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1.
J Neurosci ; 41(17): 3932-3943, 2021 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-33741721

RESUMO

The Abelson-helper integration site 1 (AHI1) gene encodes for a ciliary transition zone localizing protein that when mutated causes the human ciliopathy, Joubert syndrome. We prepared and examined neuronal cultures derived from male and female embryonic Ahi1+/+ and Ahi1-/- mice (littermates) and found that the distribution of ciliary melanin-concentrating hormone receptor-1 (MchR1) was significantly reduced in Ahi1-/- neurons; however, the total and surface expression of MchR1 on Ahi1-/- neurons was similar to controls (Ahi1+/+). This indicates that a pathway for MchR1 trafficking to the surface plasma membrane is intact, but the process of targeting MchR1 into cilia is impaired in Ahi1-deficient mouse neurons, indicating a role for Ahi1 in localizing MchR1 to the cilium. Mouse Ahi1-/- neurons that fail to accumulate MchR1 in the ciliary membrane have significant decreases in two downstream MchR1 signaling pathways [cAMP and extracellular signal-regulated kinase (Erk)] on MCH stimulation. These results suggest that the ciliary localization of MchR1 is necessary and critical for MchR1 signaling, with Ahi1 participating in regulating MchR1 localization to cilia, and further supporting cilia as critical signaling centers in neurons.SIGNIFICANCE STATEMENT Our work here demonstrates that neuronal primary cilia are powerful and focused signaling centers for the G-protein-coupled receptor (GPCR), melanin-concentrating hormone receptor-1 (MCHR1), with a role for the ciliary transition zone protein, Abelson-helper integration site 1 (AHI1), in mediating ciliary trafficking of MCHR1. Moreover, our manuscript further expands the repertoire of cilia functions on neurons, a cell type that has not received significant attention in the cilia field. Lastly, our work demonstrates the significant influence of ciliary GPCR signaling in the overall signaling of neurons.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Cílios/fisiologia , Neurônios/fisiologia , Receptores de Somatostatina/fisiologia , Transdução de Sinais/fisiologia , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/fisiopatologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Membrana Celular/fisiologia , Cerebelo/anormalidades , Cerebelo/fisiopatologia , AMP Cíclico/metabolismo , Anormalidades do Olho/genética , Anormalidades do Olho/fisiopatologia , Feminino , Doenças Renais Císticas/genética , Doenças Renais Císticas/fisiopatologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Knockout , Gravidez , Receptores de Somatostatina/genética , Retina/anormalidades , Retina/fisiopatologia , Transdução de Sinais/genética
2.
J Cell Sci ; 132(17)2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31391239

RESUMO

Mutations in the Abelson-helper integration site 1 (AHI1) gene are associated with neurological/neuropsychiatric disorders, and cause the neurodevelopmental ciliopathy Joubert syndrome (JBTS). Here, we show that deletion of the transition zone (TZ) protein Ahi1 in mouse embryonic fibroblasts (MEFs) has a small effect on cilia formation. However, Ahi1 loss in these cells results in: (1) reduced localization of the JBTS-associated protein Arl13b to the ciliary membrane, (2) decreased sonic hedgehog signaling, (3) and an abnormally elongated ciliary axoneme accompanied by an increase in ciliary IFT88 concentrations. While no changes in Arl13b levels are detected in crude cell membrane extracts, loss of Ahi1 significantly reduced the level of non-membrane-associated Arl13b and its stability via the proteasome pathway. Exogenous expression of Ahi1-GFP in Ahi1-/- MEFs restored ciliary length, increased ciliary recruitment of Arl13b and augmented Arl13b stability. Finally, Ahi1-/- MEFs displayed defects in cell motility and Pdgfr-α-dependent migration. Overall, our findings support molecular mechanisms underlying JBTS etiology that involve: (1) disruptions at the TZ resulting in defects of membrane- and non-membrane-associated proteins to localize to primary cilia, and (2) defective cell migration.This article has an associated First Person interview with the first author of the paper.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Cílios/metabolismo , Fatores de Ribosilação do ADP/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Movimento Celular/fisiologia , Fibroblastos/metabolismo , Camundongos , Camundongos Knockout , Mutação , Transporte Proteico , Transdução de Sinais
3.
Int J Mol Sci ; 22(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34502152

RESUMO

Major depressive disorder is a disabling disease with the number of affected individuals increasing each year. Current antidepressant treatments take between three to six weeks to be effective with forty percent of patients being resistant to treatment, making it necessary to search for new antidepressant treatments. Ketamine, a phencyclidine hydrochloride derivative, given intravenously, induces a rapid antidepressant effect in humans. In mice, it causes increased neurogenesis and antidepressant-like effects. However, it also produces psychomimetic effects in humans and in rodents increases the locomotor activity. In contrast, melatonin, a hormone secreted by the pineal gland and synthesized in extrapineal sites, increases new neuron formation and causes antidepressant-like effects in adult rodents with no collateral effects. Here, we assessed the effects of a non-effective dose of ketamine in combination with melatonin (KET/MEL), both on neurogenesis as well as on the antidepressant-like effect in mice. Our results showed that KET/MEL combination increased neurogenesis and produced antidepressant-like effects without altering locomotor activity after both single and triple administration protocols. Our data strongly suggest that KET/MEL combination could be used to simultaneously promote neurogenesis, reverting neuronal atrophy and inducing antidepressant-like effects.


Assuntos
Antidepressivos/uso terapêutico , Depressão/tratamento farmacológico , Ketamina/uso terapêutico , Melatonina/uso terapêutico , Animais , Antidepressivos/administração & dosagem , Antidepressivos/farmacologia , Combinação de Medicamentos , Sinergismo Farmacológico , Ketamina/administração & dosagem , Ketamina/farmacologia , Masculino , Melatonina/administração & dosagem , Melatonina/farmacologia , Camundongos , Neurogênese/efeitos dos fármacos
4.
Proc Natl Acad Sci U S A ; 108(51): 20591-6, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-22143774

RESUMO

The exchange of substances between higher organisms and the environment occurs across transporting epithelia whose basic features are tight junctions (TJs) that seal the intercellular space, and polarity, which enables cells to transport substances vectorially. In a previous study, we demonstrated that 10 nM ouabain modulates TJs, and we now show that it controls polarity as well. We gauge polarity through the development of a cilium at the apical domain of Madin-Darby canine kidney cells (MDCK, epithelial dog kidney). Ouabain accelerates ciliogenesis in an ERK1/2-dependent manner. Claudin-2, a molecule responsible for the Na(+) and H(2)O permeability of the TJs, is also present at the cilium, as it colocalizes and coprecipitates with acetylated α-tubulin. Ouabain modulates claudin-2 localization at the cilium through ERK1/2. Comparing wild-type and ouabain-resistant MDCK cells, we show that ouabain acts through Na(+),K(+)-ATPase. Taken together, our previous and present results support the possibility that ouabain constitutes a hormone that modulates the transporting epithelial phenotype, thereby playing a crucial role in metazoan life.


Assuntos
Cílios/metabolismo , Células Epiteliais/metabolismo , Ouabaína/química , Animais , Caderinas/metabolismo , Adesão Celular , Comunicação Celular , Linhagem Celular , Proliferação de Células , Claudinas/metabolismo , Cães , Imunoprecipitação , Espectroscopia de Ressonância Magnética/métodos , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Esteroides/metabolismo , Junções Íntimas , Fatores de Tempo
5.
Brain Sci ; 14(1)2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-38248261

RESUMO

Alzheimer's disease (AD), as the main cause of dementia, affects millions of people around the world, whose diagnosis is based mainly on clinical criteria. Unfortunately, the diagnosis is obtained very late, when the neurodegenerative damage is significant for most patients. Therefore, the exhaustive study of biomarkers is indispensable for diagnostic, prognostic, and even follow-up support. AD is a multifactorial disease, and knowing its underlying pathological mechanisms is crucial to propose new and valuable biomarkers. In this review, we summarize some of the main biomarkers described in AD, which have been evaluated mainly by imaging studies in cerebrospinal fluid and blood samples. Furthermore, we describe and propose neuronal precursors derived from the olfactory neuroepithelium as a potential resource to evaluate some of the widely known biomarkers of AD and to gear toward searching for new biomarkers. These neuronal lineage cells, which can be obtained directly from patients through a non-invasive and outpatient procedure, display several characteristics that validate them as a surrogate model to study the central nervous system, allowing the analysis of AD pathophysiological processes. Moreover, the ease of obtaining and harvesting endows them as an accessible and powerful resource to evaluate biomarkers in clinical practice.

6.
bioRxiv ; 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36778451

RESUMO

Frameshift mutations in Tau Tubulin Kinase 2 (TTBK2) cause spinocerebellar ataxia type 11 (SCA11), which is characterized by the progressive loss of Purkinje cells and cerebellar atrophy. Previous work showed that these TTBK2 variants generate truncated proteins that interfere with primary ciliary trafficking and with Sonic Hedgehog (SHH) signaling in mice. Nevertheless, the molecular mechanisms underlying the dominant interference of mutations remain unknown. Herein, we discover that SCA11-associated variants contain a bona fide peroxisomal targeting signal type 1. We find that their expression in RPE1 cells reduces peroxisome numbers within the cell and at the base of the cilia, disrupts peroxisome fission pathways, and impairs trafficking of ciliary SMO upon SHH signaling activation. This work uncovers a neomorphic function of SCA11-causing mutations and identifies requirements for both peroxisomes and cholesterol in trafficking of cilia-localized SHH signaling proteins. In addition, we postulate that molecular mechanisms underlying cellular dysfunction in SCA11 converge on the SHH signaling pathway.

7.
Schizophr Res ; 195: 412-420, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-28927861

RESUMO

Primary cilium (PC) is a microtubule-rich organelle that protrudes from the plasma membrane and acts as a cellular antenna sensing extracellular signals during brain development. DISC1 (Disrupted-in-Schizophrenia-1) is involved in PC formation and is considered a risk factor for neuropsychiatric disorders. We have previously described altered subcellular distribution of DISC1 and an aberrant microtubule organization in olfactory neuronal precursors (ONP) obtained from schizophrenia (SCZ) and bipolar disorder (BD) patients. Herein, we analyzed in vitro PC formation in healthy control subjects, SCZ and BD patients. The results indicated that 66.73±4.33% of ONP from control subjects showed immunostaining for the PC marker, acetylated α-tubulin. By contrast, only a small percentage of cells in culture from paranoid SCZ and BD patients showed PC staining (SCZ, 12.8±4.43%; BD, 12.32±5.86%). However, cells from an affected proband with disorganized SCZ and a subject with BD displayed a higher percentage of cells with cilia (SCZ, 42.20%; BD, 38.59%). Additionally, cilia elongation was observed in lithium-treated ONP derived from all groups, with a more evident response in cells from the BD group. The present study provides novel evidence that the molecular pathways involved in PC formation are defective in SCZ and BD, and impairment in these processes may be involved in the physiopathology of both diseases. Our observations also suggest that ONP is a patient-derived cell model with a potential use for diagnosis and high-throughput drug screening for brain diseases.


Assuntos
Transtorno Bipolar/patologia , Cílios/patologia , Neurônios/patologia , Esquizofrenia/patologia , Adenilil Ciclases/metabolismo , Adulto , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Masculino , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neurônios/efeitos dos fármacos , Fatores de Tempo , Tubulina (Proteína)/metabolismo , Ácido Valproico/farmacologia , Adulto Jovem
8.
J Neurosci Methods ; 294: 1-6, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29061345

RESUMO

BACKGROUND: Morphometric analyses of biological features have become increasingly common in recent years with such analyses being subject to a large degree of observer bias, variability, and time consumption. While commercial software packages exist to perform these analyses, they are expensive, require extensive user training, and are usually dependent on the observer tracing the morphology. NEW METHOD: To address these issues, we have developed a broadly applicable, no-cost ImageJ plugin we call 'BranchAnalysis2D/3D', to perform morphometric analyses of structures with branching morphologies, such as neuronal dendritic spines, vascular morphology, and primary cilia. RESULTS: Our BranchAnalysis2D/3D algorithm allows for rapid quantification of the length and thickness of branching morphologies, independent of user tracing, in both 2D and 3D data sets. COMPARISON WITH EXISTING METHODS: We validated the performance of BranchAnalysis2D/3D against pre-existing software packages using trained human observers and images from brain and retina. We found that the BranchAnalysis2D/3D algorithm outputs results similar to available software (i.e., Metamorph, AngioTool, Neurolucida), while allowing faster analysis times and unbiased quantification. CONCLUSIONS: BranchAnalysis2D/3D allows inexperienced observers to output results like a trained observer but more efficiently, thereby increasing the consistency, speed, and reliability of morphometric analyses.


Assuntos
Encéfalo/citologia , Imageamento Tridimensional/métodos , Microscopia Confocal/métodos , Neurônios/citologia , Software , Algoritmos , Animais , Camundongos , Variações Dependentes do Observador , Reprodutibilidade dos Testes , Retina/anatomia & histologia
9.
CNS Neurosci Ther ; 21(5): 446-53, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25620115

RESUMO

BACKGROUND: DISC1 (Disrupted-In-Schizophrenia-1) is considered a genetic risk factor for schizophrenia (SZ) and bipolar disorder (BD). DISC1 regulates microtubule stability, migration, and cAMP signaling in mammalian cell lines and mouse brain tissue. cAMP is a regulator of microtubule organization and migration in neurons. Aberrant microtubule organization has been observed in olfactory neuronal precursors (ONP) derived from patients with SZ and BD, which suggests involvement of DISC1 and cAMP. However, the biology of DISC1 in the physiopathology of psychiatric conditions remains elusive. AIMS: Herein, utilizing ONP obtained from SZ, BD patients and healthy subjects, we have studied DISC1 expression, protein levels, and subcellular distribution by qRT-PCR, immunoblotting, subcellular fractionation, and confocal microscopy. Cell migration and cAMP accumulation were assessed by Transwell and PKA competition assays. RESULTS: We found increased levels of the 75-kDa DISC1 isoform in total cell extracts of ONP from patients with SZ and BD compared with controls. Subcellular distribution showed a significant decrease of cytoplasmic DISC1 concomitant with its augmented levels in transcription sites. Moreover, significant cAMP accumulation and diminished migration were also observed in patients' cells. CONCLUSION: Alterations of DISC1 levels and its cellular distribution, which negatively modify cAMP homeostasis, microtubule organization, and cell migration, in ONP from patients with SZ and BD, suggest that their presence in early stages of brain development may impact brain maturation and function.


Assuntos
Transtorno Bipolar/fisiopatologia , Movimento Celular/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Esquizofrenia/fisiopatologia , Frações Subcelulares/metabolismo , Transtorno Bipolar/patologia , Células Cultivadas , AMP Cíclico/metabolismo , Humanos , Isomerismo , Microtúbulos/metabolismo , Microtúbulos/patologia , Mucosa Nasal/patologia , Mucosa Nasal/fisiopatologia , Células-Tronco Neurais/patologia , Células-Tronco Neurais/fisiologia , Neurônios/patologia , RNA Mensageiro/metabolismo , Esquizofrenia/patologia
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