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Studying Ciliary Signaling in Development and Disease

Periodic Reporting for period 1 - SCilS (Studying Ciliary Signaling in Development and Disease)

Reporting period: 2020-01-01 to 2021-12-31

SCilS creates an interdisciplinary and intersectoral European training network specializing in primary cilia and ciliopathies. This is a relatively new and rapidly developing research field that is in need of scientists equipped with fundamental knowledge of: (WP1) the processes that are driving development and homeostasis of these cellular antennae, that have evolved to be key signalling hubs of the cells, as they concentrate or segregate components of major cellular signalling pathways; (WP2) the critical signalling processes it regulates in different tissues, and (WP3) the biomedical consequences of its dysfunction that results in severe human genetic traits with highly heterogeneous, overlapping phenotypes, affecting as many as 1 in 400 people. SCilS gives the next generation of European cilia researchers an optimal training in (stem) cell biology, biochemistry, structural biology, cryo-electron tomography, multi-omics approaches (genomics, transcriptomics, proteomics), bio- informatics, and ultra-high-resolution imaging. Ciliary signalling is our scientific scaffold and research target, complemented by a unique career‐relevant training of young researchers by industrial and private sector partners. The training objectives will be met through academic and industrial training-by-research via individual research projects, secondments, and network-wide training sessions.
Understanding the establishment of the cilium as a dynamic signalling organelle (WP1). ESR1 studied a key IFT subunit, and determined a novel high-resolution crystal structure of its C-terminal domain, providing novel insights into the structure and function of the IFT machinery. ESR2 has investigated the effect of several different ciliopathy proteins on PCARE-induced ciliary tip expansions. Furthermore, ESR2 has determined the impact of patient mutations in the retinal ciliopathy protein INPP5E on the morphology of primary cilia in patient-derived fibroblasts, and successfully carried out a secondment in Newcastle with P8, to acquire skills in retinal organoid development in an industrial setting. ESR3 studied molecular mechanisms of cilium disassembly and has created stable hTERT RPE-1 cell lines that each express one of nine proteins known to regulate cilia disassembly. Affinity purification experiments and quantitative mass spectrometry analysis revealed several modulations in protein complex interactions during cilium disassembly. ESR4 has analysed the consequences of specific PROM1 alternative splicing on photoreceptor sensory cilia using a retinal organoid system, and was able to correlate these data to the in vivo situation using embryonic and foetal retinal samples, thus proving the concept that retinal organoids can be used to study alternative splicing.
Characterising novel ciliary signalling concepts in development and homeostasis (WP2). ESR5 targets the molecular mechanisms balancing the output of ciliary TGFβ/BMP signalling, and has scrutinized a key module in this process using patient cells, stem cell models and siRNA approaches in order to continue evaluating its role in signalling in health and disease. ESR6 has made progress in studying the interplay between primary cilia and metabolic rewiring in Polycystic Kidney Disease, and acquired skills in evaluating cilium morphology under diverse stress conditions, and the use of cilium gene ablation models (cells and mice) to distinguish the specific response of ciliary processes to cellular stress. ESR7 has provided novel insights into the role of Dlg1 and Kif13B in regulating the ciliary length and signalling capacity in kidney epithelial cells. To assess this by proximity proteomics, she successfully carried out a secondment in Nijmegen (with P1) to acquire expertise in this technique and generate relevant datasets that await further analysis. ESR8 is targeting the neuronal cilia and neurodevelopmental processes that are defective in Joubert syndrome using iPSC-derived cell lines and organoids as models. She was able to validate that the neurons cultured in the lab are ciliated, and generated iPSC homozygous knockouts of neuronal ciliopathy genes. Also, the next steps in initiating differentiated iNeurons for microelectrode arrays (MEAs) and cerebral organoid generation were initiated successfully, including acquiring expertise with several analysis methods. ESR9 is evaluating ciliary pathways and ontologies following a computational approach. In close collaboration with ESR13, she has devised a socioaffinity-like metric, which was applied to PubMed to extract disease/gene associations and (more importantly) to rank them according to the weight/uniqueness of evidence.
Determining the biomedical consequences of disrupted ciliary signalling in ciliopathies (WP3). Based on comparative transcriptomic analyses, ESR10, who is analysing altered signalling pathways in patients with renal ciliopathies, characterized an abnormal inflammatory response in urine and urinary renal epithelial cells (URECs) from patients with the renal ciliopathy, nephronophthisis (NPH), as well as in the kidneys of mouse models. ESR11 is focusing on therapeutic approaches for renal ciliopathies to characterize the effects of new molecules and their mechanism of action. Two specific compounds, including a prostaglandin-derived molecule, have a broad positive effect on ciliogenesis in URECs of patients with mutations in NPHP genes. ESR12 is focusing on ciliary signalling in cardiovascular development and disease, and characterized two new candidate genes involved in chronic heart disease using zebrafish transgenic lines. The goal of ESR13 is to provide an online system for the study of affinity proteomics data analysis to study edgetic effects resulting from mutations in ciliopathy genes. The "socioaffinity" metric to query single-wash affinity proteomics data as well as datasets when data for both wild-type and mutation(s) are available was improved. ESR14 is using zebrafish to model ciliopathy disease, and generated CRISPR knockout zebrafish lines of two ciliary genes with a potential role in cardiac regeneration. Using a novel automated medium throughput platform, she is evaluating the cardiac regeneration kinetics of mutant larvae generated after genetic ablation of cardiomyocytes.
Network wide training (WP4) is provided for the fellows in the form of six complimentary skills courses and four research training courses (all compulsory for the ESRs). These SCilS training events are not running exactly to schedule, due to COVID-19 pandemic, however we have been able to complete over 60% of the network-wide training to date. The events scheduled to take place in 2020 and in Q1-Q3 of 2021 were all organized online.
The trained ESRs and cutting-edge research output from the SCilS ITN will fuel the next generation of cilia research, and have a significant impact on the diagnosis and treatment of ciliopathies, including renal, cardiac, retinal and, neuronal ciliopathies, thus resulting in important economic and health benefits. Participation of the Ciliopathy Alliance and the International Kidney Cancer Coalition (IKCC) as partner organisations will facilitate a close connection of the ESRs to the most direct stakeholders, the ciliopathy patients and their caregivers.
Graphical abstract of the SCilS project