganho de Peso/genética
12.
J Neurosci ; 38(34): 7462-7475, 2018 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30061191

RESUMO

Cilia of olfactory sensory neurons (OSNs) are the primary site of odor binding; hence, their loss results in anosmia, a clinical manifestation of pleiotropic ciliopathies for which there are no curative therapies. We used OSN-specific Ift88 knock-out mice (Ift88osnKO) of both sexes to examine the mechanisms of ciliopathy-induced olfactory dysfunction and the potential for gene replacement to rescue odorant detection, restore olfactory circuitry, and restore odor-guided behaviors. Loss of OSN cilia in Ift88osnKO mice resulted in substantially reduced odor detection and odor-driven synaptic activity in the olfactory bulb (OB). Defects in OSN axon targeting to the OB were also observed in parallel with aberrant odor-guided behavior. Intranasal gene delivery of wild-type IFT88 to Ift88osnKO mice rescued OSN ciliation and peripheral olfactory function. Importantly, this recovery of sensory input in a limited number of mature OSNs was sufficient to restore axonal targeting in the OB of juvenile mice, and with delayed onset in adult mice. In addition, restoration of sensory input re-established course odor-guided behaviors. These findings highlight the spare capacity of the olfactory epithelium and the plasticity of primary synaptic input into the central olfactory system. The restoration of peripheral and central neuronal function supports the potential for treatment of ciliopathy-related anosmia using gene therapy.SIGNIFICANCE STATEMENT Ciliopathies, for which there are no curative therapies, are genetic disorders that alter cilia morphology and/or function in numerous tissue types, including the olfactory system, leading to sensory dysfunction. We show that in vivo intranasal gene delivery restores peripheral olfactory function in a ciliopathy mouse model, including axonal targeting in the juvenile and adult olfactory bulb. Gene therapy also demonstrated restoration of olfactory perception by rescuing odor-guided behaviors. Understanding the therapeutic window and viability for gene therapy to restore odor detection and perception may facilitate translation of therapies to ciliopathy patients with olfactory dysfunctions.


Assuntos
Ciliopatias/terapia , Terapia Genética , Transtornos do Olfato/terapia , Neurônios Receptores Olfatórios/fisiologia , Proteínas Supressoras de Tumor/uso terapêutico , Adenoviridae , Administração Intranasal , Fatores Etários , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Cílios/ultraestrutura , Feminino , Genes Reporter , Vetores Genéticos/administração & dosagem , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Odorantes , Bulbo Olfatório/fisiopatologia , Mucosa Olfatória/patologia , Percepção Olfatória/fisiologia , Neurônios Receptores Olfatórios/ultraestrutura , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologia
13.
Mol Ther ; 25(4): 904-916, 2017 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-28237838

RESUMO

Olfactory dysfunction is a pervasive but underappreciated health concern that affects personal safety and quality of life. Patients with olfactory dysfunctions have limited therapeutic options, particularly those involving congenital diseases. Bardet-Biedl syndrome (BBS) is one such disorder, where olfactory loss and other symptoms manifest from defective cilium morphology and/or function in various cell types/tissues. Olfactory sensory neurons (OSNs) of BBS mutant mice lack the capacity to build/maintain cilia, rendering the cells incapable of odor detection. Here we examined OSN cilium defects in Bbs1 mutant mice and assessed the utility of gene therapy to restore ciliation and function in young and adult mice. Bbs1 mutant mice possessed short residual OSN cilia in which BBSome protein trafficking and odorant detection were defective. Gene therapy with an adenovirus-delivered wild-type Bbs1 gene restored OSN ciliation, corrected BBSome cilium trafficking defects, and returned acute odor responses. Finally, using clinically approved AAV serotypes, we demonstrate, for the first time, the capacity of AAVs to restore ciliation and odor detection in OSNs of Bbs1 mutants. Together, our data demonstrate that OSN ciliogenesis can be promoted in differentiated cells of young and adult Bbs1 mutants and highlight the potential of gene therapy as a viable restorative treatment for congenital olfactory disorders.


Assuntos
Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/fisiopatologia , Terapia Genética , Neurônios Receptores Olfatórios/metabolismo , Alelos , Animais , Síndrome de Bardet-Biedl/terapia , Cílios/metabolismo , Cílios/patologia , Dependovirus/genética , Modelos Animais de Doenças , Expressão Ectópica do Gene , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Percepção Olfatória/genética , Fenótipo , Transporte Proteico , Transdução Genética
15.
Mol Cancer Res ; 14(1): 26-34, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26573141

RESUMO

UNLABELLED: Polycomb proteins are essential regulators of gene expression in stem cells and development. They function to reversibly repress gene transcription via posttranslational modification of histones and chromatin compaction. In many human cancers, genes that are repressed by polycomb in stem cells are subject to more stable silencing via DNA methylation of promoter CpG islands. Ewing sarcoma is an aggressive bone and soft-tissue tumor that is characterized by overexpression of polycomb proteins. This study investigates the DNA methylation status of polycomb target gene promoters in Ewing sarcoma tumors and cell lines and observes that the promoters of differentiation genes are frequent targets of CpG-island DNA methylation. In addition, the promoters of ion channel genes are highly differentially methylated in Ewing sarcoma compared with nonmalignant adult tissues. Ion channels regulate a variety of biologic processes, including proliferation, and dysfunction of these channels contributes to tumor pathogenesis. In particular, reduced expression of the voltage-gated Kv1.5 channel has been implicated in tumor progression. These data show that DNA methylation of the KCNA5 promoter contributes to stable epigenetic silencing of the Kv1.5 channel. This epigenetic repression is reversed by exposure to the DNA methylation inhibitor decitabine, which inhibits Ewing sarcoma cell proliferation through mechanisms that include restoration of the Kv1.5 channel function. IMPLICATIONS: This study demonstrates that promoters of ion channels are aberrantly methylated in Ewing sarcoma and that epigenetic silencing of KCNA5 contributes to tumor cell proliferation, thus providing further evidence of the importance of ion channel dysregulation to tumorigenesis.


Assuntos
Azacitidina/análogos & derivados , Neoplasias Ósseas/genética , Canal de Potássio Kv1.5/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Sarcoma de Ewing/genética , Azacitidina/farmacologia , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ilhas de CpG/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Decitabina , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sarcoma de Ewing/patologia
16.
J Neurosci ; 35(40): 13761-72, 2015 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-26446227

RESUMO

UNLABELLED: The olfactory epithelium (OE) is one of the few tissues to undergo constitutive neurogenesis throughout the mammalian lifespan. It is composed of multiple cell types including olfactory sensory neurons (OSNs) that are readily replaced by two populations of basal stem cells, frequently dividing globose basal cells and quiescent horizontal basal cells (HBCs). However, the precise mechanisms by which these cells mediate OE regeneration are unclear. Here, we show for the first time that the HBC subpopulation of basal stem cells uniquely possesses primary cilia that are aligned in an apical orientation in direct apposition to sustentacular cell end feet. The positioning of these cilia suggests that they function in the detection of growth signals and/or differentiation cues. To test this idea, we generated an inducible, cell type-specific Ift88 knock-out mouse line (K5rtTA;tetOCre;Ift88(fl/fl)) to disrupt cilia formation and maintenance specifically in HBCs. Surprisingly, the loss of HBC cilia did not affect the maintenance of the adult OE but dramatically impaired the regeneration of OSNs following lesion. Furthermore, the loss of cilia during development resulted in a region-specific decrease in neurogenesis, implicating HBCs in the establishment of the OE. Together, these results suggest a novel role for primary cilia in HBC activation, proliferation, and differentiation. SIGNIFICANCE STATEMENT: We show for the first time the presence of primary cilia on a quiescent population of basal stem cells, the horizontal basal cells (HBCs), in the olfactory epithelium (OE). Importantly, our data demonstrate that cilia on HBCs are necessary for regeneration of the OE following injury. Moreover, the disruption of HBC cilia alters neurogenesis during the development of the OE, providing evidence that HBCs participate in the establishment of this tissue. These data suggest that the mechanisms of penetrance for ciliopathies in the OE extend beyond that of defects in olfactory sensory neurons and may include alterations in OE maintenance and regeneration.


Assuntos
Cílios/genética , Mucosa Olfatória/lesões , Regeneração/genética , Fatores de Ribosilação do ADP/genética , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Doxiciclina/administração & dosagem , Embrião de Mamíferos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histona Desmetilases/metabolismo , Melfalan/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Proteína de Marcador Olfatório/metabolismo , Mucosa Olfatória/citologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , gama-Globulinas/metabolismo
17.
J Cell Sci ; 128(10): 1934-45, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25908845

RESUMO

Cilia are evolutionarily conserved organelles found on many mammalian cell types, including neuronal populations. Although neuronal cilia, including those on olfactory sensory neurons (OSNs), are often delineated by localization of adenylyl cyclase 3 (AC3, also known as ADCY3), the mechanisms responsible for targeting integral membrane proteins are largely unknown. Post-translational modification by small ubiquitin-like modifier (SUMO) proteins plays an important role in protein localization processes such as nuclear-cytosolic transport. Here, we identified through bioinformatic analysis that adenylyl cyclases harbor conserved SUMOylation motifs, and show that AC3 is a substrate for SUMO modification. Functionally, overexpression of the SUMO protease SENP2 prevented ciliary localization of AC3, without affecting ciliation or cilia maintenance. Furthermore, AC3-SUMO mutants did not localize to cilia. To test whether SUMOylation is sufficient for cilia entry, we compared localization of ANO2, which possesses a SUMO motif, and ANO1, which lacks SUMOylation sites and does not localize to cilia. Introduction of SUMOylation sites into ANO1 was not sufficient for ciliary entry. These data suggest that SUMOylation is necessary but not sufficient for ciliary trafficking of select constituents, further establishing the link between ciliary and nuclear import.


Assuntos
Cílios/metabolismo , Receptores Odorantes/metabolismo , Adenilil Ciclases/química , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Animais , Cães , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Dados de Sequência Molecular , Transporte Proteico , Transdução de Sinais , Sumoilação
18.
Nat Commun ; 5: 5813, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25504142

RESUMO

Cilia dysfunction underlies a class of human diseases with variable penetrance in different organ systems. Across eukaryotes, intraflagellar transport (IFT) facilitates cilia biogenesis and cargo trafficking, but our understanding of mammalian IFT is insufficient. Here we perform live analysis of cilia ultrastructure, composition and cargo transport in native mammalian tissue using olfactory sensory neurons. Proximal and distal axonemes of these neurons show no bias towards IFT kinesin-2 choice, and Kif17 homodimer is dispensable for distal segment IFT. We identify Bardet-Biedl syndrome proteins (BBSome) as bona fide constituents of IFT in olfactory sensory neurons, and show that they exist in 1:1 stoichiometry with IFT particles. Conversely, subpopulations of peripheral membrane proteins, as well as transmembrane olfactory signalling pathway components, are capable of IFT but with significantly less frequency and/or duration. Our results yield a model for IFT and cargo trafficking in native mammalian cilia and may explain the penetrance of specific ciliopathy phenotypes in olfactory neurons.


Assuntos
Axonema/metabolismo , Cílios/metabolismo , Flagelos/metabolismo , Regulação da Expressão Gênica , Neurônios Receptores Olfatórios/metabolismo , Transdução de Sinais , Adenoviridae/genética , Animais , Axonema/ultraestrutura , Transporte Biológico , Cílios/ultraestrutura , Flagelos/ultraestrutura , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinesina/genética , Cinesina/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios Receptores Olfatórios/ultraestrutura , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
19.
Circ Res ; 114(6): 982-92, 2014 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-24508725

RESUMO

RATIONALE: Kv1.5 (KCNA5) mediates the ultra-rapid delayed rectifier current that controls atrial action potential duration. Given its atrial-specific expression and alterations in human atrial fibrillation, Kv1.5 has emerged as a promising target for the treatment of atrial fibrillation. A necessary step in the development of novel agents that selectively modulate trafficking pathways is the identification of the cellular machinery controlling Kv1.5 surface density, of which little is yet known. OBJECTIVE: To investigate the role of the unconventional myosin-V (MYO5A and MYO5B) motors in determining the cell surface density of Kv1.5. METHODS AND RESULTS: Western blot analysis showed MYO5A and MYO5B expression in the heart, whereas disruption of endogenous motors selectively reduced IKur current in adult rat cardiomyocytes. Dominant negative constructs and short hairpin RNA silencing demonstrated a role for MYO5A and MYO5B in the surface trafficking of Kv1.5 and connexin-43 but not potassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2). Live-cell imaging of Kv1.5-GFP and retrospective labeling of phalloidin demonstrated motility of Kv1.5 vesicles on actin tracts. MYO5A participated in anterograde trafficking, whereas MYO5B regulated postendocytic recycling. Overexpression of mutant motors revealed a selective role for Rab11 in coupling MYO5B to Kv1.5 recycling. CONCLUSIONS: MYO5A and MYO5B control functionally distinct steps in the surface trafficking of Kv1.5. These isoform-specific trafficking pathways determine Kv1.5-encoded IKur in myocytes to regulate repolarizing current and, consequently, cardiac excitability. Therapeutic strategies that manipulate Kv1.5 selective trafficking pathways may prove useful in the treatment of arrhythmias.


Assuntos
Membrana Celular/metabolismo , Canal de Potássio Kv1.5/metabolismo , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Miosinas/fisiologia , Transporte Proteico/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Arritmias Cardíacas/fisiopatologia , Linhagem Celular , Conexina 43/análise , Canal de Potássio ERG1 , Endocitose , Canais de Potássio Éter-A-Go-Go/análise , Junções Comunicantes , Genes Reporter , Sistema de Condução Cardíaco/fisiopatologia , Transporte de Íons , Canal de Potássio Kv1.5/genética , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Cardiovasculares , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/deficiência , Miosina Tipo V/genética , Miosinas/deficiência , Miosinas/genética , Potássio/metabolismo , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , Proteínas rab de Ligação ao GTP/fisiologia
20.
Trends Biotechnol ; 31(6): 355-63, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23601268

RESUMO

Alterations in cilia formation or function underlie a growing class of pleiotropic disorders termed ciliopathies. The genetic basis of ciliopathies is remarkably complex, with an incomplete but expanding list of more than 89 loci implicated in various disorders. Current treatment of ciliopathies is limited to symptomatic therapy. However, our growing understanding of ciliopathy genetics, coupled with recent advances in gene delivery and endogenous gene and transcript repair demonstrated thus far in tissues of the eye, nose, and airway, offers hope for curative measures in the near future. This review highlights these advances, as well as the challenges that remain with the development of personalized medicine for treating a very complex spectrum of disease, penetrant in a variety of organ systems.


Assuntos
Transtornos da Motilidade Ciliar/terapia , Terapia Genética/métodos , Pesquisa Biomédica/tendências , Transtornos da Motilidade Ciliar/genética , Humanos , Medicina de Precisão/métodos
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