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1.
PLoS Genet ; 19(6): e1010796, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37315079

RESUMO

Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice.


Assuntos
Cílios , Ciliopatias , Humanos , Animais , Camundongos , Cílios/genética , Cílios/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas/genética , Aminoácidos/metabolismo , Mamíferos/metabolismo , Proteínas do Citoesqueleto/genética
2.
PLoS Genet ; 17(12): e1009711, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34962918

RESUMO

Polycystic kidney disease is an inherited degenerative disease in which the uriniferous tubules are replaced by expanding fluid-filled cysts that ultimately destroy organ function. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form, afflicting approximately 1 in 1,000 people. It primarily is caused by mutations in the transmembrane proteins polycystin-1 (Pkd1) and polycystin-2 (Pkd2). The most proximal effects of Pkd mutations leading to cyst formation are not known, but pro-proliferative signaling must be involved for the tubule epithelial cells to increase in number over time. The c-Jun N-terminal kinase (JNK) pathway promotes proliferation and is activated in acute and chronic kidney diseases. Using a mouse model of cystic kidney disease caused by Pkd2 loss, we observe JNK activation in cystic kidneys and observe increased nuclear phospho c-Jun in cystic epithelium. Genetic removal of Jnk1 and Jnk2 suppresses the nuclear accumulation of phospho c-Jun, reduces proliferation and reduces the severity of cystic disease. While Jnk1 and Jnk2 are thought to have largely overlapping functions, we find that Jnk1 loss is nearly as effective as the double loss of Jnk1 and Jnk2. Jnk pathway inhibitors are in development for neurodegeneration, cancer, and fibrotic diseases. Our work suggests that the JNK pathway should be explored as a therapeutic target for ADPKD.


Assuntos
Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/genética , Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética , Proliferação de Células/genética , Células Epiteliais/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Rim/metabolismo , Rim/patologia , Sistema de Sinalização das MAP Quinases/genética , Mutação/genética , Rim Policístico Autossômico Dominante/patologia , Transdução de Sinais/genética
3.
medRxiv ; 2023 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-38106222

RESUMO

Polycystic kidney disease (PKD) is an important cause of end stage renal disease, but treatment options are limited. While later stages of the disease have been extensively studied, mechanisms driving the initial conversion of renal tubules into cysts are not understood. To identify factors that promote the initiation of cysts we deleted polycystin-2 ( Pkd2 ) in mice and surveyed transcriptional changes before and immediately after cysts developed. We identified 74 genes which we term cyst initiation candidates (CICs). To identify conserved changes with relevance to human disease we compared these murine CICs to single cell transcriptomic data derived from patients with PKD and from healthy controls. Tumor-associated calcium signal transducer 2 ( Tacstd2 ) stood out as an epithelial-expressed gene whose levels were elevated prior to cystic transformation and further increased with disease progression. Human tissue biopsies and organoids show that TACSTD2 protein is low in normal kidney cells but is elevated in cyst lining cells. While TACSTD2 has not been studied in PKD, it has been studied in cancer where it is highly expressed in solid tumors while showing minimal expression in normal tissue. This property is being exploited by antibody drug conjugates that target TACSTD2 for the delivery of cytotoxic drugs. Our finding that Tacstd2 is highly expressed in cysts, but not normal tissue, suggests that it should be explored as a candidate for drug development in PKD. More immediately, our work suggests that PKD patients undergoing TACSTD2 treatment for cancer should be monitored for kidney effects. One Sentence Summary: The oncogene, tumor-associated calcium signal transducer 2 (Tacstd2) mRNA increased in abundance shortly after Pkd2 loss and may be a driver of cyst initiation in polycystic kidney disease.

4.
medRxiv ; 2023 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-36865301

RESUMO

Motile and non-motile cilia are critical to mammalian development and health. Assembly of these organelles depends on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). A series of human and mouse IFT74 variants were studied to understand the function of this IFT subunit. Humans missing exon 2, which codes for the first 40 residues, presented an unusual combination of ciliary chondrodysplasia and mucociliary clearance disorders while individuals carrying biallelic splice site variants developed a lethal skeletal chondrodysplasia. In mice, variants thought to remove all Ift74 function, completely block ciliary assembly and result in midgestational lethality. A mouse allele that removes the first 40 amino acids, analogous to the human exon 2 deletion, results in a motile cilia phenotype with mild skeletal abnormalities. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia could account for the motile cilia phenotype observed in human and mice.

5.
J Cell Signal ; 3(1): 62-78, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35253003

RESUMO

Polycystic kidney disease is an inherited degenerative disease in which the uriniferous tubules are replaced by expanding fluid-filled cysts that ultimately destroy organ function. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form, afflicting approximately 1 in 1,000 people and is caused by mutations in the transmembrane proteins polycystin-1 (Pkd1) and polycystin-2 (Pkd2). The mechanisms by which polycystin mutations induce cyst formation are not well understood, however pro-proliferative signaling must be involved for tubule epithelial cell number to increase over time. We recently found that the stress-activated mitogen-activated protein kinase (MAPK) pathway c-Jun N-terminal kinase (JNK) pathway is activated in cystic disease and genetically removing JNK reduces cyst growth driven by a loss of Pkd2. This review covers the current state of knowledge of signaling in ADPKD with an emphasis on the JNK pathway.

6.
Cell Signal ; 69: 109519, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31881326

RESUMO

Epithelial cells lining the ducts and tubules of the kidney nephron and collecting duct have a single non-motile cilium projecting from their surface into the lumen of the tubule. These organelles were long considered vestigial remnants left as a result of evolution from a ciliated ancestor, but we now recognize them as critical sensory antennae. In the kidney, the polycystins and fibrocystin, products of the major human polycystic kidney disease genes, localize to this organelle. The polycystins and fibrocystin, through an unknown mechanism, monitor the diameter of the kidney tubules and regulate the proliferation and differentiation of the cells lining the tubule. When the polycystins, fibrocystin or cilia themselves are defective, the cell perceives this as a pro-proliferative signal, which leads to tubule dilation and cystic disease. In addition to critical roles in preventing cyst formation in the kidney, cilia are also important in cystic and fibrotic diseases of the liver and pancreas, and ciliary defects lead to a variety of developmental abnormalities that cause structural birth defects in most organs.


Assuntos
Cílios , Fibrose Cística , Doenças Renais Císticas , Cirrose Hepática , Canais de Cátion TRPP , Animais , Cílios/genética , Cílios/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , Humanos , Doenças Renais Císticas/genética , Doenças Renais Císticas/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo
7.
Cell Rep ; 24(10): 2553-2560.e5, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30184490

RESUMO

Huntington's disease (HD) is a monogenic neurodegenerative disorder representing an ideal candidate for gene silencing with oligonucleotide therapeutics (i.e., antisense oligonucleotides [ASOs] and small interfering RNAs [siRNAs]). Using an ultra-sensitive branched fluorescence in situ hybridization (FISH) method, we show that ∼50% of wild-type HTT mRNA localizes to the nucleus and that its nuclear localization is observed only in neuronal cells. In mouse brain sections, we detect Htt mRNA predominantly in neurons, with a wide range of Htt foci observed per cell. We further show that siRNAs and ASOs efficiently eliminate cytoplasmic HTT mRNA and HTT protein, but only ASOs induce a partial but significant reduction of nuclear HTT mRNA. We speculate that, like other mRNAs, HTT mRNA subcellular localization might play a role in important neuronal regulatory mechanisms.


Assuntos
Doença de Huntington/metabolismo , Neurônios/citologia , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Feminino , Inativação Gênica , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Camundongos , Oligonucleotídeos Antissenso/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Expansão das Repetições de Trinucleotídeos/genética
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