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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.
Nat Biotechnol ; 2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083791

RESUMO

Recent spatial gene expression technologies enable comprehensive measurement of transcriptomic profiles while retaining spatial context. However, existing analysis methods do not address the limited resolution of the technology or use the spatial information efficiently. Here, we introduce BayesSpace, a fully Bayesian statistical method that uses the information from spatial neighborhoods for resolution enhancement of spatial transcriptomic data and for clustering analysis. We benchmark BayesSpace against current methods for spatial and non-spatial clustering and show that it improves identification of distinct intra-tissue transcriptional profiles from samples of the brain, melanoma, invasive ductal carcinoma and ovarian adenocarcinoma. Using immunohistochemistry and an in silico dataset constructed from scRNA-seq data, we show that BayesSpace resolves tissue structure that is not detectable at the original resolution and identifies transcriptional heterogeneity inaccessible to histological analysis. Our results illustrate BayesSpace's utility in facilitating the discovery of biological insights from spatial transcriptomic datasets.

3.
Nat Neurosci ; 24(3): 425-436, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33558695

RESUMO

We used the 10x Genomics Visium platform to define the spatial topography of gene expression in the six-layered human dorsolateral prefrontal cortex. We identified extensive layer-enriched expression signatures and refined associations to previous laminar markers. We overlaid our laminar expression signatures on large-scale single nucleus RNA-sequencing data, enhancing spatial annotation of expression-driven clusters. By integrating neuropsychiatric disorder gene sets, we showed differential layer-enriched expression of genes associated with schizophrenia and autism spectrum disorder, highlighting the clinical relevance of spatially defined expression. We then developed a data-driven framework to define unsupervised clusters in spatial transcriptomics data, which can be applied to other tissues or brain regions in which morphological architecture is not as well defined as cortical laminae. Last, we created a web application for the scientific community to explore these raw and summarized data to augment ongoing neuroscience and spatial transcriptomics research ( http://research.libd.org/spatialLIBD ).


Assuntos
Expressão Gênica , Córtex Pré-Frontal/metabolismo , Transcriptoma , Redes Reguladoras de Genes , Humanos
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
PLoS One ; 11(11): e0165342, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27902689

RESUMO

Neurons exhibit strong coupling of electrochemical and metabolic activity. Increases in intrinsic fluorescence from either oxidized flavoproteins or reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] in the mitochondria have been used as an indicator of neuronal activity for the functional mapping of neural circuits. However, this technique has not been used to investigate the flow of olfactory information within the circuitry of the main olfactory bulb (MOB). We found that intrinsic flavoprotein fluorescence signals induced by electrical stimulation of single glomeruli displayed biphasic responses within both the glomerular (GL) and external plexiform layers (EPL) of the MOB. Pharmacological blockers of mitochondrial activity, voltage-gated Na+ channels, or ionotropic glutamate receptors abolished stimulus-dependent flavoprotein responses. Blockade of GABAA receptors enhanced the amplitude and spatiotemporal spread of the flavoprotein signals, indicating an important role for inhibitory neurotransmission in shaping the spread of neural activity in the MOB. Stimulus-dependent spread of fluorescence across the GL and EPL displayed a spatial distribution consistent with that of individual glomerular microcircuits mapped by neuroanatomic tract tracing. These findings demonstrated the feasibility of intrinsic fluorescence imaging in the olfactory systems and provided a new tool to examine the functional circuitry of the MOB.


Assuntos
Flavoproteínas/metabolismo , Processamento de Imagem Assistida por Computador/métodos , NADP/metabolismo , Neurônios/fisiologia , Bulbo Olfatório/fisiologia , Transmissão Sináptica/fisiologia , Animais , Estimulação Elétrica , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neurônios/citologia , Bulbo Olfatório/citologia , Sinapses/fisiologia
12.
PLoS One ; 11(11): e0165343, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27902696

RESUMO

The mammalian main olfactory system contains several subsystems that differ not only in the receptors they express and the glomerular targets they innervate within the main olfactory bulb (MOB), but also in the strategies they use to process odor information. The canonical main olfactory system employs a combinatorial coding strategy that represents odorant identity as a pattern of glomerular activity. By contrast, the "GC-D/necklace" olfactory subsystem-formed by olfactory sensory neurons expressing the receptor guanylyl cyclase GC-D and their target necklace glomeruli (NGs) encircling the caudal MOB-is critical for the detection of a small number of semiochemicals that promote the acquisition of food preferences. The formation of these socially-transmitted food preferences requires the animal to integrate information about two types of olfactory stimuli: these specialized social chemosignals and the food odors themselves. However, the neural mechanisms with which the GC-D/necklace subsystem processes this information are unclear. We used stimulus-induced increases in intrinsic fluorescence signals to map functional circuitry associated with NGs and canonical glomeruli (CGs) in the MOB. As expected, CG-associated activity spread laterally through both the glomerular and external plexiform layers associated with activated glomeruli. Activation of CGs or NGs resulted in activity spread between the two types of glomeruli; there was no evidence of preferential connectivity between individual necklace glomeruli. These results support previous anatomical findings that suggest the canonical and GC-D/necklace subsystems are functionally connected and may integrate general odor and semiochemical information in the MOB.


Assuntos
Flavoproteínas/metabolismo , Neurônios GABAérgicos/fisiologia , NADP/metabolismo , Bulbo Olfatório/fisiologia , Condutos Olfatórios , Transmissão Sináptica/fisiologia , Animais , Estimulação Elétrica , Feminino , Neurônios GABAérgicos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Bulbo Olfatório/citologia
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