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1.
Mol Brain ; 12(1): 48, 2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31072410

RESUMO

In humans, dysfunctional primary cilia result in Bardet-Biedl syndrome (BBS), which presents with clinical features including intellectual disabilities, obesity, and retinal degeneration, and, in mouse models, the added feature of hydrocephalus. We observed increased Glial Fibrillary Acidic Protein (GFAP) immunoreactivity in BBS mouse brains. Increased GFAP expression is a hallmark of astrocyte reactivity that is associated with microglia activation and neuro-inflammation. To gain a better understanding of reactive astrocytes observed in BBS mice, we used two mouse models of BBS8, a BBSome protein, to characterize the reactive astrocyte phenotype. The finding of reactive astrocytes in young BBS mouse brains led us to hypothesize that loss of BBSome function leads to reactive astrocytes prior to hydrocephalus and obesity. By using two mouse models of BBS8, a congenital BBS8 knockout with hydrocephalus, and a tamoxifen-inducible BBS8 knockout without hydrocephalus, we were able to molecularly phenotype the reactive astrocytes. Molecular phenotype of reactive astrocytes shows differential regulation of inducers of Pan, A1 neurotoxic, and A2 neuroprotective astrocytes that are significantly altered in brains of both congenital and induced knockouts of BBS8, but without microglia activation. We find evidence for neuroinflammation in the brains of congenital knockout mice, but not in induced knockout mice. Protein levels of GFAP, SERPINA3N and post-synaptic density 95 (PSD95) are significantly increased in congenital knockout mice, but remain unchanged in induced knockout mice. Thus, despite the reactive astrocyte phenotype being present in both models, the molecular signature of reactive astrocytes in BBS8 mice models are distinct. Together, these findings suggest that BBS8, and by extension the BBSome, plays a role in neuro-astrocyte functions independent of hydrocephalus, and its dysregulation is associated with astrocyte reactivity without microglia activation. (Total word count 278).

2.
Diabetes ; 68(8): 1591-1603, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31127052

RESUMO

The BBSome, a complex of eight Bardet-Biedl syndrome (BBS) proteins involved in cilia function, has emerged as an important regulator of energy balance, but the underlying cellular and molecular mechanisms are not fully understood. Here, we show that the control of energy homeostasis by the anorexigenic proopiomelanocortin (POMC) neurons and orexigenic agouti-related peptide (AgRP) neurons require intact BBSome. Targeted disruption of the BBSome by Bbs1 gene deletion in POMC or AgRP neurons increases body weight and adiposity. We demonstrate that obesity in mice lacking the Bbs1 gene in POMC neurons is associated with hyperphagia. Mechanistically, we present evidence implicating the BBSome in the trafficking of G protein-coupled neuropeptide Y Y2 receptor (NPY2R) and serotonin 5-hydroxytryptamine (HT)2C receptor (5-HT2CR) to cilia and plasma membrane, respectively. Consistent with this, loss of the BBSome reduced cell surface expression of the 5-HT2CR, interfered with serotonin-evoked increase in intracellular calcium and membrane potential, and blunted the anorectic and weight-reducing responses evoked by the 5-HT2cR agonist, lorcaserin. Finally, we show that disruption of the BBSome causes the 5-HT2CR to be stalled in the late endosome. Our results demonstrate the significance of the hypothalamic BBSome for the control of energy balance through regulation of trafficking of important metabolic receptors.

3.
Hum Gene Ther ; 30(8): 967-974, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31106594

RESUMO

In a screen of 1,000 consecutively ascertained families, we recently found that mutations in the gene RPGR are the third most common cause of all inherited retinal disease. As the two most frequent disease-causing genes, ABCA4 and USH2A, are far too large to fit into clinically relevant adeno-associated virus (AAV) vectors, RPGR is an obvious early target for AAV-based ocular gene therapy. In generating plasmids for this application, we discovered that those containing wild-type RPGR sequence, which includes the highly repetitive low complexity region ORF15, were extremely unstable (i.e., they showed consistent accumulation of genomic changes during plasmid propagation). To develop a stable RPGR gene transfer vector, we used a bioinformatics approach to identify predicted regions of genomic instability within ORF15 (i.e., potential non-B DNA conformations). Synonymous substitutions were made in these regions to reduce the repetitiveness and increase the molecular stability while leaving the encoded amino acid sequence unchanged. The resulting construct was subsequently packaged into AAV serotype 5, and the ability to drive transcript expression and functional protein production was demonstrated via subretinal injection in rat and pull-down assays, respectively. By making synonymous substitutions within the repetitive region of RPGR, we were able to stabilize the plasmid and subsequently generate a clinical-grade gene transfer vector (IA-RPGR). Following subretinal injection in rat, we demonstrated that the augmented transcript was expressed at levels similar to wild-type constructs. By performing in vitro pull-down experiments, we were able to show that IA-RPGR protein product retained normal protein binding properties (i.e., analysis revealed normal binding to PDE6D, INPP5E, and RPGRIP1L). In summary, we have generated a stable RPGR gene transfer vector capable of producing functional RPGR protein, which will facilitate safety and toxicity studies required for progression to an Investigational New Drug application.

4.
Proc Natl Acad Sci U S A ; 116(4): 1353-1360, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30622176

RESUMO

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease with severe vision impairment leading to blindness. About 10-15% of RP cases are caused by mutations in the RPGR gene, with RPGR mutations accounting for 70% of X-linked RP cases. The mechanism by which RPGR mutations cause photoreceptor cell dysfunction is not well understood. In this study, we show that the two isoforms of RPGR (RPGR1-19 and RPGRORF15) interact with endogenous PDE6D, INPP5E, and RPGRIP1L. The RPGR1-19 isoform contains two PDE6D binding sites with the C-terminal prenylation site being the predominant PDE6D binding site. The C terminus of RPGR1-19 that contains the prenylation site regulates its interaction with PDE6D, INPP5E, and RPGRIP1L. Only the RPGR1-19 isoform localizes to cilia in cultured RPE1 cells. Missense variations found in RPGR patients disrupt the interaction between RPGR isoforms and their endogenous interactors INPP5E, PDE6D, and RPGRIP1L. We evaluated a RPGR missense variation (M58K) found in a family with X-linked retinitis pigmentosa (XLRP) and show that this missense variation disrupts the interaction of RPGR isoforms with their endogenous interactors. The M58K variation also disrupts the ciliary localization of the RPGR1-19 isoform. Using this assay, we also show that some of the RPGR missense variants reported in the literature might not actually be disease causing. Our data establishes an in vitro assay that can be used to validate the potential pathogenicity of RPGR missense variants.


Assuntos
Proteínas do Olho/genética , Mutação de Sentido Incorreto/genética , Proteínas Nucleares/genética , Domínios e Motivos de Interação entre Proteínas/genética , Retinite Pigmentosa/genética , Animais , Sítios de Ligação/genética , Linhagem Celular , Cílios/genética , Células HEK293 , Humanos , Camundongos , Isoformas de Proteínas/genética
5.
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.

6.
Front Physiol ; 9: 708, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29971011

RESUMO

Osteoarthritis (OA) is a debilitating inflammation related disease characterized by joint pain and effusion, loss of mobility, and deformity that may result in functional joint failure and significant impact on quality of life. Once thought of as a simple "wear and tear" disease, it is now widely recognized that OA has a considerable metabolic component and is related to chronic inflammation. Defects associated with primary cilia have been shown to be cause OA-like changes in Bardet-Biedl mice. We examined the role of dysfunctional primary cilia in OA in mice through the regulation of the previously identified degradative and pro-inflammatory molecular pathways common to OA. We observed an increase in the presence of pro-inflammatory markers TGFß-1 and HTRA1 as well as cartilage destructive protease MMP-13 but a decrease in DDR-2. We observed a morphological difference in cartilage thickness in Bbs1 M390R/M390R mice compared to wild type (WT). We did not observe any difference in OARSI or Mankin scores between WT and Bbs1M390R/M390R mice. Primary cilia appear to be involved in the upregulation of biomarkers, including pro-inflammatory markers common to OA.

8.
J Biol Chem ; 293(25): 9854-9868, 2018 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-29743238

RESUMO

Elevation of intraocular pressure (IOP) is a serious adverse effect of glucocorticoid (GC) therapy. Increased extracellular matrix (ECM) accumulation and endoplasmic reticulum (ER) stress in the trabecular meshwork (TM) is associated with GC-induced IOP elevation. However, the molecular mechanisms by which GCs induce ECM accumulation and ER stress in the TM have not been determined. Here, we show that a potent GC, dexamethasone (Dex), activates transforming growth factor ß (TGFß) signaling, leading to GC-induced ECM accumulation, ER stress, and IOP elevation. Dex increased both the precursor and bioactive forms of TGFß2 in conditioned medium and activated TGFß-induced SMAD signaling in primary human TM cells. Dex also activated TGFß2 in the aqueous humor and TM of a mouse model of Dex-induced ocular hypertension. We further show that Smad3-/- mice are protected from Dex-induced ocular hypertension, ER stress, and ECM accumulation. Moreover, treating WT mice with a selective TGFß receptor kinase I inhibitor, LY364947, significantly decreased Dex-induced ocular hypertension. Of note, knockdown of the ER stress-induced activating transcription factor 4 (ATF4), or C/EBP homologous protein (CHOP), completely prevented Dex-induced TGFß2 activation and ECM accumulation in TM cells. These observations suggested that chronic ER stress promotes Dex-induced ocular hypertension via TGFß signaling. Our results indicate that TGFß2 signaling plays a central role in GC-induced ocular hypertension and provides therapeutic targets for GC-induced ocular hypertension.

9.
PLoS One ; 13(2): e0192755, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29444170

RESUMO

Nephronophthisis-related ciliopathies (NPHP-RC) are a group of disorders that present with end-stage renal failure in childhood/adolescence, kidney cysts, retinal degeneration, and cerebellar hypoplasia. One disorder that shares clinical features with NPHP-RC is Bardet-Biedl Syndrome (BBS). Serologically defined colon cancer antigen 8 (SDCCAG8; also known as NPHP10 and BBS16) is an NPHP gene that is also associated with BBS. To better understand the patho-mechanisms of NPHP and BBS caused by loss of SDCCAG8 function, we characterized an SDCCAG8 mouse model (Sdccag8Tn(sb-Tyr)2161B.CA1C2Ove) generated by Sleeping Beauty Transposon (SBT)-mediated insertion mutagenesis. Consistent with the previously reported, independent SDCCAG8 mouse models, our mutant mice display pre-axial polydactyly in their hind limbs. In addition, we report patterning defects in the secondary palate, brain abnormalities, as well as neonatal lethality associated with developmental defects in the lung in our mouse model. The neonatal lethality phenotype is genetic background dependent and rescued by introducing 129S6/SvEvTac background. Genetic modifier(s) responsible for this effect were mapped to a region between SNPs rs3714172 and rs3141832 on chromosome 11. While determining the precise genetic lesion in our mouse model, we found that SBT insertion resulted in a deletion of multiple exons from both Sdccag8 and its neighboring gene Akt3. We ascribe the patterning defects in the limb and the secondary palate as well as lung abnormalities to loss of SDCCAG8, while the developmental defects in the brain are likely due to the loss of AKT3. This mouse model may be useful to study features not observed in other SDCCAG8 models but cautions are needed in interpreting data.


Assuntos
Autoantígenos/genética , Genótipo , Proteínas de Neoplasias/genética , Fenótipo , Animais , Padronização Corporal/genética , Membro Posterior/anormalidades , Camundongos , Camundongos Mutantes , Modelos Animais , Mutagênese , Palato/anormalidades , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-akt/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
PLoS Genet ; 13(10): e1007057, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29049287

RESUMO

Genetic mutations disrupting the structure and function of primary cilia cause various inherited retinal diseases in humans. Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, pleiotropic ciliopathy characterized by retinal degeneration, obesity, postaxial polydactyly, intellectual disability, and genital and renal abnormalities. To gain insight into the mechanisms of retinal degeneration in BBS, we developed a congenital knockout mouse of Bbs8, as well as conditional mouse models in which function of the BBSome (a protein complex that mediates ciliary trafficking) can be temporally inactivated or restored. We demonstrate that BBS mutant mice have defects in retinal outer segment morphogenesis. We further demonstrate that removal of Bbs8 in adult mice affects photoreceptor function and disrupts the structural integrity of the outer segment. Notably, using a mouse model in which a gene trap inhibiting Bbs8 gene expression can be removed by an inducible FLP recombinase, we show that when BBS8 is restored in immature retinas with malformed outer segments, outer segment extension can resume normally and malformed outer segment discs are displaced distally by normal outer segment structures. Over time, the retinas of the rescued mice become morphologically and functionally normal, indicating that there is a window of plasticity when initial retinal outer segment morphogenesis defects can be ameliorated.


Assuntos
Morfogênese/fisiologia , Células Fotorreceptoras/metabolismo , Transporte Proteico/fisiologia , Animais , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/patologia , Cílios/metabolismo , Camundongos , Camundongos Knockout , Modelos Animais , Morfogênese/genética , Mutação/genética , Transporte Proteico/genética , Retina/metabolismo , Retina/fisiologia
11.
Proc Natl Acad Sci U S A ; 114(42): 11199-11204, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-28973933

RESUMO

Primary open-angle glaucoma (POAG) is a leading cause of irreversible vision loss worldwide, with elevated intraocular pressure (IOP) a major risk factor. Myocilin (MYOC) dominant gain-of-function mutations have been reported in ∼4% of POAG cases. MYOC mutations result in protein misfolding, leading to endoplasmic reticulum (ER) stress in the trabecular meshwork (TM), the tissue that regulates IOP. We use CRISPR-Cas9-mediated genome editing in cultured human TM cells and in a MYOC mouse model of POAG to knock down expression of mutant MYOC, resulting in relief of ER stress. In vivo genome editing results in lower IOP and prevents further glaucomatous damage. Importantly, using an ex vivo human organ culture system, we demonstrate the feasibility of human genome editing in the eye for this important disease.


Assuntos
Sistemas CRISPR-Cas , Proteínas do Citoesqueleto/genética , Proteínas do Olho/genética , Edição de Genes , Terapia Genética/métodos , Glaucoma de Ângulo Aberto/terapia , Glicoproteínas/genética , Animais , Linhagem Celular , Glaucoma de Ângulo Aberto/genética , Humanos , Técnicas In Vitro , Camundongos
13.
PLoS Genet ; 13(7): e1006936, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28753627

RESUMO

Mutations in BBS6 cause two clinically distinct syndromes, Bardet-Biedl syndrome (BBS), a syndrome caused by defects in cilia transport and function, as well as McKusick-Kaufman syndrome, a genetic disorder characterized by congenital heart defects. Congenital heart defects are rare in BBS, and McKusick-Kaufman syndrome patients do not develop retinitis pigmentosa. Therefore, the McKusick-Kaufman syndrome allele may highlight cellular functions of BBS6 distinct from the presently understood functions in the cilia. In support, we find that the McKusick-Kaufman syndrome disease-associated allele, BBS6H84Y; A242S, maintains cilia function. We demonstrate that BBS6 is actively transported between the cytoplasm and nucleus, and that BBS6H84Y; A242S, is defective in this transport. We developed a transgenic zebrafish with inducible bbs6 to identify novel binding partners of BBS6, and we find interaction with the SWI/SNF chromatin remodeling protein Smarcc1a (SMARCC1 in humans). We demonstrate that through this interaction, BBS6 modulates the sub-cellular localization of SMARCC1 and find, by transcriptional profiling, similar transcriptional changes following smarcc1a and bbs6 manipulation. Our work identifies a new function for BBS6 in nuclear-cytoplasmic transport, and provides insight into the disease mechanism underlying the congenital heart defects in McKusick-Kaufman syndrome patients.


Assuntos
Anormalidades Múltiplas/genética , Síndrome de Bardet-Biedl/genética , Chaperoninas do Grupo II/genética , Cardiopatias Congênitas/genética , Hidrocolpos/genética , Polidactilia/genética , Fatores de Transcrição/genética , Doenças Uterinas/genética , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/patologia , Transporte Ativo do Núcleo Celular/genética , Animais , Animais Geneticamente Modificados/genética , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/patologia , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Cílios/metabolismo , Cílios/patologia , Citoplasma/metabolismo , Modelos Animais de Doenças , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/patologia , Humanos , Hidrocolpos/metabolismo , Hidrocolpos/patologia , Camundongos , Mutação , Polidactilia/metabolismo , Polidactilia/patologia , Transporte Proteico/genética , Fatores de Transcrição/biossíntese , Doenças Uterinas/metabolismo , Doenças Uterinas/patologia , Peixe-Zebra/genética
14.
Ophthalmology ; 124(9): 1314-1331, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28559085

RESUMO

PURPOSE: To devise a comprehensive multiplatform genetic testing strategy for inherited retinal disease and to describe its performance in 1000 consecutive families seen by a single clinician. DESIGN: Retrospective series. PARTICIPANTS: One thousand consecutive families seen by a single clinician. METHODS: The clinical records of all patients seen by a single retina specialist between January 2010 and June 2016 were reviewed, and all patients who met the clinical criteria for a diagnosis of inherited retinal disease were included in the study. Each patient was assigned to 1 of 62 diagnostic categories, and this clinical diagnosis was used to define the scope and order of the molecular investigations that were performed. The number of nucleotides evaluated in a given subject ranged from 2 to nearly 900 000. MAIN OUTCOME MEASURES: Sensitivity and false genotype rate. RESULTS: Disease-causing genotypes were identified in 760 families (76%). These genotypes were distributed across 104 different genes. More than 75% of these 104 genes have coding sequences small enough to be packaged efficiently into an adeno-associated virus. Mutations in ABCA4 were the most common cause of disease in this cohort (173 families), whereas mutations in 80 genes caused disease in 5 or fewer families (i.e., 0.5% or less). Disease-causing genotypes were identified in 576 of the families without next-generation sequencing (NGS). This included 23 families with mutations in the repetitive region of RPGR exon 15 that would have been missed by NGS. Whole-exome sequencing of the remaining 424 families revealed mutations in an additional 182 families, and whole-genome sequencing of 4 of the remaining 242 families revealed 2 additional genotypes that were invisible by the other methods. Performing the testing in a clinically focused tiered fashion would be 6.1% more sensitive and 17.7% less expensive and would have a significantly lower average false genotype rate than using whole-exome sequencing to assess more than 300 genes in all patients (7.1% vs. 128%; P < 0.001). CONCLUSIONS: Genetic testing for inherited retinal disease is now more than 75% sensitive. A clinically directed tiered testing strategy can increase sensitivity and improve statistical significance without increasing cost.


Assuntos
Oftalmopatias Hereditárias/genética , Proteínas do Olho/genética , Mutação , Doenças Retinianas/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Análise Mutacional de DNA , Exoma/genética , Saúde da Família , Feminino , Testes Genéticos , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Linhagem , Estudos Retrospectivos , Sensibilidade e Especificidade , Análise de Sequência de DNA , Estados Unidos
15.
J Med Genet ; 54(9): 633-639, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28536242

RESUMO

BACKGROUND: Azoospermia is diagnosed when sperm cells are completely absent in the ejaculate even after centrifugation. It is identified in approximately 1% of all men and in 10%-20% of infertile males. Non-obstructive azoospermia (NOA) is characterised by the absence of sperm due to either a Sertoli cell-only pattern, maturation arrest, hypospermatogenesis or mixed patterns. NOA is a severe form of male infertility, with limited treatment options and low fertility success rates. In the majority of patients, the cause for NOA is not known and mutations in only a few genes were shown to be causative. AIM: We investigated the cause of maturation arrest in five azoospermic infertile men of a large consanguineous Bedouin family. METHODS AND RESULTS: Using whole genome genotyping and exome sequencing we identified a 4 bp deletion frameshift mutation in TDRD9 as the causative mutation with a Lod Score of 3.42. We demonstrate that the mutation results in a frameshift as well as exon skipping. Immunofluorescent staining with anti-TDRD9 antibody directed towards the N terminus demonstrated the presence of the protein in testicular biopsies of patients with an intracellular distribution comparable to a control biopsy. The mutation does not cause female infertility. CONCLUSION: This is the first report of a recessive deleterious mutation in TDRD9 in humans. The clinical phenotype recapitulates that observed in the Tdrd9 knockout mice where this gene was demonstrated to participate in long interspersed element-1 retrotransposon silencing. If this function is preserved in human, our data underscore the importance of maintaining DNA stability in the human male germ line.


Assuntos
Azoospermia/genética , DNA Helicases/genética , Mutação da Fase de Leitura , Azoospermia/patologia , DNA Helicases/análise , DNA Helicases/química , Genes Recessivos , Humanos , Masculino , Fenótipo , Domínios Proteicos , Processamento de RNA , Testículo/química , Testículo/patologia
16.
Invest Ophthalmol Vis Sci ; 58(4): 2054-2062, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28384726

RESUMO

Purpose: Primary open-angle glaucoma (POAG) is particularly common in older individuals and associated with pathologic degeneration of the trabecular meshwork (TM). We have shown previously that transplantation of induced pluripotent stem cell (iPSC) derived TM cells restores aqueous humor dynamics in young transgenic mice expressing a pathogenic form of human myocilin (Tg-MYOCY437H). This study was designed to determine if this approach is feasible in older mice with more pronounced TM dysfunction. Methods: Mouse iPSC were differentiated toward a TM cell phenotype (iPSC-TM) and injected into the anterior chamber of 6-month-old Tg-MYOCY437H or control mice. IOP and aqueous humor outflow facility were recorded for up to 3 months. Transmission electron microscopy, Western blot, and immunohistochemistry were performed to analyze TM morphology, quantify endoplasmic reticulum (ER) stress, and assess TM cellularity. Results: A 12 weeks after transplantation, IOP in iPSC-TM recipients was statistically lower and outflow facility was significantly improved compared to untreated controls. The number of endogenous TM cells increased significantly in iPSC-TM recipients along with the appearance of TM cells immmunopositive for a marker of cellular division. Morphologically, transplantation of iPSC-TM preserves ER structure 12 weeks after transplantation. However, myocilin and calnexin expression levels remain elevated in transplanted eyes of these 9-month-old Tg-MYOCY437H mice, indicating that ER stress persists within the TM. Conclusions: Transplantation of iPSC-TM can restore IOP and outflow facility in aged Tg-MYOCY437H mice. This type of stem cell-based therapy is a promising possibility for restoration of IOP control in some glaucoma patients.


Assuntos
Humor Aquoso/metabolismo , Transplante de Células/métodos , Glaucoma de Ângulo Aberto/cirurgia , Células-Tronco Pluripotentes Induzidas/transplante , Pressão Intraocular/fisiologia , Malha Trabecular/transplante , Animais , Western Blotting , Calnexina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Proteínas do Olho/metabolismo , Seguimentos , Glaucoma de Ângulo Aberto/patologia , Glaucoma de Ângulo Aberto/fisiopatologia , Glicoproteínas/metabolismo , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Malha Trabecular/citologia
17.
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
18.
Med Res Arch ; 5(9)2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29457131

RESUMO

Upwards of 90% of individuals with Bardet-Biedl syndrome (BBS) display rod-cone dystrophy with early macular involvement. BBS is an autosomal recessive, genetically heterogeneous, pleiotropic ciliopathy for which 21 causative genes have been discovered to date. In addition to retinal degeneration, the cardinal features of BBS include obesity, cognitive impairment, renal anomalies, polydactyly, and hypogonadism. Here, we review the genes, proteins, and protein complexes involved in BBS and the BBS model organisms available for the study of retinal degeneration. We include comprehensive lists for all known BBS genes, their known phenotypes, and the model organisms available. We also review the molecular mechanisms believed to lead to retinal degeneration. We provide an overview of the mode of inheritance and describe the relationships between BBS genes and Joubert syndrome, Leber Congenital Amaurosis, Senior-Løken syndrome, and non-syndromic retinitis pigmentosa. Finally, we propose ways that new advances in technology will allow us to better understand the role of different BBS genes in retinal formation and function.

19.
Bone ; 101: 179-190, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27170093

RESUMO

Bardet-Biedl Syndrome (BBS) is an autosomal recessive disorder and is classified as one of the ciliopathy. The patients manifest a characteristic craniofacial dysmorphology but the effects of Bbs3 deficiency in the developmental process during the craniofacial pathogenesis are still incompletely understood. Here, we analyzed a cranial development of a BBS model Bbs3-/- mouse. It was previously reported that these mutant mice exhibit a dome-shape cranium. We show that Bbs3-/- mouse embryos present mid-facial hypoplasia and solitary central upper incisor. Morphologically, these mutant mice show synchondrosis of the cranial base midline due to the failure to fuse in association with loss of intrasphenoidal synchondrosis. The cranial base was laterally expanded and longitudinally shortened. In the developing cartilaginous primordium of cranial base, cells present in the midline were less in Bbs3-/- embryos. Expression of BBS3 was observed specifically in a cell population lying between condensed ectomesenchyme in the midline and the ventral midbrain at this stage. Finally, siRNA-based knockdown of Bbs3 in ATDC5 cells impaired migration in culture. Our data suggest that BBS3 is required for the development of cranial base via regulation of cell migration toward the midline where they promote the condensation of ectomesenchyme and form the future cartilaginous templates of cranial base.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Síndrome de Bardet-Biedl/metabolismo , Base do Crânio/crescimento & desenvolvimento , Base do Crânio/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Fatores de Ribosilação do ADP/genética , Animais , Síndrome de Bardet-Biedl/genética , Feminino , Imunofluorescência , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Fenótipo , Microtomografia por Raio-X , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
20.
Proc Natl Acad Sci U S A ; 113(25): E3492-500, 2016 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-27274060

RESUMO

Glaucoma is a common cause of vision loss or blindness and reduction of intraocular pressure (IOP) has been proven beneficial in a large fraction of glaucoma patients. The IOP is maintained by the trabecular meshwork (TM) and the elevation of IOP in open-angle glaucoma is associated with dysfunction and loss of the postmitotic cells residing within this tissue. To determine if IOP control can be maintained by replacing lost TM cells, we transplanted TM-like cells derived from induced pluripotent stem cells into the anterior chamber of a transgenic mouse model of glaucoma. Transplantation led to significantly reduced IOP and improved aqueous humor outflow facility, which was sustained for at least 9 wk. The ability to maintain normal IOP engendered survival of retinal ganglion cells, whose loss is ultimately the cause for reduced vision in glaucoma. In vivo and in vitro analyses demonstrated higher TM cellularity in treated mice compared with littermate controls and indicated that this increase is primarily because of a proliferative response of endogenous TM cells. Thus, our study provides in vivo demonstration that regeneration of the glaucomatous TM is possible and points toward novel approaches in the treatment of this disease.


Assuntos
Modelos Animais de Doenças , Glaucoma/terapia , Transplante de Células-Tronco , Malha Trabecular/patologia , Animais , Proteínas do Citoesqueleto/genética , Proteínas do Olho/genética , Glaucoma/patologia , Glaucoma/fisiopatologia , Glicoproteínas/genética , Humanos , Pressão Intraocular , Camundongos , Camundongos Transgênicos , Mutação
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