scholarly journals Loss of CEP162 function at the primary cilium delays ciliogenesis and causes retinal ciliopathy in humans

2021 ◽  
Author(s):  
Nafisa Nuzhat ◽  
Kristof Van Schil ◽  
Sandra Liakopoulos ◽  
Miriam Bauwens ◽  
Alfredo Dueñas Rey ◽  
...  
Keyword(s):  
Transition Zone ◽  
Primary Cilium ◽  
Outer Segment ◽  
Retinal Dystrophy ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Photoreceptor Outer Segment ◽  
Zone Formation ◽  
Mother Centriole

Ciliopathies often comprise retinal degeneration since the photoreceptor outer segment is an adapted primary cilium. CEP162 is a distal end centriolar protein required for proper transition zone assembly during ciliogenesis and whose loss causes ciliopathy in zebrafish. CEP162 has so far not been implicated in human disease. Here, we identified a homozygous CEP162 frameshift variant, c.1935dupA (p.(E646R*5)), in retinitis pigmentosa patients from two unrelated Moroccan families, likely representing a founder allele. We found that even though mRNA levels were reduced, the truncated CEP162-E646R*5 protein was expressed and localized to the mitotic spindle during mitosis, but not at the basal body of the cilium. In CEP162 knockdown cells, expression of the truncated CEP162-E646R*5 protein is unable to restore ciliation indicating its loss of function at the cilium. In patient fibroblasts, cilia overcome the absence of CEP162 from the primary cilium by delaying ciliogenesis through the persistence of CP110 at the mother centriole. The patient fibroblasts are ultimately able to extend some abnormally long cilia that are missing key transition zone components. Defective transition zone formation likely disproportionately affects the long-living ciliary outer segment of photoreceptors resulting in retinal dystrophy. CEP162 is expressed in human retina, and we show that wild-type CEP162, but not truncated CEP162-E646R*5, specifically localizes to the distal end of centrioles of mouse photoreceptor cilia. Together, our genetic, cell-based, and in vivo modeling establish that CEP162 deficiency causes retinal ciliopathy in humans.

scholarly journals TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chengwu Xiao ◽  
Wei Zhang ◽  
Meimian Hua ◽  
Huan Chen ◽  
Bin Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Prognostic Indicator ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Protein Levels ◽  
Kaplan Meier ◽  
Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

scholarly journals SLC38A8 mutations result in arrested retinal development with loss of cone photoreceptor specialization

2020 ◽  
Vol 29 (18) ◽  
pp. 2989-3002 ◽  
Author(s):  
Helen J Kuht ◽  
Jinu Han ◽  
Gail D E Maconachie ◽  
Sung Eun Park ◽  
Seung-Tae Lee ◽  
...  
Keyword(s):  
Outer Segment ◽  
Transmembrane Domain ◽  
Retinal Development ◽  
Anterior Segment ◽  
Cone Photoreceptor ◽  
Photoreceptor Outer Segment ◽  
Multi Centre Study ◽  
Photoreceptor Layer ◽  
Foveal Hypoplasia

Abstract Foveal hypoplasia, optic nerve decussation defects and anterior segment dysgenesis is an autosomal recessive disorder arising from SLC38A8 mutations. SLC38A8 is a putative glutamine transporter with strong expression within the photoreceptor layer in the retina. Previous studies have been limited due to lack of quantitative data on retinal development and nystagmus characteristics. In this multi-centre study, a custom-targeted next generation sequencing (NGS) gene panel was used to identify SLC38A8 mutations from a cohort of 511 nystagmus patients. We report 16 novel SLC38A8 mutations. The sixth transmembrane domain is most frequently disrupted by missense SLC38A8 mutations. Ninety percent of our cases were initially misdiagnosed as PAX6-related phenotype or ocular albinism prior to NGS. We characterized the retinal development in vivo in patients with SLC38A8 mutations using high-resolution optical coherence tomography. All patients had severe grades of arrested retinal development with lack of a foveal pit and no cone photoreceptor outer segment lengthening. Loss of foveal specialization features such as outer segment lengthening implies reduced foveal cone density, which contributes to reduced visual acuity. Unlike other disorders (such as albinism or PAX6 mutations) which exhibit a spectrum of foveal hypoplasia, SLC38A8 mutations have arrest of retinal development at an earlier stage resulting in a more under-developed retina and severe phenotype.

scholarly journals The presynaptic glycine transporter GlyT2 is regulated by the Hedgehog pathway in vitro and in vivo

2020 ◽  
Author(s):  
A. de la Rocha-Muñoz ◽  
E. Núñez ◽  
S. Gómez-López ◽  
B. López-Corcuera ◽  
J. de Juan-Sanz ◽  
...  
Keyword(s):  
Hedgehog Signaling ◽  
Hedgehog Pathway ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Transport Activity ◽  
Spinal Cord Neurons ◽  
Glycine Transporter ◽  
Glycinergic Neurotransmission

ABSTRACTThe identity of a glycinergic synapse is maintained presynaptically by the activity of a surface glycine transporter, GlyT2, which recaptures glycine back to presynaptic terminals to preserve vesicular glycine content. GlyT2 loss-of-function mutations cause Hyperekplexia, a rare neurological disease in which loss of glycinergic neurotransmission causes generalized stiffness and strong motor alterations. However, the molecular underpinnings controlling GlyT2 activity remain poorly understood. In this work, we identify the Hedgehog pathway as a robust controller of GlyT2 expression and transport activity. Modulating the activation state of the Hedgehog pathway in vitro in rodent primary spinal cord neurons or in vivo in zebrafish embryos induced a selective control in GlyT2 expression, regulating GlyT2 transport activity. Our results indicate that activation of Hedgehog reduces GlyT2 expression by decreasing its mRNA levels and increasing its ubiquitination and degradation. This work describes a new molecular link between the Hedgehog signaling pathway and presynaptic glycine availability.

Vegf regulates embryonic erythroid development through Gata1 modulation

Blood ◽  
2010 ◽  
Vol 116 (12) ◽  
pp. 2141-2151 ◽  
Author(s):  
Benjamin Drogat ◽  
Joanna Kalucka ◽  
Laura Gutiérrez ◽  
Hamida Hammad ◽  
Steven Goossens ◽  
...  
Keyword(s):  
Target Genes ◽  
Erythroid Differentiation ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Erythroid Progenitor ◽  
Expression Levels ◽  
Conditional Deletion ◽  
Differentiation Block ◽  
Erythroid Development

Abstract To determine the role of vascular endothelial growth factor (Vegf) in embryonic erythroid development we have deleted or overexpressed Vegf specifically in the erythroid lineage using the EpoR-iCre transgenic line in combination with Cre/loxP conditional gain and loss of function Vegf alleles. ROSA26 promoter-based expression of the Vegf164 isoform in the early erythroid lineage resulted in a differentiation block of primitive erythroid progenitor (EryP) development and a partial block in definitive erythropoiesis between the erythroid burst-forming unit and erythroid colony-forming unit stages. Decreased mRNA expression levels of the key erythroid transcription factor Gata1 were causally linked to this phenotype. Conditional deletion of Vegf within the erythroid lineage was associated with increased Gata1 levels and increased erythroid differentiation. Expression of a ROSA26-based GATA2 transgene rescued Gata1 mRNA levels and target genes and restored erythroid differentiation in our Vegf gain of function model. These results demonstrate that Vegf modulates Gata1 expression levels in vivo and provides new molecular insight into Vegf's ability to modulate erythropoiesis.

scholarly journals mRNAs Encoding Telomerase Components and Regulators Are Controlled by UPF Genes in Saccharomyces cerevisiae

2003 ◽  
Vol 2 (1) ◽  
pp. 134-142 ◽  
Author(s):  
Jeffrey N. Dahlseid ◽  
Jodi Lew-Smith ◽  
Michael J. Lelivelt ◽  
Shinichiro Enomoto ◽  
Amanda Ford ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomere Length ◽  
Dna Sequences ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Telomeric Silencing ◽  
Expression Of Genes ◽  
Mutant Strains ◽  
Genes Encoding

ABSTRACT Telomeres, the chromosome ends, are maintained by a balance of activities that erode and replace the terminal DNA sequences. Furthermore, telomere-proximal genes are often silenced in an epigenetic manner. In Saccharomyces cerevisiae, average telomere length and telomeric silencing are reduced by loss of function of UPF genes required in the nonsense-mediated mRNA decay (NMD) pathway. Because NMD controls the mRNA levels of several hundred wild-type genes, we tested the hypothesis that NMD affects the expression of genes important for telomere functions. In upf mutants, high-density oligonucleotide microarrays and Northern blots revealed that the levels of mRNAs were increased for genes encoding the telomerase catalytic subunit (Est2p), in vivo regulators of telomerase (Est1p, Est3p, Stn1p, and Ten1p), and proteins that affect telomeric chromatin structure (Sas2p and Orc5p). We investigated whether overexpressing these genes could mimic the telomere length and telomeric silencing phenotypes seen previously in upf mutant strains. Increased dosage of STN1, especially in combination with increased dosage of TEN1, resulted in reduced telomere length that was indistinguishable from that in upf mutants. Increased levels of STN1 together with EST2 resulted in reduced telomeric silencing like that of upf mutants. The half-life of STN1 mRNA was not altered in upf mutant strains, suggesting that an NMD-controlled transcription factor regulates the levels of STN1 mRNA. Together, these results suggest that NMD maintains the balance of gene products that control telomere length and telomeric silencing primarily by maintaining appropriate levels of STN1, TEN1, and EST2 mRNA.

scholarly journals Knockout of myoc Provides Evidence for the Role of Myocilin in Zebrafish Sex Determination Associated with Wnt Signalling Downregulation

Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 98
Author(s):  
Raquel Atienzar-Aroca ◽  
José-Daniel Aroca-Aguilar ◽  
Susana Alexandre-Moreno ◽  
Jesús-José Ferre-Fernández ◽  
Juan-Manuel Bonet-Fernández ◽  
...  
Keyword(s):  
Sex Determination ◽  
Anterior Segment ◽  
Premature Termination Codon ◽  
Wnt Signalling ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Ocular Tissues ◽  
Key Factor

Myocilin is a secreted glycoprotein with a poorly understood biological function and it is mainly known as the first glaucoma gene. To explore the normal role of this protein in vivo we developed a myoc knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. This line carries a homozygous variant (c.236_239delinsAAAGGGGAAGGGGA) that is predicted to result in a loss-of-function of the protein because of a premature termination codon p.(V75EfsX60) that resulted in a significant reduction of myoc mRNA levels. Immunohistochemistry showed the presence of myocilin in wild-type embryonic (96 h post-fertilization) anterior segment eye structures and caudal muscles. The protein was also detected in different adult ocular and non-ocular tissues. No gross macroscopic or microscopic alterations were identified in the KO zebrafish, but, remarkably, we observed absence of females among the adult KO animals and apoptosis in the immature juvenile gonad (28 dpf) of these animals, which is characteristic of male development. Transcriptomic analysis showed that adult KO males overexpressed key genes involved in male sex determination and presented differentially expressed Wnt signalling genes. These results show that myocilin is required for ovary differentiation in zebrafish and provides in vivo support for the role of myocilin as a Wnt signalling pathway modulator. In summary, this myoc KO zebrafish line can be useful to investigate the elusive function of this protein, and it provides evidence for the unexpected function of myocilin as a key factor in zebrafish sex determination.

scholarly journals Genetically-modified X. laevis indicate distinct roles for prominin-1 and photoreceptor cadherin in outer segment morphogenesis and retinal dystrophy

2020 ◽  
Author(s):  
Brittany J Carr ◽  
Paloma Stanar ◽  
Orson L Moritz
Keyword(s):  
Outer Segment ◽  
Retinal Dystrophy ◽  
Macular Dystrophy ◽  
Direct Effects ◽  
Fusion Event ◽  
Photoreceptor Outer Segment ◽  
Structural Support ◽  
Degenerative Disorders ◽  
And Function ◽  
Null Mutants

ABSTRACTMutations in prominin-1 (prom1) and photoreceptor cadherin (cdhr1) are associated with inherited retinal degenerative disorders such as retinitis pigmentosa, cone-rod dystrophy, and juvenile macular dystrophy. The proteins encoded by these genes are hypothesized to regulate photoreceptor outer segment disc morphogenesis, but their functions remain unknown. We used CRISPR/Cas9 to generate prom1-, cdhr1-, and prom1 + cdhr1-null X. laevis and then documented the effects of these mutations on photoreceptor structure and function. Prom1-null mutations resulted in dysmorphic photoreceptors comprised of overgrown and disorganized disc membranes. Cones were more severely affected than rods; outer segments were elongated and fragmented, and ERG response was impaired. Autofluorescent deposits in the outer segment layer of aging prom1-null animals indicate that secondary toxic effects to the retina or RPE drive retinal degeneration for this mutation, instead of direct effects on outer segment disc morphogenesis. Cdhr1-null photoreceptors did not appear grossly dysmorphic, but ultrastructural analysis revealed that some disc membranes were overgrown or aligned vertically within the plasma membrane. Prom1 + cdhr1-null mutants did not differ significantly from prom1-null mutants. Our results indicate that neither prom1 nor cdhr1 are necessary for outer segment disc membrane evagination or the membrane fusion event involved in disc sealing. Rather, they are necessary for higher-order organization of the nascent outer segment discs. Prom1 may align and reinforce interactions between the disc leading edges, a function more critical in cone photoreceptors for structural support. Cdhr1 may help to align nascent discs and maintain horizontal disc orientation prior to fusion.

scholarly journals Photoreceptor discs form through peripherin-dependent suppression of ciliary ectosome release

2017 ◽  
Vol 216 (5) ◽  
pp. 1489-1499 ◽  
Author(s):  
Raquel Y. Salinas ◽  
Jillian N. Pearring ◽  
Jin-Dong Ding ◽  
William J. Spencer ◽  
Ying Hao ◽  
...  
Keyword(s):  
Primary Cilium ◽  
Outer Segment ◽  
Genetic Material ◽  
Photoreceptor Cells ◽  
Specific Protein ◽  
Photoreceptor Outer Segment ◽  
Extracellular Signals ◽  
Membrane Surfaces ◽  
Light Capture ◽  
Innate Ability

The primary cilium is a highly conserved organelle housing specialized molecules responsible for receiving and processing extracellular signals. A recently discovered property shared across many cilia is the ability to release small vesicles called ectosomes, which are used for exchanging protein and genetic material among cells. In this study, we report a novel role for ciliary ectosomes in building the elaborate photoreceptor outer segment filled with hundreds of tightly packed “disc” membranes. We demonstrate that the photoreceptor cilium has an innate ability to release massive amounts of ectosomes. However, this process is suppressed by the disc-specific protein peripherin, which enables retained ectosomes to be morphed into discs. This new function of peripherin is performed independently from its well-established role in maintaining the high curvature of disc edges, and each function is fulfilled by a separate part of peripherin’s molecule. Our findings explain how the outer segment structure evolved from the primary cilium to provide photoreceptor cells with vast membrane surfaces for efficient light capture.

scholarly journals Dominant role of the MyD88-dependent signaling pathway in mediating early endotoxin-induced murine ileus

2010 ◽  
Vol 299 (2) ◽  
pp. G531-G538 ◽  
Author(s):  
Bettina M. Buchholz ◽  
Timothy R. Billiar ◽  
Anthony J. Bauer
Keyword(s):  
Signaling Pathways ◽  
Inflammatory Mediators ◽  
Dominant Role ◽  
Gastrointestinal Transit ◽  
Adaptor Protein ◽  
Primary Response ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Downstream Signaling

TLR4 ligation by pathogen-associated molecular patterns, such as Gram-negative bacteria-derived LPS, triggers a nonhematopoietic cell-mediated ileus during early endotoxemia. Our objective was to investigate the quantitative contributions of the two downstream signaling pathways of TLR4, namely the adapter proteins myeloid differentiation primary response gene 88 (MyD88) and Toll-IL-1-resistance (TIR) domain-containing adaptor-inducing IFN-β (TRIF). Six hours after intraperitoneal injection of highly purified LPS (UP-LPS, 5 mg/kg), in vivo gastrointestinal transit and intestinal muscularis gene transcripts of inflammatory mediators chemokine (C-X-C motif) ligand 10, synonymous IP-10 (CXCL10), granulomonocyte colony stimulating factor (GM-CSF, synonymous CSF-2), IL-1β, IL-6, IL-10, and inducible NO synthase (iNOS) were assessed in mice with transgenic loss-of-function for MyD88 or TRIF. LPS-induced MyD88 and TRIF mRNA upregulation was quantified within the intestinal muscularis of TLR4-competent and TLR4-mutant mice, and MyD88 mRNA levels were additionally measured in TLR4 bone marrow chimeras. MyD88 deficiency completely protected mice from early endotoxin-induced ileus, while TRIF deficiency partially ameliorated ileus severity. LPS induction of the primary downstream signaling element MyD88 was TLR4 dependent and was derived in equal amounts from both the hematopoietic and the nonhematopoietic cells. Conversely, no induction of TRIF mRNA was detectable. Significant gene induction of all inflammatory mediators was dependent on intracellular signal transduction by MyD88, while the TRIF MyD88-independent pathway predominantly regulated the molecular levels of CXCL10. In summary, MyD88 and TRIF are nonredundant signaling pathways in early endotoxin-induced rodent ileus, but MyD88 is the essential adaptor molecule for transduction of early TLR4-induced ileus and inflammatory signaling. The dependency of ileus on individual adaptor protein pathways is also reflected in the manifestation of specific molecular inflammatory events within the intestinal muscularis externa.

scholarly journals Aurora Kinase A proximity interactome reveals centriolar satellites as regulators of its function during primary cilium biogenesis

2020 ◽  
Author(s):  
Melis D. Arslanhan ◽  
Navin Rauniyar ◽  
John R. Yates ◽  
Elif N. Firat-Karalar
Keyword(s):  
Primary Cilium ◽  
Full Range ◽  
Aurora Kinase ◽  
Loss Of Function ◽  
Aurora Kinase A ◽  
Cellular Processes ◽  
Spatiotemporal Regulation ◽  
Pleiotropic Functions ◽  
Kinase A

AbstractAurora kinase A (AURKA) is a conserved kinase that plays crucial roles in numerous cellular processes. Although AURKA overexpression is frequent in human cancers, its pleiotropic functions and complex spatiotemporal regulation have presented challenges in its therapeutic targeting. An essential step to overcome these challenges is the identification of the full range of AURKA regulators and substrates, which are often weak and transient. Previous proteomic studies were limited in monitoring dynamic and non-mitotic AURKA interactions. Here, we generated the first in vivo proximity interactome of AURKA, which consisted of over 100 proteins involving multiple biological processes and cellular compartments. Importantly, AURKA had extensive proximity interactions to centriolar satellites, key regulators of the primary cilium. Affinity pulldown and phosphoproteomics experiments confirmed this proximity relationship at the physical level. Loss-of-function experiments defined satellites as negative regulators of AURKA activity, abundance and localization in quiescent cells. Notably, loss of satellites increased AURKA activation at the basal body and resulted in defective cilium assembly and enhanced cilium disassembly. Collectively, our results provide a powerful resource for dissecting AURKA function and regulation and uncover proteostatic regulation of AURKA by centriolar satellites as a new regulatory mechanism for its non-mitotic functions.

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