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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.

01 september 2023, Contains fulltext : 296660.pdf (Publisher’s version ) (Open Access), Despite comprehensive research efforts over the last decades, the pathomechanisms of age-related macular degeneration (AMD) remain far from being understood. Large-scale genome wide association studies (GWAS) were able to provide a defined set of genetic aberrations which contribute to disease risk, with the strongest contributors mapping to distinct regions on chromosome 1 and 10. While the chromosome 1 locus comprises factors of the complement system with well-known functions, the role of the 10q26-locus in AMD-pathophysiology remains enigmatic. 10q26 harbors a cluster of three functional genes, namely PLEKHA1, ARMS2 and HTRA1, with most of the AMD-associated genetic variants mapping to the latter two genes. High linkage disequilibrium between ARMS2 and HTRA1 has kept association studies from reliably defining the risk-causing gene for long and only very recently the genetic risk region has been narrowed to ARMS2, suggesting that this is the true AMD gene at this locus. However, genetic associations alone do not suffice to prove causality and one or more of the 14 SNPs on this haplotype may be involved in long-range control of gene expression, leaving HTRA1 and PLEKHA1 still suspects in the pathogenic pathway. Both, ARMS2 and HTRA1 have been linked to extracellular matrix homeostasis, yet their exact molecular function as well as their role in AMD pathogenesis remains to be uncovered. The transcriptional regulation of the 10q26 locus adds an additional level of complexity, given, that gene-regulatory as well as epigenetic alterations may influence expression levels from 10q26 in diseased individuals. Here, we provide a comprehensive overview on the 10q26 locus and its three gene products on various levels of biological complexity and discuss current and future research strategies to shed light on one of the remaining enigmatic spots in the AMD landscape.

Inherited retinal diseases (IRDs) are a heterogeneous group of blinding disorders, which result in dysfunction or death of the light-sensing cone and rod photoreceptors. Despite individual IRDs (Inherited retinal disease) being rare, collectively, they affect up to 1:2000 people worldwide, causing a significant socioeconomic burden, especially when cone-mediated central vision is affected. This study uses the Pde6ccpfl1 mouse model of achromatopsia, a cone-specific vision loss IRD (Inherited retinal disease), to investigate the potential gene-independent therapeutic benefits of a histone demethylase inhibitor GSK-J4 on cone cell survival. We investigated the effects of GSK-J4 treatment on cone cell survival in vivo and ex vivo and changes in cone-specific gene expression via single-cell RNA sequencing. A single intravitreal GSK-J4 injection led to transcriptional changes in pathways involved in mitochondrial dysfunction, endoplasmic reticulum stress, among other key epigenetic pathways, highlighting the complex interplay between methylation and acetylation in healthy and diseased cones. Furthermore, continuous administration of GSK-J4 in retinal explants increased cone survival. Our results suggest that IRD (Inherited retinal disease)-affected cones respond positively to epigenetic modulation of histones, indicating the potential of this approach in developing a broad class of novel therapies to slow cone degeneration.

Item does not contain fulltext, PURPOSE: Current prediction models for advanced age-related macular degeneration (AMD) are based on a restrictive set of risk factors. The objective of this study was to develop a comprehensive prediction model applying a machine learning algorithm allowing selection of the most predictive risk factors automatically. DESIGN: Two population-based cohort studies. PARTICIPANTS: The Rotterdam Study I (RS-I; training set) included 3838 participants 55 years of age or older, with a median follow-up period of 10.8 years, and 108 incident cases of advanced AMD. The Antioxydants, Lipids Essentiels, Nutrition et Maladies Oculaires (ALIENOR) study (test set) included 362 participants 73 years of age or older, with a median follow-up period of 6.5 years, and 33 incident cases of advanced AMD. METHODS: The prediction model used the bootstrap least absolute shrinkage and selection operator (LASSO) method for survival analysis to select the best predictors of incident advanced AMD in the training set. Predictive performance of the model was assessed using the area under the receiver operating characteristic curve (AUC). MAIN OUTCOME MEASURES: Incident advanced AMD (atrophic, neovascular, or both), based on standardized interpretation of retinal photographs. RESULTS: The prediction model retained (1) age, (2) a combination of phenotypic predictors (based on the presence of intermediate drusen, hyperpigmentation in one or both eyes, and Age-Related Eye Disease Study simplified score), (3) a summary genetic risk score based on 49 single nucleotide polymorphisms, (4) smoking, (5) diet quality, (6) education, and (7) pulse pressure. The cross-validated AUC estimation in RS-I was 0.92 (95% confidence interval [CI], 0.88-0.97) at 5 years, 0.92 (95% CI, 0.90-0.95) at 10 years, and 0.91 (95% CI, 0.88-0.94) at 15 years. In ALIENOR, the AUC reached 0.92 at 5 years (95% CI, 0.87-0.98). In terms of calibration, the model tended to underestimate the cumulative incidence of advanced AMD for the

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes., QC 20200304

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 : 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.

In age-related macular degeneration (AMD), both systemic and local zinc levels decline. Elevation of zinc in clinical studies delayed the progression to end-stage AMD. However, the molecular pathways underpinning this beneficial effect are not yet identified. In this study, we used differentiated primary human fetal retinal pigment epithelium (RPE) cultures and long-term zinc supplementation to carry out a combined transcriptome, proteome and secretome analysis from three genetically different human donors. After combining significant differences, we identified the complex molecular networks using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA). The cell cultures from the three donors showed extensive pigmentation, development of microvilli and basal infoldings and responded to zinc supplementation with an increase in transepithelial electrical resistance (TEER) (apical supplementation: 443.2 ± 79.3%, basal supplementation: 424.9 ± 116.8%, compared to control: 317.5 ± 98.2%). Significant changes were observed in the expression of 1044 genes, 151 cellular proteins and 124 secreted proteins. Gene set enrichment analysis revealed changes in specific molecular pathways related to cell adhesion/polarity, extracellular matrix organization, protein processing/transport, and oxidative stress response by zinc and identified a key upstream regulator effect similar to that of TGFB1.

Contains fulltext : 226200.pdf (publisher's version ) (Open Access)

Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.

Contains fulltext : 205500.pdf (publisher's version ) (Open Access), Age-related macular degeneration (AMD) is a common, progressive multifactorial vision-threatening disease and many genetic and environmental risk factors have been identified. The risk of AMD is influenced by lifestyle and diet, which may be reflected by an altered metabolic profile. Therefore, measurements of metabolites could identify biomarkers for AMD, and could aid in identifying high-risk individuals. Hypothesis-free technologies such as metabolomics have a great potential to uncover biomarkers or pathways that contribute to disease pathophysiology. To date, only a limited number of metabolomic studies have been performed in AMD. Here, we aim to contribute to the discovery of novel biomarkers and metabolic pathways for AMD using a targeted metabolomics approach of 188 metabolites. This study focuses on non-advanced AMD, since there is a need for biomarkers for the early stages of disease before severe visual loss has occurred. Targeted metabolomics was performed in 72 patients with early or intermediate AMD and 72 control individuals, and metabolites predictive for AMD were identified by a sparse partial least squares discriminant analysis. In our cohort, we identified four metabolite variables that were most predictive for early and intermediate stages of AMD. Increased glutamine and phosphatidylcholine diacyl C28:1 levels were detected in non-advanced AMD cases compared to controls, while the rate of glutaminolysis and the glutamine to glutamate ratio were reduced in non-advanced AMD. The association of glutamine with non-advanced AMD corroborates a recent report demonstrating an elevated glutamine level in early AMD using a different metabolomics technique. In conclusion, this study indicates that metabolomics is a suitable method for the discovery of biomarker candidates for AMD. In the future, larger metabolomics studies could add to the discovery of novel biomarkers in yet unknown AMD pathways and expand our insights in AMD pathophysiology.

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 : 190748.pdf (Publisher’s version ) (Open Access), Biomarkers can help unravel mechanisms of disease and identify new targets for therapy. They can also be useful in clinical practice for monitoring disease progression, evaluation of treatment efficacy, and risk assessment in multifactorial diseases, such as age-related macular degeneration (AMD). AMD is a highly prevalent progressive retinal disorder for which multiple genetic and environmental risk factors have been described, but the exact etiology is not yet fully understood. Many compounds have been evaluated for their association with AMD. We performed an extensive literature review of all compounds measured in serum, plasma, vitreous, aqueous humor, and urine of AMD patients. Over 3600 articles were screened, resulting in more than 100 different compounds analyzed in AMD studies, involved in neovascularization, immunity, lipid metabolism, extracellular matrix, oxidative stress, diet, hormones, and comorbidities (such as kidney disease). For each compound, we provide a short description of its function and discuss the results of the studies in relation to its usefulness as AMD biomarker. In addition, biomarkers identified by hypothesis-free techniques, including metabolomics, proteomics, and epigenomics, are covered. In summary, compounds belonging to the oxidative stress pathway, the complement system, and lipid metabolism are the most promising biomarker candidates for AMD. We hope that this comprehensive survey of the literature on systemic and ocular fluid compounds as potential biomarkers in AMD will provide a stepping stone for future research and possible implementation in clinical practice.

Contains fulltext : 200479.pdf (Publisher’s version ) (Open Access), There is an urgency to find new treatment strategies that could prevent or delay the onset or progression of AMD. Different classes of lipids and lipoproteins metabolism genes have been associated with AMD in a multiple ways, but despite the ever-increasing knowledge base, we still do not understand fully how circulating lipids or local lipid metabolism contribute to AMD. It is essential to clarify whether dietary lipids, systemic or local lipoprotein metabolismtrafficking of lipids in the retina should be targeted in the disease. In this article, we critically evaluate what has been reported in the literature and identify new directions needed to bring about a significant advance in our understanding of the role for lipids in AMD. This may help to develop potential new treatment strategies through targeting the lipid homeostasis.