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1.
Cells ; 8(8)2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31443299

RESUMO

Primary cilia are microtubule-based organelles protruding from the surface of almost all vertebrate cells. This organelle represents the cell's antenna which acts as a communication hub to transfer extracellular signals into intracellular responses during development and in tissue homeostasis. Recently, it has been shown that loss of cilia negatively regulates autophagy, the main catabolic route of the cell, probably utilizing the autophagic machinery localized at the peri-ciliary compartment. On the other side, autophagy influences ciliogenesis in a context-dependent manner, possibly to ensure that the sensing organelle is properly formed in a feedback loop model. In this review we discuss the recent literature and propose that the autophagic machinery and the ciliary proteins are functionally strictly related to control both autophagy and ciliogenesis. Moreover, we report examples of diseases associated with autophagic defects which cause cilia abnormalities, and propose and discuss the hypothesis that, at least some of the clinical manifestations observed in human diseases associated to ciliary disfunction may be the result of a perturbed autophagy.

2.
PLoS One ; 14(5): e0216705, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31095607

RESUMO

The cilium is an essential organelle at the surface of mammalian cells whose dysfunction causes a wide range of genetic diseases collectively called ciliopathies. The current rate at which new ciliopathy genes are identified suggests that many ciliary components remain undiscovered. We generated and rigorously analyzed genomic, proteomic, transcriptomic and evolutionary data and systematically integrated these using Bayesian statistics into a predictive score for ciliary function. This resulted in 285 candidate ciliary genes. We generated independent experimental evidence of ciliary associations for 24 out of 36 analyzed candidate proteins using multiple cell and animal model systems (mouse, zebrafish and nematode) and techniques. For example, we show that OSCP1, which has previously been implicated in two distinct non-ciliary processes, causes ciliogenic and ciliopathy-associated tissue phenotypes when depleted in zebrafish. The candidate list forms the basis of CiliaCarta, a comprehensive ciliary compendium covering 956 genes. The resource can be used to objectively prioritize candidate genes in whole exome or genome sequencing of ciliopathy patients and can be accessed at http://bioinformatics.bio.uu.nl/john/syscilia/ciliacarta/.

3.
EMBO Mol Med ; 11(5)2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30979712

RESUMO

Mitochondrial diseases (MDs) are a heterogeneous group of devastating and often fatal disorders due to defective oxidative phosphorylation. Despite the recent advances in mitochondrial medicine, effective therapies are still not available for these conditions. Here, we demonstrate that the microRNAs miR-181a and miR-181b (miR-181a/b) regulate key genes involved in mitochondrial biogenesis and function and that downregulation of these miRNAs enhances mitochondrial turnover in the retina through the coordinated activation of mitochondrial biogenesis and mitophagy. We thus tested the effect of miR-181a/b inactivation in different animal models of MDs, such as microphthalmia with linear skin lesions and Leber's hereditary optic neuropathy. We found that miR-181a/b downregulation strongly protects retinal neurons from cell death and significantly ameliorates the disease phenotype in all tested models. Altogether, our results demonstrate that miR-181a/b regulate mitochondrial homeostasis and that these miRNAs may be effective gene-independent therapeutic targets for MDs characterized by neuronal degeneration.

4.
Gastroenterology ; 2018 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-30452922

RESUMO

BACKGROUND AND AIMS: Wilson disease is an inherited disorder of copper metabolism that leads to copper accumulation and toxicity in liver and brain. It is caused by mutations in the ATPase copper transporting beta gene (ATP7B), which encodes a protein that transports copper out of heaptocytes into the bile. We studied ATP7B-deficient cells and animals to identify strategies to reduce copper toxicity in patients with Wilson disease. METHODS: We used RNA-seq to compare gene expression patterns between wild-type and ATP7B-knockout HepG2 cells exposed to copper. We collected blood and liver tissues from Atp7b-/- and Atp7b+/- (control) rats (LPP) and mice; some mice were given 5 daily injections of an autophagy inhibitor (spautin-1) or vehicle. We obtained liver biopsies from 2 patients with Wilson disease in Italy and liver tissues from patients without Wilson disease (control). Liver tissues were analyzed by immunohistochemistry, immunofluorescence, cell viability, and apoptosis assays, as well as by electron and confocal microscopy. Proteins were knocked down in cell lines using small interfering RNAs. Levels of copper were measured in cell lysates, blood samples, liver homogenates, and subcellular fractions by spectroscopy. RESULTS: Following exposure to copper, ATP7B-knockout cells had significant increases in expression of 103 genes that regulate autophagy (including MAP1LC3A, known as LC3), compared with wild-type cells. Electron and confocal microscopy revealed more autophagic structures in the cytoplasm of ATP7B-knockout cells than wild-type cells following copper exposure. Hepatocytes in liver tissues from patients with Wilson disease, as well as Atp7b-/- mice and rats (but not controls), had multiple autophagosomes. In ATP7B-knockout cells, mTOR had reduced activity and was dissociated from lysosomes; this resulted in translocation of the mTOR substrate transcription factor EB (TFEB) to the nucleus, and activation of autophagy-related genes. In wild-type HepG2 cells (but not ATP7B-knockout cells), exposure to copper and amino acids induced recruitment of mTOR to lysosomes. Pharmacologic inhibitors of autophagy or knockdown of autophagy proteins ATG7 and ATG13 induced and accelerated death of ATP7B-knockout HepG2 cells, compared to wild-type cells. Autophagy protected ATP7B-knockout cells from copper-induced death. CONCLUSION: ATP7B-deficient hepatocytes, such as in those in patients with Wilson disease, activate autophagy in response to copper overload to prevent copper-induced apoptosis. Agents designed to activate this autophagic pathway might reduce copper toxicity in patients with Wilson disease.

5.
Sci Rep ; 7(1): 1224, 2017 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-28450740

RESUMO

Protein synthesis is traditionally associated with specific cytoplasmic compartments. We now show that OFD1, a centrosomal/basal body protein, interacts with components of the Preinitiation complex of translation (PIC) and of the eukaryotic Initiation Factor (eIF)4F complex and modulates the translation of specific mRNA targets in the kidney. We demonstrate that OFD1 cooperates with the mRNA binding protein Bicc1 to functionally control the protein synthesis machinery at the centrosome where also the PIC and eIF4F components were shown to localize in mammalian cells. Interestingly, Ofd1 and Bicc1 are both involved in renal cystogenesis and selected targets were shown to accumulate in two models of inherited renal cystic disease. Our results suggest a possible role for the centrosome as a specialized station to modulate translation for specific functions of the nearby ciliary structures and may provide functional clues for the understanding of renal cystic disease.


Assuntos
Centrossomo/metabolismo , Regulação da Expressão Gênica , Biossíntese de Proteínas , Mapeamento de Interação de Proteínas , Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células HEK293 , Células HeLa , Humanos
6.
J Med Genet ; 54(6): 371-380, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28289185

RESUMO

Oral-facial-digital syndromes (OFDS) gather rare genetic disorders characterised by facial, oral and digital abnormalities associated with a wide range of additional features (polycystic kidney disease, cerebral malformations and several others) to delineate a growing list of OFDS subtypes. The most frequent, OFD type I, is caused by a heterozygous mutation in the OFD1 gene encoding a centrosomal protein. The wide clinical heterogeneity of OFDS suggests the involvement of other ciliary genes. For 15 years, we have aimed to identify the molecular bases of OFDS. This effort has been greatly helped by the recent development of whole-exome sequencing (WES). Here, we present all our published and unpublished results for WES in 24 cases with OFDS. We identified causal variants in five new genes (C2CD3, TMEM107, INTU, KIAA0753 and IFT57) and related the clinical spectrum of four genes in other ciliopathies (C5orf42, TMEM138, TMEM231 and WDPCP) to OFDS. Mutations were also detected in two genes previously implicated in OFDS. Functional studies revealed the involvement of centriole elongation, transition zone and intraflagellar transport defects in OFDS, thus characterising three ciliary protein modules: the complex KIAA0753-FOPNL-OFD1, a regulator of centriole elongation; the Meckel-Gruber syndrome module, a major component of the transition zone; and the CPLANE complex necessary for IFT-A assembly. OFDS now appear to be a distinct subgroup of ciliopathies with wide heterogeneity, which makes the initial classification obsolete. A clinical classification restricted to the three frequent/well-delineated subtypes could be proposed, and for patients who do not fit one of these three main subtypes, a further classification could be based on the genotype.


Assuntos
Face/anormalidades , Síndromes Orofaciodigitais/genética , Anormalidades Múltiplas/genética , Transtornos da Motilidade Ciliar/genética , Encefalocele/genética , Feminino , Heterozigoto , Humanos , Masculino , Mutação/genética , Doenças Renais Policísticas/genética , Proteínas/genética , Retinite Pigmentosa
7.
Hum Mol Genet ; 26(1): 19-32, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27798113

RESUMO

Defects in OFD1 underlie the clinically complex ciliopathy, Oral-Facial-Digital syndrome Type I (OFD Type I). Our understanding of the molecular, cellular and clinical consequences of impaired OFD1 originates from its characterised roles at the centrosome/basal body/cilia network. Nonetheless, the first described OFD1 interactors were components of the TIP60 histone acetyltransferase complex. We find that OFD1 can also localise to chromatin and its reduced expression is associated with mis-localization of TIP60 in patient-derived cell lines. TIP60 plays important roles in controlling DNA repair. OFD Type I cells exhibit reduced histone acetylation and altered chromatin dynamics in response to DNA double strand breaks (DSBs). Furthermore, reduced OFD1 impaired DSB repair via homologous recombination repair (HRR). OFD1 loss also adversely impacted upon the DSB-induced G2-M checkpoint, inducing a hypersensitive and prolonged arrest. Our findings show that OFD Type I patient cells have pronounced defects in the DSB-induced histone modification, chromatin remodelling and DSB-repair via HRR; effectively phenocopying loss of TIP60. These data extend our knowledge of the molecular and cellular consequences of impaired OFD1, demonstrating that loss of OFD1 can negatively impact upon important nuclear events; chromatin plasticity and DNA repair.


Assuntos
Cromatina/metabolismo , Cílios/patologia , Reparo do DNA/genética , Síndromes Orofaciodigitais/genética , Síndromes Orofaciodigitais/patologia , Proteínas/metabolismo , Recombinação Genética/genética , Acetilação , Pontos de Checagem do Ciclo Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Cromatina/genética , Cílios/enzimologia , Quebras de DNA de Cadeia Dupla , Fibroblastos , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Síndromes Orofaciodigitais/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/genética , RNA Interferente Pequeno/genética
9.
Sci Rep ; 6: 27315, 2016 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-27265476

RESUMO

Non-coding RNAs provide additional regulatory layers to gene expression as well as the potential to being exploited as therapeutic tools. Non-coding RNA-based therapeutic approaches have been attempted in dominant diseases, however their use for treatment of genetic diseases caused by insufficient gene dosage is currently more challenging. SINEUPs are long antisense non-coding RNAs that up-regulate translation in mammalian cells in a gene-specific manner, although, so far evidence of SINEUP efficacy has only been demonstrated in in vitro systems. We now show that synthetic SINEUPs effectively and specifically increase protein levels of a gene of interest in vivo. We demonstrated that SINEUPs rescue haploinsufficient gene dosage in a medakafish model of a human disorder leading to amelioration of the disease phenotype. Our results demonstrate that SINEUPs act through mechanisms conserved among vertebrates and that SINEUP technology can be successfully applied in vivo as a new research and therapeutic tool for gene-specific up-regulation of endogenous functional proteins.


Assuntos
Produtos Biológicos/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Terapia Genética/métodos , Microftalmia/terapia , RNA Longo não Codificante/administração & dosagem , Anormalidades da Pele/terapia , Animais , Produtos Biológicos/metabolismo , Modelos Animais de Doenças , Humanos , Oryzias , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Resultado do Tratamento
10.
Cilia ; 5: 12, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27141300

RESUMO

Oral-facial-digital syndromes (OFDS) represent a heterogeneous group of rare developmental disorders affecting the mouth, the face and the digits. Additional signs may involve brain, kidneys and other organs thus better defining the different clinical subtypes. With the exception of OFD types I and VIII, which are X-linked, the majority of OFDS is transmitted as an autosomal recessive syndrome. A number of genes have already found to be mutated in OFDS and most of the encoded proteins are predicted or proven to be involved in primary cilia/basal body function. Preliminary data indicate a physical interaction among some of those proteins and future studies will clarify whether all OFDS proteins are part of a network functionally connected to cilia. Mutations in some of the genes can also lead to other types of ciliopathies with partially overlapping phenotypes, such as Joubert syndrome (JS) and Meckel syndrome (MKS), supporting the concept that cilia-related diseases might be a continuous spectrum of the same phenotype with different degrees of severity. To date, seven of the described OFDS still await a molecular definition and two unclassified forms need further clinical and molecular validation. Next-generation sequencing (NGS) approaches are expected to shed light on how many OFDS geneticists should consider while evaluating oral-facial-digital cases. Functional studies will establish whether the non-ciliary functions of the transcripts mutated in OFDS might contribute to any of the phenotypic abnormalities observed in OFDS.

11.
Nat Genet ; 48(6): 648-56, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27158779

RESUMO

Cilia use microtubule-based intraflagellar transport (IFT) to organize intercellular signaling. Ciliopathies are a spectrum of human diseases resulting from defects in cilia structure or function. The mechanisms regulating the assembly of ciliary multiprotein complexes and the transport of these complexes to the base of cilia remain largely unknown. Combining proteomics, in vivo imaging and genetic analysis of proteins linked to planar cell polarity (Inturned, Fuzzy and Wdpcp), we identified and characterized a new genetic module, which we term CPLANE (ciliogenesis and planar polarity effector), and an extensive associated protein network. CPLANE proteins physically and functionally interact with the poorly understood ciliopathy-associated protein Jbts17 at basal bodies, where they act to recruit a specific subset of IFT-A proteins. In the absence of CPLANE, defective IFT-A particles enter the axoneme and IFT-B trafficking is severely perturbed. Accordingly, mutation of CPLANE genes elicits specific ciliopathy phenotypes in mouse models and is associated with ciliopathies in human patients.


Assuntos
Ciliopatias/metabolismo , Flagelos/fisiologia , Proteínas/fisiologia , Animais , Humanos , Camundongos , Mutação , Fenótipo , Ligação Proteica , Transporte Proteico , Proteínas/genética
12.
PLoS One ; 11(4): e0153757, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27124303

RESUMO

We report molecular genetic analysis of 42 affected individuals referred with a diagnosis of aniridia who previously screened as negative for intragenic PAX6 mutations. Of these 42, the diagnoses were 31 individuals with aniridia and 11 individuals referred with a diagnosis of Gillespie syndrome (iris hypoplasia, ataxia and mild to moderate developmental delay). Array-based comparative genomic hybridization identified six whole gene deletions: four encompassing PAX6 and two encompassing FOXC1. Six deletions with plausible cis-regulatory effects were identified: five that were 3' (telomeric) to PAX6 and one within a gene desert 5' (telomeric) to PITX2. Sequence analysis of the FOXC1 and PITX2 coding regions identified two plausibly pathogenic de novo FOXC1 missense mutations (p.Pro79Thr and p.Leu101Pro). No intragenic mutations were detected in PITX2. FISH mapping in an individual with Gillespie-like syndrome with an apparently balanced X;11 reciprocal translocation revealed disruption of a gene at each breakpoint: ARHGAP6 on the X chromosome and PHF21A on chromosome 11. In the other individuals with Gillespie syndrome no mutations were identified in either of these genes, or in HCCS which lies close to the Xp breakpoint. Disruption of PHF21A has previously been implicated in the causation of intellectual disability (but not aniridia). Plausibly causative mutations were identified in 15 out of 42 individuals (12/32 aniridia; 3/11 Gillespie syndrome). Fourteen of these mutations presented in the known aniridia genes; PAX6, FOXC1 and PITX2. The large number of individuals in the cohort with no mutation identified suggests greater locus heterogeneity may exist in both isolated and syndromic aniridia than was previously appreciated.


Assuntos
Aniridia/genética , Ataxia Cerebelar/genética , Deficiência Intelectual/genética , Fator de Transcrição PAX6/genética , Cromossomos Humanos Par 11/genética , Cromossomos Humanos X/genética , Hibridização Genômica Comparativa/métodos , Feminino , Fatores de Transcrição Forkhead/genética , Proteínas Ativadoras de GTPase/genética , Testes Genéticos/métodos , Histona Desacetilases/genética , Proteínas de Homeodomínio/genética , Humanos , Masculino , Mutação/genética , Fatores de Transcrição/genética
13.
Hum Mol Genet ; 25(3): 497-513, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26643951

RESUMO

Oral-facial-digital (OFD) syndromes are rare heterogeneous disorders characterized by the association of abnormalities of the face, the oral cavity and the extremities, some due to mutations in proteins of the transition zone of the primary cilia or the closely associated distal end of centrioles. These two structures are essential for the formation of functional cilia, and for signaling events during development. We report here causal compound heterozygous mutations of KIAA0753/OFIP in a patient with an OFD VI syndrome. We show that the KIAA0753/OFIP protein, whose sequence is conserved in ciliated species, associates with centrosome/centriole and pericentriolar satellites in human cells and forms a complex with FOR20 and OFD1. The decreased expression of any component of this ternary complex in RPE1 cells causes a defective recruitment onto centrosomes and satellites. The OFD KIAA0753/OFIP mutant loses its capacity to interact with FOR20 and OFD1, which may be the molecular basis of the defect. We also show that KIAA0753/OFIP has microtubule-stabilizing activity. OFD1 and FOR20 are known to regulate the integrity of the centriole distal end, confirming that this structural element is a target of importance for pathogenic mutations in ciliopathies.


Assuntos
Centríolos/metabolismo , Centrossomo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Síndromes Orofaciodigitais/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Centríolos/ultraestrutura , Centrossomo/ultraestrutura , Cílios/genética , Cílios/metabolismo , Cílios/patologia , Sequência Conservada , Feminino , Expressão Gênica , Heterozigoto , Humanos , Recém-Nascido , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Mutação , Síndromes Orofaciodigitais/genética , Síndromes Orofaciodigitais/patologia , Ligação Proteica , Proteínas/genética , Alinhamento de Sequência
14.
J Mol Biol ; 427(10): 1887-902, 2015 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-25796437

RESUMO

Ultradian oscillators are cyclically expressed genes with a period of less than 24h, found in the major signalling pathways. The Notch effector hairy and enhancer of split Hes genes are ultradian oscillators. The physiological signals that synchronise and entrain Hes oscillators remain poorly understood. We investigated whether cellular metabolism modulates Hes1 cyclic expression. We demonstrated that, in mouse myoblasts (C2C12), Hes1 oscillation depends on reactive oxygen species (ROS), which are generated by the mitochondria electron transport chain and by NADPH oxidases NOXs. In vitro, the regulation of Hes1 by ROS occurs via the calcium-mediated signalling. The modulation of Hes1 by ROS was relevant in vivo, since perturbing ROS homeostasis was sufficient to alter Medaka (Oryzias latipes) somitogenesis, a process that is dependent on Hes1 ultradian oscillation during embryo development. Moreover, in a Medaka model for human microphthalmia with linear skin lesions syndrome, in which mitochondrial ROS homeostasis was impaired, we documented important somitogenesis defects and the deregulation of Hes homologues genes involved in somitogenesis. Notably, both molecular and developmental defects were rescued by antioxidant treatments. Our studies provide the first evidence of a coupling between cellular redox metabolism and an ultradian biological oscillator with important pathophysiological implication for somitogenesis.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Relógios Biológicos/fisiologia , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Oryzias/genética , Espécies Reativas de Oxigênio/metabolismo , Somitos/patologia , Animais , Antioxidantes/farmacologia , Cálcio/metabolismo , Células Cultivadas , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Embrião não Mamífero/metabolismo , Humanos , Hibridização In Situ , Camundongos , Microftalmia/metabolismo , Microftalmia/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , NADPH Oxidases/metabolismo , Oryzias/embriologia , Transdução de Sinais , Anormalidades da Pele/metabolismo , Anormalidades da Pele/patologia , Somitos/embriologia , Somitos/metabolismo , Síndrome , Fatores de Transcrição HES-1
15.
BMC Genomics ; 15: 658, 2014 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-25102769

RESUMO

BACKGROUND: Cilia are microtubule-based organelles protruding from almost all mammalian cells which, when dysfunctional, result in genetic disorders called "ciliopathies". High-throughput studies have revealed that cilia are composed of thousands of proteins. However, despite many efforts, much remains to be determined regarding the biological functions of this increasingly important complex organelle. RESULTS: We have derived an online tool, from a systematic network-based approach to dissect the cilia/centrosome complex interactome (CCCI). The tool integrates all current available data into a model which provides an "interaction" perspective on ciliary function. We generated a network of interactions between human proteins organized into functionally relevant "communities", which can be defined as groups of genes that are both highly inter-connected and strongly co-expressed. We then combined sequence and co-expression data in order to identify the transcription factors responsible for regulating genes within their respective communities. Our analyses have discovered communities significantly specialized for delegating specific biological functions such as mRNA processing, protein translation, folding and degradation processes that had never been associated with ciliary proteins until now. CONCLUSIONS: CCCI will allow us to clarify the roles of previously unknown ciliary functions, elucidate the molecular mechanisms underlying ciliary-associated phenotypes, and apply our knowledge of the functional roles of relatively uncharacterized molecular entities to disease phenotypes and new clinical applications.


Assuntos
Centrossomo/metabolismo , Cílios/metabolismo , Biologia Computacional/métodos , Humanos , Internet , Transporte Proteico , Fatores de Transcrição/metabolismo , Transcriptoma
16.
Nat Genet ; 46(8): 905-11, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24997988

RESUMO

Centrioles are microtubule-based, barrel-shaped structures that initiate the assembly of centrosomes and cilia. How centriole length is precisely set remains elusive. The microcephaly protein CPAP (also known as MCPH6) promotes procentriole growth, whereas the oral-facial-digital (OFD) syndrome protein OFD1 represses centriole elongation. Here we uncover a new subtype of OFD with severe microcephaly and cerebral malformations and identify distinct mutations in two affected families in the evolutionarily conserved C2CD3 gene. Concordant with the clinical overlap, C2CD3 colocalizes with OFD1 at the distal end of centrioles, and C2CD3 physically associates with OFD1. However, whereas OFD1 deletion leads to centriole hyperelongation, loss of C2CD3 results in short centrioles without subdistal and distal appendages. Because C2CD3 overexpression triggers centriole hyperelongation and OFD1 antagonizes this activity, we propose that C2CD3 directly promotes centriole elongation and that OFD1 acts as a negative regulator of C2CD3. Our results identify regulation of centriole length as an emerging pathogenic mechanism in ciliopathies.


Assuntos
Centríolos/genética , Proteínas Associadas aos Microtúbulos/genética , Síndromes Orofaciodigitais/genética , Linhagem Celular , Pré-Escolar , Predisposição Genética para Doença , Células HEK293 , Humanos , Masculino , Microcefalia/genética , Proteínas/genética
17.
Orphanet J Rare Dis ; 9: 74, 2014 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-24884629

RESUMO

BACKGROUND: Oral-facial-digital type 1 syndrome (OFD1; OMIM 311200) belongs to the expanding group of disorders ascribed to ciliary dysfunction. With the aim of contributing to the understanding of the role of primary cilia in the central nervous system (CNS), we performed a thorough characterization of CNS involvement observed in this disorder. METHODS: A cohort of 117 molecularly diagnosed OFD type I patients was screened for the presence of neurological symptoms and/or cognitive/behavioral abnormalities on the basis of the available information supplied by the collaborating clinicians. Seventy-one cases showing CNS involvement were further investigated through neuroimaging studies and neuropsychological testing. RESULTS: Seventeen patients were molecularly diagnosed in the course of this study and five of these represent new mutations never reported before. Among patients displaying neurological symptoms and/or cognitive/behavioral abnormalities, we identified brain structural anomalies in 88.7%, cognitive impairment in 68%, and associated neurological disorders and signs in 53% of cases. The most frequently observed brain structural anomalies included agenesis of the corpus callosum and neuronal migration/organisation disorders as well as intracerebral cysts, porencephaly and cerebellar malformations. CONCLUSIONS: Our results support recent published findings indicating that CNS involvement in this condition is found in more than 60% of cases. Our findings correlate well with the kind of brain developmental anomalies described in other ciliopathies. Interestingly, we also described specific neuropsychological aspects such as reduced ability in processing verbal information, slow thought process, difficulties in attention and concentration, and notably, long-term memory deficits which may indicate a specific role of OFD1 and/or primary cilia in higher brain functions.


Assuntos
Doenças do Sistema Nervoso Central/fisiopatologia , Síndromes Orofaciodigitais/fisiopatologia , Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso Central/psicologia , Estudos de Coortes , Feminino , Humanos , Imagem por Ressonância Magnética , Mutação , Testes Neuropsicológicos , Síndromes Orofaciodigitais/genética , Síndromes Orofaciodigitais/psicologia
18.
J Clin Invest ; 124(5): 2059-70, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24691443

RESUMO

Cilia are critical mediators of paracrine signaling; however, it is unknown whether proteins that contribute to ciliopathies converge on multiple paracrine pathways through a common mechanism. Here, we show that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In WT cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. Furthermore, overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from Ofd1-deficient mice. Finally, we tested the hypothesis that other proteasome-dependent pathways not known to be associated with ciliopathies are defective in the absence of ciliopathy proteins. We found that loss of BBS1, BBS4, or OFD1 led to decreased NF-κB activity and concomitant IκBß accumulation and that these defects were ameliorated with SFN treatment. Taken together, our data indicate that basal body proteasomal regulation governs paracrine signaling pathways and suggest that augmenting proteasomal function might benefit ciliopathy patients.


Assuntos
Proteínas I-kappa B/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismo , Proteólise , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Anticarcinógenos/farmacologia , Linhagem Celular , Transtornos da Motilidade Ciliar/tratamento farmacológico , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/metabolismo , Transtornos da Motilidade Ciliar/patologia , Humanos , Proteínas I-kappa B/genética , Isotiocianatos/farmacologia , Ácido Mevalônico/análogos & derivados , Ácido Mevalônico/farmacologia , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas/genética , Proteínas de Peixe-Zebra/genética
19.
Hum Genet ; 133(3): 367-77, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24178751

RESUMO

Oral-facial-digital syndrome type VI (OFD VI) is a recessive ciliopathy defined by two diagnostic criteria: molar tooth sign (MTS) and one or more of the following: (1) tongue hamartoma (s) and/or additional frenula and/or upper lip notch; (2) mesoaxial polydactyly of one or more hands or feet; (3) hypothalamic hamartoma. Because of the MTS, OFD VI belongs to the "Joubert syndrome related disorders". Its genetic aetiology remains largely unknown although mutations in the TMEM216 gene, responsible for Joubert (JBS2) and Meckel-Gruber (MKS2) syndromes, have been reported in two OFD VI patients. To explore the molecular cause(s) of OFD VI syndrome, we used an exome sequencing strategy in six unrelated families followed by Sanger sequencing. We identified a total of 14 novel mutations in the C5orf42 gene in 9/11 families with positive OFD VI diagnostic criteria including a severe fetal case with microphthalmia, cerebellar hypoplasia, corpus callosum agenesis, polydactyly and skeletal dysplasia. C5orf42 mutations have already been reported in Joubert syndrome confirming that OFD VI and JBS are allelic disorders, thus enhancing our knowledge of the complex, highly heterogeneous nature of ciliopathies.


Assuntos
Proteínas de Membrana/genética , Síndromes Orofaciodigitais/diagnóstico , Síndromes Orofaciodigitais/genética , Anormalidades Múltiplas , Adolescente , Adulto , Alelos , Doenças Cerebelares/diagnóstico , Doenças Cerebelares/genética , Cerebelo/anormalidades , Criança , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/genética , Exoma , Anormalidades do Olho/diagnóstico , Anormalidades do Olho/genética , Feminino , Hamartoma/diagnóstico , Hamartoma/genética , Humanos , Doenças Hipotalâmicas/diagnóstico , Doenças Hipotalâmicas/genética , Doenças Renais Císticas/diagnóstico , Doenças Renais Císticas/genética , Masculino , Mutação , Malformações do Sistema Nervoso/diagnóstico , Malformações do Sistema Nervoso/genética , Fenótipo , Polidactilia/diagnóstico , Polidactilia/genética , Retina/anormalidades , Análise de Sequência de DNA , Adulto Jovem
20.
Nature ; 502(7470): 254-7, 2013 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-24089205

RESUMO

The primary cilium is a microtubule-based organelle that functions in sensory and signalling pathways. Defects in ciliogenesis can lead to a group of genetic syndromes known as ciliopathies. However, the regulatory mechanisms of primary ciliogenesis in normal and cancer cells are incompletely understood. Here we demonstrate that autophagic degradation of a ciliopathy protein, OFD1 (oral-facial-digital syndrome 1), at centriolar satellites promotes primary cilium biogenesis. Autophagy is a catabolic pathway in which cytosol, damaged organelles and protein aggregates are engulfed in autophagosomes and delivered to lysosomes for destruction. We show that the population of OFD1 at the centriolar satellites is rapidly degraded by autophagy upon serum starvation. In autophagy-deficient Atg5 or Atg3 null mouse embryonic fibroblasts, OFD1 accumulates at centriolar satellites, leading to fewer and shorter primary cilia and a defective recruitment of BBS4 (Bardet-Biedl syndrome 4) to cilia. These defects are fully rescued by OFD1 partial knockdown that reduces the population of OFD1 at centriolar satellites. More strikingly, OFD1 depletion at centriolar satellites promotes cilia formation in both cycling cells and transformed breast cancer MCF7 cells that normally do not form cilia. This work reveals that removal of OFD1 by autophagy at centriolar satellites represents a general mechanism to promote ciliogenesis in mammalian cells. These findings define a newly recognized role of autophagy in organelle biogenesis.


Assuntos
Autofagia , Centríolos/metabolismo , Cílios/fisiologia , Proteínas/metabolismo , Animais , Autofagia/genética , Linhagem Celular , Cílios/genética , Cílios/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Transporte Proteico , Proteínas/genética
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