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01 april 2023, Item does not contain fulltext, NPHP1 (Nephrocystin 1) is a protein that localizes to the transition zone of the cilium, a small organelle that projects from the plasma membrane of most cells and allows for integration and coordination of signalling pathways during development and homeostasis. Loss of NPHP1 function due to biallelic NPHP1 gene mutations can lead to the development of ciliopathies - a heterogeneous spectra of disorders characterized by ciliary dysfunction. Here we report the generation of an NPHP1-null hiPSC line (UCSFi001-A-68) via CRISPR/Cas9-mediated non-homologous end joining in the UCSFi001-A background, for study of the role that this protein plays in different tissues.

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Contains fulltext : 289452.pdf (Publisher’s version ) (Open Access)

Radboud University, 05 juni 2023, Promotores : Collin, R.W.J., Roepman, R., Item does not contain fulltext

Contains fulltext : 293272.pdf (Publisher’s version ) (Open Access), Leber congenital amaurosis (LCA) is a group of inherited retinal diseases characterized by early-onset, rapid loss of photoreceptor cells. Despite the discovery of a growing number of genes associated with this disease, the molecular mechanisms of photoreceptor cell degeneration of most LCA subtypes remain poorly understood. Here, using retina-specific affinity proteomics combined with ultrastructure expansion microscopy, we reveal the structural and molecular defects underlying LCA type 5 (LCA5) with nanoscale resolution. We show that LCA5-encoded lebercilin, together with retinitis pigmentosa 1 protein (RP1) and the intraflagellar transport (IFT) proteins IFT81 and IFT88, localized at the bulge region of the photoreceptor outer segment (OS), a region crucial for OS membrane disc formation. Next, we demonstrate that mutant mice deficient in lebercilin exhibited early axonemal defects at the bulge region and the distal OS, accompanied by reduced levels of RP1 and IFT proteins, affecting membrane disc formation and presumably leading to photoreceptor death. Finally, adeno-associated virus-based LCA5 gene augmentation partially restored the bulge region, preserved OS axoneme structure and membrane disc formation, and resulted in photoreceptor cell survival. Our approach thus provides a next level of assessment of retinal (gene) therapy efficacy at the molecular level.

Contains fulltext : 293760.pdf (Publisher’s version ) (Open Access), Mutations in the lebercilin-encoding gene LCA5 cause one of the most severe forms of Leber congenital amaurosis, an early-onset retinal disease that results in severe visual impairment. Here, we report on the generation of a patient-specific cellular model to study LCA5-associated retinal disease. CRISPR-Cas9 technology was used to correct a homozygous nonsense variant in LCA5 (c.835C>T; p.Q279∗) in patient-derived induced pluripotent stem cells (iPSCs). The absence of off-target editing in gene-corrected (isogenic) control iPSCs was demonstrated by whole-genome sequencing. We differentiated the patient, gene-corrected, and unrelated control iPSCs into three-dimensional retina-like cells, so-called retinal organoids. We observed opsin and rhodopsin mislocalization to the outer nuclear layer in patient-derived but not in the gene-corrected or unrelated control organoids. We also confirmed the rescue of lebercilin expression and localization along the ciliary axoneme within the gene-corrected organoids. Here, we show the potential of combining precise single-nucleotide gene editing with the iPSC-derived retinal organoid system for the generation of a cellular model of early-onset retinal disease.

Item does not contain fulltext, Depriving cells of nutrients triggers an energetic crisis, which is resolved by metabolic rewiring and organelle reorganization. Primary cilia are microtubule-based organelles at the cell surface, capable of integrating multiple metabolic and signalling cues, but their precise sensory function is not fully understood. Here we show that primary cilia respond to nutrient availability and adjust their length via glutamine-mediated anaplerosis facilitated by asparagine synthetase (ASNS). Nutrient deprivation causes cilia elongation, mediated by reduced mitochondrial function, ATP availability and AMPK activation independently of mTORC1. Of note, glutamine removal and replenishment is necessary and sufficient to induce ciliary elongation or retraction, respectively, under nutrient stress conditions both in vivo and in vitro by restoring mitochondrial anaplerosis via ASNS-dependent glutamate generation. Ift88-mutant cells lacking cilia show reduced glutamine-dependent mitochondrial anaplerosis during metabolic stress, due to reduced expression and activity of ASNS at the base of cilia. Our data indicate a role for cilia in responding to, and possibly sensing, cellular glutamine levels via ASNS during metabolic stress.

01 juli 2023, Contains fulltext : 294778.pdf (Publisher’s version ) (Open Access), PURPOSE: Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) regulates cell growth in response to nutritional status. Central to the mTORC1 function is the Rag-GTPase heterodimer. One component of the Rag heterodimer is RagC (Ras-related GTP-binding protein C), which is encoded by the RRAGC gene. METHODS: Genetic testing via trio exome sequencing was applied to identify the underlying disease cause in 3 infants with dilated cardiomyopathy, hepatopathy, and brain abnormalities, including pachygyria, polymicrogyria, and septo-optic dysplasia. Studies in patient-derived skin fibroblasts and in a HEK293 cell model were performed to investigate the cellular consequences. RESULTS: We identified 3 de novo missense variants in RRAGC (NM_022157.4: c.269C>A, p.(Thr90Asn), c.353C>T, p.(Pro118Leu), and c.343T>C, p.(Trp115Arg)), which were previously reported as occurring somatically in follicular lymphoma. Studies of patient-derived fibroblasts carrying the p.(Thr90Asn) variant revealed increased cell size, as well as dysregulation of mTOR-related p70S6K (ribosomal protein S6 kinase 1) and transcription factor EB signaling. Moreover, subcellular localization of mTOR was decoupled from metabolic state. We confirmed the key findings for all RRAGC variants described in this study in a HEK293 cell model. CONCLUSION: The above results are in line with a constitutive overactivation of the mTORC1 pathway. Our study establishes de novo missense variants in RRAGC as cause of an early-onset mTORopathy with unfavorable prognosis.

Contains fulltext : 299175.pdf (Publisher’s version ) (Closed access), Radboud University, 18 december 2023, Promotores : Roepman, R., Collin, R.W.J. Co-promotor : Garanto Iglesias, A., 271 p.

Radboud University, 16 juni 2022, Promotores : Roepman, R., Nadif Kasri, N., Contains fulltext : 250092.pdf (Publisher’s version ) (Open Access)

Radboud University, 06 september 2022, Promotores : Drenth, J.P.H., Roepman, R., Contains fulltext : 252881.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 282683.pdf (Publisher’s version ) (Open Access), Retinitis pigmentosa (RP) is a genetically heterogeneous form of inherited retinal disease that leads to progressive visual impairment. One genetic subtype of RP, RP54, has been linked to mutations in PCARE (photoreceptor cilium actin regulator). We have recently shown that PCARE recruits WASF3 to the tip of a primary cilium, and thereby activates an Arp2/3 complex which results in the remodeling of actin filaments that drives the expansion of the ciliary tip membrane. On the basis of these findings, and the lack of proper photoreceptor development in mice lacking Pcare, we postulated that PCARE plays an important role in photoreceptor outer segment disk formation. In this study, we aimed to decipher the relationship between predicted structural and function amino acid motifs within PCARE and its function. Our results show that PCARE contains a predicted helical coiled coil domain together with evolutionary conserved binding sites for photoreceptor kinase MAK (type RP62), as well as EVH1 domain-binding linear motifs. Upon deletion of the helical domain, PCARE failed to localize to the cilia. Furthermore, upon deletion of the EVH1 domain-binding motifs separately or together, co-expression of mutant protein with WASF3 resulted in smaller ciliary tip membrane expansions. Finally, inactivation of the lipid modification on the cysteine residue at amino acid position 3 also caused a moderate decrease in the sizes of ciliary tip expansions. Taken together, our data illustrate the importance of amino acid motifs and domains within PCARE in fulfilling its physiological function.

Contains fulltext : 251964.pdf (Publisher’s version ) (Open Access), Being able to see the beauty of this world is a wonderful thing unfortunately unavailable to people with inherited blindness. In this issue of PLOS Biology, Mercey and colleagues present optimized expansion microscopy for retinal tissue, which represents a huge step forward in our ability to study these blinding conditions.

Radboud University, 08 december 2022, Promotor : Roepman, R. Co-promotor : Aslanyan, M.G., Contains fulltext : 284465.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 245085.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 243899.pdf (Publisher’s version ) (Open Access), Inherited retinal diseases (IRDs) cause progressive loss of light-sensitive photoreceptors in the eye and can lead to blindness. Gene-based therapies for IRDs have shown remarkable progress in the past decade, but the vast majority of forms remain untreatable. In the era of personalised medicine, induced pluripotent stem cells (iPSCs) emerge as a valuable system for cell replacement and to model IRD because they retain the specific patient genome and can differentiate into any adult cell type. Three-dimensional (3D) iPSCs-derived retina-like tissue called retinal organoid contains all major retina-specific cell types: amacrine, bipolar, horizontal, retinal ganglion cells, Müller glia, as well as rod and cone photoreceptors. Here, we describe the main applications of retinal organoids and provide a comprehensive overview of the state-of-art analysis methods that apply to this model system. Finally, we will discuss the outlook for improvements that would bring the cellular model a step closer to become an established system in research and treatment development of IRDs.

Contains fulltext : 241056.pdf (Publisher’s version ) (Open Access)

Radboud University, 26 maart 2021, Promotor : Roepman, R., Contains fulltext : 230657.pdf (publisher's version ) (Open Access)

Contains fulltext : 246467.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 241056.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 229376.pdf (publisher's version ) (Open Access), Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45(-/-) mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

Contains fulltext : 226792.pdf (publisher's version ) (Open Access)

Contains fulltext : 225431.pdf (Publisher’s version ) (Closed access), Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy characterized by a pathognomonic hindbrain malformation. All known JBTS genes encode proteins involved in the structure or function of primary cilia, ubiquitous antenna-like organelles essential for cellular signal transduction. Here, we used the recently identified JBTS-associated protein armadillo repeat motif-containing 9 (ARMC9) in tandem-affinity purification and yeast 2-hybrid screens to identify a ciliary module whose dysfunction underlies JBTS. In addition to the known JBTS-associated proteins CEP104 and CSPP1, we identified coiled-coil domain containing 66 (CCDC66) and TOG array regulator of axonemal microtubules 1 (TOGARAM1) as ARMC9 interaction partners. We found that TOGARAM1 variants cause JBTS and disrupt TOGARAM1 interaction with ARMC9. Using a combination of protein interaction analyses, characterization of patient-derived fibroblasts, and analysis of CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrated that dysfunction of ARMC9 or TOGARAM1 resulted in short cilia with decreased axonemal acetylation and polyglutamylation, but relatively intact transition zone function. Aberrant serum-induced ciliary resorption and cold-induced depolymerization in ARMC9 and TOGARAM1 patient cell lines suggest a role for this new JBTS-associated protein module in ciliary stability.

Contains fulltext : 214147.pdf (publisher's version ) (Open Access)

Contains fulltext : 218985.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 206876.pdf (publisher's version ) (Open Access)

Contains fulltext : 208460.pdf (publisher's version ) (Open Access), The light sensing outer segments of photoreceptors (PRs) are renewed every ten days due to their high photoactivity, especially of the cones during daytime vision. This demands a tremendous amount of energy, as well as a high turnover of their main biosynthetic compounds, membranes, and proteins. Therefore, a refined proteostasis network (PN), regulating the protein balance, is crucial for PR viability. In many inherited retinal diseases (IRDs) this balance is disrupted leading to protein accumulation in the inner segment and eventually the death of PRs. Various studies have been focusing on therapeutically targeting the different branches of the PR PN to restore the protein balance and ultimately to treat inherited blindness. This review first describes the different branches of the PN in detail. Subsequently, insights are provided on how therapeutic compounds directed against the different PN branches might slow down or even arrest the appalling, progressive blinding conditions. These insights are supported by findings of PN modulators in other research disciplines.

Contains fulltext : 204035.pdf (publisher's version ) (Open Access)

Contains fulltext : 204265.pdf (publisher's version ) (Open Access), 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/.

Contains fulltext : 196363.pdf (Publisher’s version ) (Open Access), BACKGROUND: Nephronophthisis is an autosomal recessive ciliopathy and important cause of end-stage renal disease (ESRD) in children and young adults. Diagnostic delay is frequent. This study investigates clinical characteristics, initial symptoms, and genetic defects in a cohort with nephronophthisis-related ciliopathy, to improve early detection and genetic counseling. METHODS: Forty patients from 36 families with nephronophthisis-related ciliopathy were recruited at university medical centers and online. Comprehensive clinical and genotypic data were recorded. Patients without molecular diagnosis were offered genetic analysis. RESULTS: Of 40 patients, 45% had isolated nephronophthisis, 48% syndromic diagnosis, and 7% nephronophthisis with extrarenal features not constituting a recognizable syndrome. Patients developed ESRD at median 13 years (range 5-47). Median age of symptom onset was 9 years in both isolated and syndromic forms (range 5-26 vs. 5-33). Common presenting symptoms were fatigue (42%), polydipsia/polyuria (33%), and hypertension (21%). Renal ultrasound showed small-to-normal-sized kidneys, increased echogenicity (65%), cysts (43%), and abnormal corticomedullary differentiation (32%). Renal biopsies in eight patients showed nonspecific signs of chronic kidney disease (CKD). Twenty-three patients (58%) had genetic diagnosis upon inclusion. Thirteen of those without a genetic diagnosis gave consent for genetic testing, and a cause was identified in five (38%). CONCLUSIONS: Nephronophthisis is genetically and phenotypically heterogeneous and should be considered in children and young adults presenting with persistent fatigue and polyuria, and in all patients with unexplained CKD. As symptom onset can occur into adulthood, presymptomatic monitoring of kidney function in syndromic ciliopathy patients should continue until at least age 30.

Radboud University, 10 september 2018, Promotores : Hoenderop, J.G.J., Roepman, R. Co-promotor : Arjona, F.J., Contains fulltext : 194281.pdf (publisher's version ) (Open Access)

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Item does not contain fulltext, Most genetically distinct inherited retinal degenerations are primary photoreceptor degenerations. We selected a severe early onset form of Leber congenital amaurosis (LCA), caused by mutations in the gene LCA5, in order to test the efficacy of gene augmentation therapy for a ciliopathy. The LCA5-encoded protein, Lebercilin, is essential for the trafficking of proteins and vesicles to the photoreceptor outer segment. Using the AAV serotype AAV7m8 to deliver a human LCA5 cDNA into an Lca5 null mouse model of LCA5, we show partial rescue of retinal structure and visual function. Specifically, we observed restoration of rod-and-cone-driven electroretinograms in about 25% of injected eyes, restoration of pupillary light responses in the majority of treated eyes, an approximately 20-fold decrease in target luminance necessary for visually guided behavior, and improved retinal architecture following gene transfer. Using LCA5 patient-derived iPSC-RPEs, we show that delivery of the LCA5 cDNA restores lebercilin protein and rescues cilia quantity. The results presented in this study support a path forward aiming to develop safety and efficacy trials for gene augmentation therapy in human subjects with LCA5 mutations. They also provide the framework for measuring the effects of intervention in ciliopathies and other severe, early-onset blinding conditions.

Contains fulltext : 193089.pdf (publisher's version ) (Open Access), Synapse development and neuronal activity represent fundamental processes for the establishment of cognitive function. Structural organization as well as signalling pathways from receptor stimulation to gene expression regulation are mediated by synaptic activity and misregulated in neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability (ID). Deleterious mutations in the PTCHD1 (Patched domain containing 1) gene have been described in male patients with X-linked ID and/or ASD. The structure of PTCHD1 protein is similar to the Patched (PTCH1) receptor; however, the cellular mechanisms and pathways associated with PTCHD1 in the developing brain are poorly determined. Here we show that PTCHD1 displays a C-terminal PDZ-binding motif that binds to the postsynaptic proteins PSD95 and SAP102. We also report that PTCHD1 is unable to rescue the canonical sonic hedgehog (SHH) pathway in cells depleted of PTCH1, suggesting that both proteins are involved in distinct cellular signalling pathways. We find that Ptchd1 deficiency in male mice (Ptchd1(-/y)) induces global changes in synaptic gene expression, affects the expression of the immediate-early expression genes Egr1 and Npas4 and finally impairs excitatory synaptic structure and neuronal excitatory activity in the hippocampus, leading to cognitive dysfunction, motor disabilities and hyperactivity. Thus our results support that PTCHD1 deficiency induces a neurodevelopmental disorder causing excitatory synaptic dysfunction.

Contains fulltext : 196394.pdf (publisher's version ) (Open Access)

Contains fulltext : 199977.pdf (publisher's version ) (Open Access), Background: Mainzer-Saldino syndrome (MZSDS) is a skeletal ciliopathy and part of the short-rib thoracic dysplasia (SRTD) group of ciliary disorders. The main characteristics of MZSDS are short limbs, mild narrow thorax, blindness, and renal failure. Thus far, variants in two genes are associated with MZSDS: IFT140, and IFT172. In this study, we describe a 1-year-old girl presenting with mild skeletal abnormalities, Leber congenital amaurosis, and bilateral hearing difficulties. For establishing an accurate diagnosis, we combined clinical, molecular, and functional analyses. Methods: We performed diagnostic whole-exome sequencing (WES) analysis to determine the genetic cause of the disease and analyzed two gene panels, containing all currently known genes in vision disorders, and in hearing impairment. Upon detection of the likely causative variants, ciliary phenotyping was performed in patient urine-derived renal epithelial cells (URECs) and rescue experiments were performed in CRISPR/Cas9-derived Ift140 knock out cells to determine the pathogenicity of the detected variants in vitro. Cilium morphology, cilium length, and intraflagellar transport (IFT) were evaluated by immunocytochemistry. Results: Diagnostic WES revealed two novel compound heterozygous variants in IFT140, encoding IFT140. Thorough investigation of WES data did not reveal any variants in candidate genes associated with hearing impairment. Patient-derived URECs revealed an accumulation of IFT-B protein IFT88 at the ciliary tip in 41% of the cells indicative of impaired retrograde IFT, while this was absent in cilia from control URECs. Furthermore, transfection of CRISPR/Cas9-derived Ift140 knock out cells with an IFT140 construct containing the patient mutation p.Tyr923Asp resulted in a significantly higher percentage of IFT88 tip accumulation than transfection with the wild-type IFT140 construct. Conclusions: By combining the clinical, genetic, and functional data from this study, we could conclud

Contains fulltext : 196646.pdf (Publisher’s version ) (Open Access), Retinitis pigmentosa (RP) encompasses a group of inherited retinal dystrophies characterized by the primary degeneration of rod and cone photoreceptors. RP is a leading cause of visual disability, with a worldwide prevalence of 1:4000. Although the majority of RP cases are non-syndromic, 20-30% of patients with RP also have an associated non-ocular condition. RP typically manifests with night blindness in adolescence, followed by concentric visual field loss, reflecting the principal dysfunction of rod photoreceptors; central vision loss occurs later in life due to cone dysfunction. Photoreceptor function measured with an electroretinogram is markedly reduced or even absent. Optical coherence tomography (OCT) and fundus autofluorescence (FAF) imaging show a progressive loss of outer retinal layers and altered lipofuscin distribution in a characteristic pattern. Over the past three decades, a vast number of disease-causing variants in more than 80 genes have been associated with non-syndromic RP. The wide heterogeneity of RP makes it challenging to describe the clinical findings and pathogenesis. In this review, we provide a comprehensive overview of the clinical characteristics of RP specific to genetically defined patient subsets. We supply a unique atlas with color fundus photographs of most RP subtypes, and we discuss the relevant considerations with respect to differential diagnoses. In addition, we discuss the genes involved in the pathogenesis of RP, as well as the retinal processes that are affected by pathogenic mutations in these genes. Finally, we review management strategies for patients with RP, including counseling, visual rehabilitation, and current and emerging therapeutic options.

Radboud University, 20 april 2018, Promotores : Drenth, J.P.H., Roepman, R., Contains fulltext : 187337.pdf (publisher's version ) (Open Access)

Retinitis pigmentosa (RP) encompasses a group of inherited retinal dystrophies characterized by the primary degeneration of rod and cone photoreceptors. RP is a leading cause of visual disability, with a worldwide prevalence of 1:4000. Although the majority of RP cases are non-syndromic, 20–30% of patients with RP also have an associated non-ocular condition. RP typically manifests with night blindness in adolescence, followed by concentric visual field loss, reflecting the principal dysfunction of rod photoreceptors; central vision loss occurs later in life due to cone dysfunction. Photoreceptor function measured with an electroretinogram is markedly reduced or even absent. Optical coherence tomography (OCT) and fundus autofluorescence (FAF) imaging show a progressive loss of outer retinal layers and altered lipofuscin distribution in a characteristic pattern. Over the past three decades, a vast number of disease-causing variants in more than 80 genes have been associated with non-syndromic RP. The wide heterogeneity of RP makes it challenging to describe the clinical findings and pathogenesis. In this review, we provide a comprehensive overview of the clinical characteristics of RP specific to genetically defined patient subsets. We supply a unique atlas with color fundus photographs of most RP subtypes, and we discuss the relevant considerations with respect to differential diagnoses. In addition, we discuss the genes involved in the pathogenesis of RP, as well as the retinal processes that are affected by pathogenic mutations in these genes. Finally, we review management strategies for patients with RP, including counseling, visual rehabilitation, and current and emerging therapeutic options.

Radboud University, 03 december 2018, Promotores : Collin, R.W.J., Roepman, R. Co-promotor : Garanto Iglesias, A., Contains fulltext : 197551.pdf (publisher's version ) (Open Access)

Contains fulltext : 197525.pdf (Publisher’s version ) (Open Access)

Radboud University, 25 januari 2018, Promotores : Roepman, R., Cremers, F.P.M. Co-promotor : Arts, H.H., Contains fulltext : 181326.pdf (publisher's version ) (Open Access)

Contains fulltext : 191425.pdf (publisher's version ) (Open Access)