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 , Retinose Pigmentar/genética , Tubulina (Proteína)/metabolismo , Aumento de Peso/genéticaRESUMO
Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.
Assuntos
Padronização Corporal/genética , Cílios/genética , Síndrome de Kartagener/genética , Proteínas/genética , Animais , Movimento Celular/genética , Chlamydomonas/genética , Cílios/patologia , Proteínas do Citoesqueleto , Citoesqueleto/genética , Modelos Animais de Doenças , Extremidades/crescimento & desenvolvimento , Extremidades/patologia , Predisposição Genética para Doença , Humanos , Síndrome de Kartagener/patologia , Camundongos , Microtúbulos/genética , Mutação , Tubo Neural/crescimento & desenvolvimento , Tubo Neural/patologia , Transdução de Sinais/genéticaRESUMO
Nephronophthisis (NPHP) is a ciliopathy in which genetic modifiers may underlie the variable penetrance of clinical features. To identify modifiers, a screen was conducted on C. elegans nphp-4(tm925) mutants. Mutations in ten loci exacerbating nphp-4(tm925) ciliary defects were obtained. Four loci have been identified, three of which are established ciliopathy genes mks-1, mks-2, and mks-5. The fourth allele (yhw66) is a missense mutation (S316F) in OSM-3, a kinesin required for cilia distal segment assembly. While osm-3(yhw66) mutants alone have no overt cilia phenotype, nphp-4(tm925);osm-3(yhw66) double mutants lack distal segments and are dye-filling (Dyf) and osmotic avoidance (Osm) defective, similar to osm-3(mn357) null mutants. In osm-3(yhw66) mutants anterograde intraflagellar transport (IFT) velocity is reduced. Furthermore, expression of OSM-3(S316F)::GFP reduced IFT velocities in nphp-4(tm925) mutants, but not in wild type animals. In silico analysis indicates the S316F mutation may affect a phosphorylation site. Putative phospho-null OSM-3(S316F) and phospho-mimetic OSM-3(S316D) proteins accumulate at the cilia base and tip respectively. FRAP analysis indicates that the cilia entry rate of OSM-3(S316F) is slower than OSM-3 and that in the presence of OSM-3(S316F), OSM-3 and OSM-3(S316D) rates decrease. In the presence OSM-3::GFP or OSM-3(S316D)::GFP, OSM-3(S316F)::tdTomato redistributes along the cilium and accumulates in the cilia tip. OSM-3(S316F) and OSM-3(S316D) are functional as they restore cilia distal segment formation in osm-3(mn357) null mutants; however, only OSM-3(S316F) rescues the osm-3(mn357) null Dyf phenotype. Despite rescue of cilia length in osm-3(mn357) null mutants, neither OSM-3(S316F) nor OSM-3(S316D) restores ciliary defects in nphp-4(tm925);osm-3(yhw66) double mutants. Thus, these OSM-3 mutations cause NPHP-4 dependent and independent phenotypes. These data indicate that in addition to regulating cilia protein entry or exit, NPHP-4 influences localization and function of a distal ciliary kinesin. Moreover, data suggest human OSM-3 homolog (Kif17) could act as a modifying locus affecting disease penetrance or expressivity in NPHP patients.
Assuntos
Alelos , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Cílios/metabolismo , Transtornos da Motilidade Ciliar/genética , Encefalocele/genética , Epistasia Genética , Testes Genéticos , Cinesinas/genética , Doenças Renais Policísticas/genética , Animais , Teste de Complementação Genética , Mutagênese/genética , Mutação/genética , Fenótipo , Fosforilação , Estrutura Terciária de Proteína , Transporte Proteico , Retinose PigmentarRESUMO
BACKGROUND: Joubert syndrome (JBTS) and related disorders are defined by cerebellar malformation (molar tooth sign), together with neurological symptoms of variable expressivity. The ciliary basis of Joubert syndrome related disorders frequently extends the phenotype to tissues such as the eye, kidney, skeleton and craniofacial structures. RESULTS: Using autozygome and exome analyses, we identified a null mutation in KIAA0556 in a multiplex consanguineous family with hallmark features of mild Joubert syndrome. Patient-derived fibroblasts displayed reduced ciliogenesis potential and abnormally elongated cilia. Investigation of disease pathophysiology revealed that Kiaa0556 (-/-) null mice possess a Joubert syndrome-associated brain-restricted phenotype. Functional studies in Caenorhabditis elegans nematodes and cultured human cells support a conserved ciliary role for KIAA0556 linked to microtubule regulation. First, nematode KIAA0556 is expressed almost exclusively in ciliated cells, and the worm and human KIAA0556 proteins are enriched at the ciliary base. Second, C. elegans KIAA0056 regulates ciliary A-tubule number and genetically interacts with an ARL13B (JBTS8) orthologue to control cilium integrity. Third, human KIAA0556 binds to microtubules in vitro and appears to stabilise microtubule networks when overexpressed. Finally, human KIAA0556 biochemically interacts with ciliary proteins and p60/p80 katanins. The latter form a microtubule-severing enzyme complex that regulates microtubule dynamics as well as ciliary functions. CONCLUSIONS: We have identified KIAA0556 as a novel microtubule-associated ciliary base protein mutated in Joubert syndrome. Consistent with the mild patient phenotype, our nematode, mice and human cell data support the notion that KIAA0556 has a relatively subtle and variable cilia-related function, which we propose is related to microtubule regulation.
Assuntos
Corpos Basais/metabolismo , Cerebelo/anormalidades , Proteínas Associadas aos Microtúbulos/genética , Mutação , Retina/anormalidades , Fatores de Ribosilação do ADP/metabolismo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Adenosina Trifosfatases/metabolismo , Adulto , Animais , Corpos Basais/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Células Cultivadas , Cerebelo/patologia , Criança , Pré-Escolar , Cílios/genética , Cílios/patologia , Exoma , Anormalidades do Olho/genética , Anormalidades do Olho/patologia , Feminino , Humanos , Katanina , Doenças Renais Císticas/genética , Doenças Renais Císticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Linhagem , Ligação Proteica , Retina/patologiaRESUMO
It has been known for decades that neurons throughout the brain possess solitary, immotile, microtubule based appendages called primary cilia. Only recently have studies tried to address the functions of these cilia and our current understanding remains poor. To determine if neuronal cilia have a role in behavior we specifically disrupted ciliogenesis in the cortex and hippocampus of mice through conditional deletion of the Intraflagellar Transport 88 (Ift88) gene. The effects on learning and memory were analyzed using both Morris Water Maze and fear conditioning paradigms. In comparison to wild type controls, cilia mutants displayed deficits in aversive learning and memory and novel object recognition. Furthermore, hippocampal neurons from mutants displayed an altered paired-pulse response, suggesting that loss of IFT88 can alter synaptic properties. A variety of other behavioral tests showed no significant differences between conditional cilia mutants and controls. This type of conditional allele approach could be used to distinguish which behavioral features of ciliopathies arise due to defects in neural development and which result from altered cell physiology. Ultimately, this could lead to an improved understanding of the basis for the cognitive deficits associated with human cilia disorders such as Bardet-Biedl syndrome, and possibly more common ailments including depression and schizophrenia.
Assuntos
Cílios/metabolismo , Medo , Aprendizagem em Labirinto , Neurogênese/genética , Animais , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Cílios/genética , Depressão/genética , Depressão/patologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Neurônios/patologia , Esquizofrenia/genética , Esquizofrenia/patologia , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: Cilia are found on nearly every cell type in the mammalian body, and have been historically classified as either motile or immotile. Motile cilia are important for fluid and cellular movement; however, the roles of non-motile or primary cilia in most tissues remain unknown. Several genetic syndromes, called the ciliopathies, are associated with defects in cilia structure or function and have a wide range of clinical presentations. Much of what we know about the formation and maintenance of cilia comes from model systems like C. elegans and Chalmydomonas. Studies of mammalian cilia in live tissues have been hampered by difficulty visualizing them. RESULTS: To facilitate analyses of mammalian cilia function we generated an inducible CiliaGFP mouse by targeting mouse cDNA encoding a cilia-localized protein somatostatin receptor 3 fused to GFP (Sstr3::GFP) into the ROSA26 locus. In this system, Sstr3::GFP is expressed from the ubiquitous ROSA26 promoter after Cre mediated deletion of an upstream Neo cassette flanked by lox P sites. Fluorescent cilia labeling was observed in a variety of live tissues and after fixation. Both cell-type specific and temporally regulated cilia labeling were obtained using multiple Cre lines. The analysis of renal cilia in anesthetized live mice demonstrates that cilia commonly lay nearly parallel to the apical surface of the tubule. In contrast, in more deeply anesthetized mice the cilia display a synchronized, repetitive oscillation that ceases upon death, suggesting a relationship to heart beat, blood pressure or glomerular filtration. CONCLUSIONS: The ability to visualize cilia in live samples within the CiliaGFP mouse will greatly aid studies of ciliary function. This mouse will be useful for in vivo genetic and pharmacological screens to assess pathways regulating cilia motility, signaling, assembly, trafficking, resorption and length control and to study cilia regulated physiology in relation to ciliopathy phenotypes.
RESUMO
Although primary cilia are well established as important sensory and signaling structures, their function in most tissues remains unknown. Obesity is a feature associated with some syndromes of cilia dysfunction, such as Bardet-Biedl syndrome (BBS) and Alström syndrome, as well as in several cilia mutant mouse models. Recent data indicate that obesity in BBS mutant mice is due to defects in leptin receptor trafficking and leptin resistance. Furthermore, induction of cilia loss in leptin-responsive proopiomelanocortin neurons results in obesity, implicating cilia on hypothalamic neurons in regulating feeding behavior. Here, we directly test the importance of the cilium as a mediator of the leptin response. In contrast to the current dogma, a longitudinal study of conditional Ift88 cilia mutant mice under different states of adiposity indicates that leptin resistance is present only when mutants are obese. Our studies show that caloric restriction leads to an altered anticipatory feeding behavior that temporarily abrogates the anorectic actions of leptin despite normalized circulating leptin levels. Interestingly, preobese Bbs4 mutant mice responded to the anorectic effects of leptin and did not display other phenotypes associated with defective leptin signaling. Furthermore, thermoregulation and activity measurements in cilia mutant mice are inconsistent with phenotypes previously observed in leptin deficient ob/ob mice. Collectively, these data indicate that cilia are not directly involved in leptin responses and that a defect in the leptin signaling axis is not the initiating event leading to hyperphagia and obesity associated with cilia dysfunction.
Assuntos
Cílios/patologia , Leptina/metabolismo , Obesidade/metabolismo , Animais , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/patologia , Composição Corporal , Modelos Animais de Doenças , Comportamento Alimentar , Camundongos , Camundongos Obesos , Camundongos Transgênicos , Atividade Motora , Mutação , Neurônios/metabolismo , Obesidade/genética , Obesidade/patologia , Fenótipo , Transdução de Sinais , TemperaturaRESUMO
Disrupting the function of cilia in mouse kidneys results in rapid or slow progression of cystic disease depending on whether the animals are juveniles or adults, respectively. Renal injury can also markedly accelerate the renal cyst formation that occurs after disruption of cilia in adult mice. Rates of cell proliferation are markedly higher in juvenile than adult kidneys and increase after renal injury, suggesting that cell proliferation may enhance the development of cysts. Here, we induced cilia loss in the kidneys of adult mice in the presence or absence of a Cux-1 transgene, which maintains cell proliferation. By using this model, we were able to avoid additional factors such as inflammation and dedifferentiation, which associate with renal injury and may also influence the rate of cystogenesis. After induction of cilia loss, cystic disease was not more pronounced in adult mice with the Cux-1 transgene compared with those without the transgene. In conclusion, these data suggest that proliferation is unlikely to be the sole mechanism underlying the rapid cystogenesis observed after injury in mice that lose cilia function in adulthood.
Assuntos
Cílios/patologia , Doenças Renais Císticas/etiologia , Doenças Renais Císticas/patologia , Túbulos Renais Proximais/patologia , Animais , Proliferação de Células , Cílios/fisiologia , Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/fisiologia , Tamoxifeno/farmacologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Astrocytes provide a principal pathway for glutamate uptake in the mammalian brain, a task accomplished by the powerful action of excitatory amino acid transporters (EAAT) 1 and 2. These transporters are synthesized within the endoplasmic reticulum and are then trafficked to the plasma membrane. The characteristics of their intracellular traffic within astrocytes have not been investigated. We monitored the trafficking of secretory vesicles laden with the recombinant fluorescent protein chimera of EAAT2 in cultured astrocytes. Such vesicles appeared as fluorescent puncta, and their trafficking parameters were obtained using original algorithms, which we describe here in detail. We determined the maximal displacement, average instantaneous speed, and trajectory angle of individual puncta/vesicles, with angles near 0° indicating radial movement directly away from or toward the nucleus and angles near 90° indicating tangential movement. Analysis of these trafficking parameters demonstrated that trafficking of EAAT2-laden vesicles has typical characteristics expected of the trafficking of secretory vesicles in cultured astrocytes. The distribution of trajectory angles for directional vesicles, i.e. those with a maximal displacement greater than 1 µm within the 40-s time-lapse imaging, was found to be unimodal, with angles near 0° being the most prominent (mode 7°). These measurements are in good agreement with previous measurements of trajectory angles of similar trafficking vesicles carrying cannabinoid receptor 1, evidencing the validity and robustness of our analytical approach and algorithms.
RESUMO
Disruption of the primary cilium is associated with a growing number of human diseases collectively termed ciliopathies. Ciliopathies present with a broad range of clinical features consistent with the near ubiquitous nature of the organelle and its role in diverse signaling pathways throughout development and adult homeostasis. The clinical features associated with cilia dysfunction can include such phenotypes as polycystic kidneys, skeletal abnormalities, blindness, anosmia, and obesity. Although the clinical relevance of the primary cilium is evident, the effects that cilia dysfunction has on the cell and how this contributes to disease remains poorly understood. Here, we show that loss of ciliogenesis genes such as Ift88 and Kif3a lead to increases in post-translational modifications on cytosolic microtubules. This effect was observed in cilia mutant kidney cells grown in vitro and in vivo in cystic kidneys. The hyper-acetylation of microtubules resulting from cilia loss is associated with both altered microtubule stability and increased α-tubulin acetyl-transferase activity. Intriguingly, the effect on microtubules was also evident in renal samples from patients with autosomal recessive polycystic kidneys. These findings indicate that altered microtubule post-translational modifications may influence some of the phenotypes observed in ciliopathies.
Assuntos
Cílios/metabolismo , Doenças Renais Císticas/metabolismo , Microtúbulos/metabolismo , Acetilação , Animais , Linhagem Celular , Proliferação de Células , Imunofluorescência , Humanos , Immunoblotting , Técnicas In Vitro , Masculino , Camundongos , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/metabolismoRESUMO
Astrocytes can release various gliotransmitters in response to stimuli that cause increases in intracellular Ca(2+) levels; this secretion occurs via a regulated exocytosis pathway. Indeed, astrocytes express protein components of the vesicular secretory apparatus. However, the detailed temporal characteristics of vesicular fusions in astrocytes are not well understood. In order to start addressing this issue, we used total internal reflection fluorescence microscopy (TIRFM) to visualize vesicular fusion events in astrocytes expressing the fluorescent synaptobrevin 2 derivative, synapto-pHluorin. Although our cultured astrocytes from visual cortex express synaptosome-associated protein of 23 kDa (SNAP23), but not of 25 kDa (SNAP25), these glial cells exhibited a slow burst of exocytosis under mechanical stimulation; the expression of SNAP25B did not affect bursting behaviour. The relative amount of two distinct types of events observed, transient and full fusions, depended on the applied stimulus. Expression of exogenous synaptotagmin 1 (Syt1) in astrocytes endogenously expressing Syt4, led to a greater proportion of transient fusions when astrocytes were stimulated with bradykinin, a stimulus otherwise resulting in more full fusions. Additionally, we studied the stability of the transient fusion pore by measuring its dwell time, relation to vesicular size, flickering and decay slope; all of these characteristics were secretagogue dependent. The expression of SNAP25B or Syt1 had complex effects on transient fusion pore stability in a stimulus-specific manner. SNAP25B obliterated the appearance of flickers and reduced the dwell time when astrocytes were mechanically stimulated, while astrocytes expressing SNAP25B and stimulated with bradykinin had a reduction in decay slope. Syt1 reduced the dwell time when astrocytes were stimulated either mechanically or with bradykinin. Our detailed study of temporal characteristics of astrocytic exocytosis will not only aid the general understanding of this process, but also the interpretation of the events at the tripartite synapse, both in health and disease.
Assuntos
Astrócitos , Proteína 2 Associada à Membrana da Vesícula , Astrócitos/metabolismo , Células Cultivadas , Exocitose , Fusão de Membrana , Vesículas Secretórias/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Skin and hair follicle morphogenesis and homeostasis require the integration of multiple signaling pathways, including Hedgehog (Hh) and Wingless (Wnt), and oriented cell divisions, all of which have been associated with primary cilia. Although studies have shown that disrupting dermal cilia causes follicular arrest and attenuated Hh signaling, little is known about the role of epidermal cilia. Here, epidermal cilia function was analyzed using conditional alleles of the ciliogenic genes Ift88 and Kif3a. At birth, epidermal cilia mutants appeared normal, but developed basaloid hyperplasia and ingrowths into the dermis of the ventrum with age. In addition, follicles in the tail were disorganized and had excess sebaceous gland lobules. Epidermal cilia mutants displayed fewer long-term label-retaining cells, suggesting altered stem cell homeostasis. Abnormal proliferation and differentiation were evident from lineage-tracing studies and showed an expansion of follicular cells into the interfollicular epidermis, as is seen during wound repair. These phenotypes were not associated with changes in canonical Wnt activity or oriented cell division. However, nuclear accumulation of the ΔNp63 transcription factor, which is involved in stratification, keratinocyte differentiation and wound repair, was increased, whereas the Hh pathway was repressed. Intriguingly, the phenotypes were not typical of those associated with loss of Hh signaling but exhibited similarities with those of mice in which ΔNp63 is overexpressed in the epidermis. Collectively, these data indicate that epidermal primary cilia may function in stress responses and epidermal homeostasis involving pathways other than those typically associated with primary cilia.
Assuntos
Cílios/fisiologia , Células Epidérmicas , Folículo Piloso/fisiologia , Homeostase/fisiologia , Fenômenos Fisiológicos da Pele , Animais , Animais Recém-Nascidos , Cílios/genética , Cílios/metabolismo , Epiderme/metabolismo , Epiderme/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Homeostase/genética , Integrases/genética , Integrases/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Fenômenos Fisiológicos da Pele/genética , Transgenes/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Levetiracetam is an FDA-approved drug used to treat epilepsy and other disorders of the nervous system. Although it is known that levetiracetam binds the synaptic vesicle protein SV2A, how drug binding affects synaptic functioning remains unknown. Here we report that levetiracetam reverses the effects of excess SV2A in autaptic hippocampal neurons. Expression of an SV2A-EGFP fusion protein produced a â¼1.5-fold increase in synaptic levels of SV2, and resulted in reduced synaptic release probability. The overexpression phenotype parallels that seen in neurons from SV2 knockout mice, which experience severe seizures. Overexpression of SV2A also increased synaptic levels of the calcium-sensor protein synaptotagmin, an SV2-binding protein whose stability and trafficking are regulated by SV2. Treatment with levetiracetam rescued normal neurotransmission and restored normal levels of SV2 and synaptotagmin at the synapse. These results indicate that changes in SV2 expression in either direction impact neurotransmission, and suggest that levetiracetam may modulate SV2 protein interactions.
Assuntos
Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Piracetam/análogos & derivados , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Animais , Proteínas de Fluorescência Verde/metabolismo , Levetiracetam , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotransmissores/metabolismo , Fenótipo , Piracetam/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Sinaptotagminas/metabolismoRESUMO
Carbon nanotubes have electrical, mechanical and chemical properties that make them one of the most promising materials for applications in neuroscience. Single-walled and multi-walled carbon nanotubes have been increasingly used as scaffolds for neuronal growth and more recently for neural stem cell growth and differentiation. They are also used in interfaces with neurons, where they can detect neuronal electrical activity and also deliver electrical stimulation to these cells. The emerging picture is that carbon nanotubes do not have obvious adverse effects on mammalian health. Thus in the near future they could be used in brain-machine interfaces.
Assuntos
Nanotecnologia/métodos , Nanotubos de Carbono/estatística & dados numéricos , Neurociências , Animais , Humanos , Nanotubos de Carbono/química , Nanotubos de Carbono/ultraestruturaRESUMO
Astrocytes possess GPCRs (G-protein-coupled receptors) for neuroactive substances and can respond via these receptors to signals originating from neurons as well as astrocytes. Like many transmembrane proteins, GPCRs exist in a dynamic equilibrium between receptors expressed at the plasma membrane and those present within intracellular trafficking compartments. The characteristics of GPCR trafficking within astrocytes have not been investigated. We therefore monitored the trafficking of recombinant fluorescent protein chimeras of the CB1R (cannabinoid receptor 1) that is thought to be expressed natively in astrocytes. CB1R chimeras displayed a marked punctate intracellular localization when expressed in cultured rat visual cortex astrocytes, an expression pattern reminiscent of native CB1R expression in these cells. Based upon trafficking characteristics, we found the existence of two populations of vesicular CB1R puncta: (i) relatively immobile puncta with movement characteristic of diffusion and (ii) mobile puncta with movement characteristic of active transport along cytoskeletal elements. The predominant direction of active transport is oriented radially to/from the nuclear region, which can be abolished by disruption of the microtubule cytoskeleton. CB1R puncta are localized within intracellular acidic organelles, mainly co-localizing with endocytic compartments. Constitutive trafficking of CB1R to and from the plasma membrane is an energetically costly endeavour whose function is at present unclear in astrocytes. However, given that intracellular CB1Rs can engage cell signalling pathways, it is likely that this process plays an important regulatory role.
Assuntos
Astrócitos/metabolismo , Simulação de Dinâmica Molecular , Receptor CB1 de Canabinoide/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Astrócitos/citologia , Células Cultivadas , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Córtex Visual/citologia , Córtex Visual/metabolismoRESUMO
We used conductive nanotube films as substrates with which we could systematically vary the conductance to see how this property affects neuronal growth. Here we show that nanotube substrates in a narrow range of conductivity promote the outgrowth of neurites with a decrease in the number of growth cones as well as an increase in cell body area, while at higher conductance these effects disappear.
Assuntos
Nanotubos de Carbono/química , Nanotubos de Carbono/ultraestrutura , Neurônios/citologia , Neurônios/fisiologia , Animais , Animais Recém-Nascidos , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condutividade Elétrica , Ratos , Ratos Sprague-DawleyRESUMO
We report the use of chemically functionalized water soluble single-walled carbon nanotube (SWNT) graft copolymers to inhibit endocytosis. The graft copolymers were prepared by the functionalization of SWNTs with polyethylene glycol. When added to the culturing medium, these functionalized water soluble SWNTs were able to increase the length of various neuronal processes, neurites, as previously reported. Here we have determined that SWNTs are able to block stimulated membrane endocytosis in neurons, which could then explain the previously noted extended neurite length.
Assuntos
Endocitose , Nanotecnologia/métodos , Nanotubos de Carbono/química , Neurônios/fisiologia , Água/química , Animais , Membrana Celular/metabolismo , Modelos Biológicos , Nanotubos/química , Neuritos/metabolismo , Neurônios/metabolismo , Fosfopiruvato Hidratase/metabolismo , Polímeros/química , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Astrocytes can respond to a variety of stimuli by elevating their cytoplasmic Ca2+ concentration and can in turn release glutamate to signal adjacent neurons. The majority of this Ca2+ is derived from internal stores while a portion also comes from outside of the cell. Astrocytes use Ca2+ entry through store-operated Ca2+ channels to refill their internal stores. Therefore, we investigated what role this store-operated Ca2+ entry plays in astrocytic Ca2+ responses and subsequent glutamate release. Astrocytes express canonical transient receptor potential (TRPC) channels that have been implicated in mediating store-operated Ca2+ entry. Here, we show that astrocytes in culture and freshly isolated astrocytes from visual cortex express TRPC1, TRPC4, and TRPC5. Indirect immunocytochemistry reveals that these proteins are present throughout the cell; the predominant expression of functionally tested TRPC1, however, is on the plasma membrane. Labeling in freshly isolated astrocytes reveals changes in TRPC expression throughout development. Using an antibody against TRPC1 we were able to block the function of TRPC1 channels and determine their involvement in mechanically and agonist-evoked Ca2+ entry in cultured astrocytes. Blocking TRPC1 was also found to reduce mechanically induced Ca2+-dependent glutamate release. These data indicate that Ca2+ entry through TRPC1 channels contributes to Ca2+ signaling in astrocytes and the consequent glutamate release from these cells.
Assuntos
Astrócitos/metabolismo , Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Líquido Intracelular/metabolismo , Dinâmica não Linear , Canais de Cátion TRPC/fisiologia , Trifosfato de Adenosina/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/ultraestrutura , Cálcio/farmacologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Indóis/farmacologia , Líquido Intracelular/efeitos dos fármacos , Metoxi-Hidroxifenilglicol/análogos & derivados , Metoxi-Hidroxifenilglicol/farmacologia , Estimulação Física , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Transfecção/métodos , Córtex Visual/citologiaRESUMO
Astrocytes play an important role in cell-cell signaling in the mammalian central nervous system. The ability of astrocytes to communicate with surrounding cells through gap-junctional coupling or signaling via the release of transmitters makes characterization of these cells difficult in vitro and even more so in vivo. To simplify the complexity of common in vitro systems, introduced by intercellular communication between astrocytes, we developed a novel cell culturing method, in which purified rat visual cortical astrocytes were grown in spatially defined cell-adhesion wells which we termed micropits. We showed that astrocytes cultured in micropit regions were viable and exhibited similar characteristics of Ca(2+) dynamics and astrocytic marker expression to those of cells cultured in non-micropit regions. Examination of intracellular Ca(2+) oscillations in solitary astrocytes cultured in micropits revealed less variable oscillations than those of non-micropit grouped astrocytes, which were in contact with their neighbors. Solitary cells in micropit regions can undergo ATP-mediated astrocyte-microglia signaling, demonstrating that this culturing method can also be used to investigate glial-glial interactions in a spatially well-defined microenvironment.
RESUMO
Astrocytes can release the excitatory transmitter glutamate which is capable of modulating activity in nearby neurons. This astrocytic glutamate release can occur through six known mechanisms: (i) reversal of uptake by glutamate transporters (ii) anion channel opening induced by cell swelling, (iii) Ca2+-dependent exocytosis, (iv) glutamate exchange via the cystine-glutamate antiporter, (v) release through ionotropic purinergic receptors and (vi) functional unpaired connexons, "hemichannels", on the cell surface. Although these various pathways have been defined, it is not clear how often and to what extent astrocytes employ different mechanisms. It will be necessary to determine whether the same glutamate release mechanisms that operate under physiological conditions operate during pathological conditions or whether there are specific release mechanisms that operate under particular conditions.