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Dominant mutations in the rhodopsin gene (Rho) contribute to 25% of autosomal dominant retinitis pigmentosa (adRP), characterized by photoreceptor loss and progressive blindness. One such mutation, Rho∆I256, carries a 3-bp deletion, resulting in the loss of one of two isoleucines at codons 255 and 256. Our investigation, using recombinant expression in HEK293 and COS-7 cells, revealed that Rho∆I256, akin to the known adRP mutation RhoP23H, induces the formation of rhodopsin protein (RHO) aggregates at the perinuclear region. Co-expression of Rho∆I256 or RhoP23H with wild-type RhoWT, mimicking the heterozygous genotype of adRP patients, demonstrated the dominant-negative effect, as all isoforms were retained in perinuclear aggregates, impeding membrane trafficking. In retinal explants from WT mice, mislocalization of labeled adRP isoforms at the outer nuclear layer was observed. Further analysis revealed that RHO∆I256 aggregates are retained at the endoplasmic reticulum (ER), undergo ER-associated degradation (ERAD), and colocalize with the AAA-ATPase escort chaperone valosin-containing protein (VCP). These aggregates are polyubiquitinated and partially colocalized with the 20S proteasome subunit beta-5 (PSMB5). Pharmacological inhibition of proteasome- or VCP activity increased RHO∆I256 aggregate size. In summary, RHO∆I256 exhibits dominant pathogenicity by sequestering normal RHOWT in ER aggregates, preventing its membrane trafficking and following the ERAD degradation.

The intraflagellar transport (IFT) machinery is essential for cilia assembly, maintenance, and trans-localization of signaling proteins. The IFT machinery consists of two large multiprotein complexes, one of which is the IFT-B. TTC30A and TTC30B are integral components of this complex and were previously shown to have redundant functions in the context of IFT, preventing the disruption of IFT-B and, thus, having a severe ciliogenesis defect upon loss of one paralog. In this study, we re-analyzed the paralog-specific protein complexes and discovered a potential involvement of TTC30A or TTC30B in ciliary signaling. Specifically, we investigated a TTC30A-specific interaction with protein kinase A catalytic subunit α, a negative regulator of Sonic hedgehog (Shh) signaling. Defects in this ciliary signaling pathway are often correlated to synpolydactyly, which, intriguingly, is also linked to a rare TTC30 variant. For an in-depth analysis of this unique interaction and the influence on Shh, TTC30A or B single- and double-knockout hTERT-RPE1 were employed, as well as rescue cells harboring wildtype TTC30 or the corresponding mutation. We could show that mutant TTC30A inhibits the ciliary localization of Smoothened. This observed effect is independent of Patched1 but associated with a distinct phosphorylated PKA substrate accumulation upon treatment with forskolin. This rather prominent phenotype was attenuated in mutant TTC30B. Mass spectrometry analysis of wildtype versus mutated TTC30A or TTC30B uncovered differences in protein complex patterns and identified an impaired TTC30A–IFT57 interaction as the possible link leading to synpolydactyly. We could observe no impact on cilia assembly, leading to the hypothesis that a slight decrease in IFT-B binding can be compensated, but mild phenotypes, like synpolydactyly, can be induced by subtle signaling changes. Our systematic approach revealed the paralog-specific influence of TTC30A KO and mutated TTC30A on the activity of PRKACA and the uptake of Smoothened into the cilium, resulting in a downregulation of Shh. This downregulation, combined with interactome alterations, suggests a potential mechanism of how mutant TTC30A is linked to synpolydactyly.

The human Usher syndrome (USH) is the most common form of a sensory hereditary ciliopathy characterized by progressive vision and hearing loss. Mutations in the genes ADGRV1 and CIB2 have been associated with two distinct sub-types of USH, namely, USH2C and USH1J. The proteins encoded by the two genes belong to very distinct protein families: the adhesion G protein-coupled receptor ADGRV1 also known as the very large G protein-coupled receptor 1 (VLGR1) and the Ca2+- and integrin-binding protein 2 (CIB2), respectively. In the absence of tangible knowledge of the molecular function of ADGRV1 and CIB2, pathomechanisms underlying USH2C and USH1J are still unknown. Here, we aimed to enlighten the cellular functions of CIB2 and ADGRV1 by the identification of interacting proteins, a knowledge that is commonly indicative of cellular functions. Applying affinity proteomics by tandem affinity purification in combination with mass spectrometry, we identified novel potential binding partners of the CIB2 protein and compared these with the data set we previously obtained for ADGRV1. Surprisingly, the interactomes of both USH proteins showed a high degree of overlap indicating their integration in common networks, cellular pathways and functional modules which we confirmed by GO term analysis. Validation of protein interactions revealed that ADGRV1 and CIB2 mutually interact. In addition, we showed that the USH proteins also interact with the TRiC/CCT chaperonin complex and the Bardet Biedl syndrome (BBS) chaperonin-like proteins. Immunohistochemistry on retinal sections demonstrated the co-localization of the interacting partners at the photoreceptor cilia, supporting the role of USH proteins ADGRV1 and CIB2 in primary cilia function. The interconnection of protein networks involved in the pathogenesis of both syndromic retinal dystrophies BBS and USH suggest shared pathomechanisms for both syndromes on the molecular level.

Neurons build vast gap junction-coupled networks (GJ-nets) that are permeable to ions or small molecules, enabling lateral signaling. Herein, we investigate (1) the effect of blinding diseases on GJ-nets in mouse retinas and (2) the impact of electrical stimulation on GJ permeability. GJ permeability was traced in the acute retinal explants of blind retinal degeneration 1 (rd1) mice using the GJ tracer neurobiotin. The tracer was introduced via the edge cut method into the GJ-net, and its spread was visualized in histological preparations (fluorescent tagged) using microscopy. Sustained stimulation was applied to modulate GJ permeability using a single large electrode. Our findings are: (1) The blind rd1 retinas displayed extensive intercellular coupling via open GJs. Three GJ-nets were identified: horizontal, amacrine, and ganglion cell networks. (2) Sustained stimulation significantly diminished the tracer spread through the GJs in all the cell layers, as occurs with pharmaceutical inhibition with carbenoxolone. We concluded that the GJ-nets of rd1 retinas remain coupled and functional after blinding disease and that their permeability is regulatable by sustained stimulation. These findings are essential for understanding molecular signaling in diseases over coupled networks and therapeutic approaches using electrical implants, such as eliciting visual sensations or suppressing cortical seizures.

How pancreatic islets of Langerhans are built during development is incompletely understood. Here the authors find that Synaptotagmin-13 mediates remodeling of cell-matrix adhesion to regulate endocrine cell egression and islet morphogenesis.

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.

Since its invention, super-resolution microscopy has become a popular tool for advanced imaging of biological structures, allowing visualisation of subcellular structures at a spatial scale below the diffraction limit. Thus, it is not surprising that recently, different super-resolution techniques are being applied in neuroscience, e.g. to resolve the clustering of neurotransmitter receptors and protein complex composition in presynaptic terminals. Still, the vast majority of these experiments were carried out either in cell cultures or very thin tissue sections, while there are only a few examples of super-resolution imaging in deeper layers (30 - 50 µm) of biological samples. In that context, the mammalian whole-mount retina has rarely been studied with super-resolution microscopy. Here, we aimed at establishing a stimulated-emission-depletion (STED) microscopy protocol for imaging whole-mount retina. To this end, we developed sample preparation including horizontal slicing of retinal tissue, an immunolabeling protocol with STED-compatible fluorophores and optimised the image acquisition settings. We labelled subcellular structures in somata, dendrites, and axons of retinal ganglion cells in the inner mouse retina. By measuring the full width at half maximum of the thinnest filamentous structures in our preparation, we achieved a resolution enhancement of two or higher compared to conventional confocal images. When combined with horizontal slicing of the retina, these settings allowed visualisation of putative GABAergic horizontal cell synapses in the outer retina. Taken together, we successfully established a STED protocol for reliable super-resolution imaging in the whole-mount mouse retina at depths between 30 and 50 µm, which enables investigating, for instance, protein complex composition and cytoskeletal ultrastructure at retinal synapses in health and disease.

Bardet–Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate.

Establishment and maintenance of the primary cilium as a signaling-competent organelle requires a high degree of fine tuning, which is at least in part achieved by a variety of post-translational modifications. One such modification is ubiquitination. The small and highly conserved ubiquitin protein possesses a unique versatility in regulating protein function via its ability to build mono and polyubiquitin chains onto target proteins. We aimed to take an unbiased approach to generate a comprehensive blueprint of the ciliary ubiquitinome by deploying a multi-proteomics approach using both ciliary-targeted ubiquitin affinity proteomics, as well as ubiquitin-binding domain-based proximity labelling in two different mammalian cell lines. This resulted in the identification of several key proteins involved in signaling, cytoskeletal remodeling and membrane and protein trafficking. Interestingly, using two different approaches in IMCD3 and RPE1 cells, respectively, we uncovered several novel mechanisms that regulate cilia function. In our IMCD3 proximity labeling cell line model, we found a highly enriched group of ESCRT-dependent clathrin-mediated endocytosis-related proteins, suggesting an important and novel role for this pathway in the regulation of ciliary homeostasis and function. In contrast, in RPE1 cells we found that several structural components of caveolae (CAV1, CAVIN1, and EHD2) were highly enriched in our cilia affinity proteomics screen. Consistently, the presence of caveolae at the ciliary pocket and ubiquitination of CAV1 specifically, were found likely to play a role in the regulation of ciliary length in these cells. Cilia length measurements demonstrated increased ciliary length in RPE1 cells stably expressing a ubiquitination impaired CAV1 mutant protein. Furthermore, live cell imaging in the same cells revealed decreased CAV1 protein turnover at the cilium as the possible cause for this phenotype. In conclusion, we have generated a comprehensive list of cilia-specific proteins that are subject to regulation via ubiquitination which can serve to further our understanding of cilia biology in health and disease.

Abstract Objective The mammalian Notch ligand DLL1 has essential functions during development. To visualise DLL1 in tissues, for sorting and enrichment of DLL1-expressing cells, and to efficiently purify DLL1 protein complexes we tagged DLL1 in mice with AcGFPHA or Strep/FLAG. Results We generated constructs to express DLL1 that carried C-terminal in-frame an AcGFPHA tag flanked by loxP sites followed by a Strep/FLAG (SF) tag out of frame. Cre-mediated recombination replaced AcGFP-HA by SF. The AcGFPHAstopSF cassette was added to DLL1 for tests in cultured cells and introduced into endogenous DLL1 in mice by homologous recombination. Tagged DLL1 protein was detected by antibodies against GFP and HA or Flag, respectively, both in CHO cells and embryo lysates. In CHO cells the AcGFP fluorophore fused to DLL1 was functional. In vivo AcGFP expression was below the level of detection by direct fluorescence. However, the SF tag allowed us to specifically purify DLL1 complexes from embryo lysates. Homozygous mice expressing AcGFPHA or SF-tagged DLL1 revealed a vertebral column phenotype reminiscent of disturbances in AP polarity during somitogenesis, a process most sensitive to reduced DLL1 function. Thus, even small C-terminal tags can impinge on sensitive developmental processes requiring DLL1 activity.

Extracellular vesicles (EV) are known to be released from the primary cilium, but the role ciliary proteins play in EV biogenesis remains unexplored. Here, the authors demonstrate increased secretion of small EVs with altered cargo composition from cells with known ciliarelated mutations. Wnt related molecules made up a majority of altered cargo

Background: The electroretinogram (ERG) is an essential diagnostic tool for visual function, both in clinical and research settings. Here, we establish an advanced in vitro approach to assess cell-type-specific ERG signal components. Methods: Retinal explant cultures, maintained under entirely controlled conditions, were derived from wild-type mice and rd10 rod- and cpfl1 cone-degeneration mouse models. Local micro-ERG (µERG) and simultaneous ganglion cell (GC) recordings were obtained from the retinal explants using multi-electrode arrays. Band-pass filtering was employed to distinguish photoreceptor, bipolar cell, amacrine cell (AC), and GC responses. Results: Scotopic and photopic stimulation discriminated between rod and cone responses in wild-type and mutant retina. The 25 kHz sampling rate allowed the visualization of oscillatory potentials (OPs) in extraordinary detail, revealing temporal correlations between OPs and GC responses. Pharmacological isolation of different retinal circuits found that OPs are generated by inner retinal AC electrical synapses. Importantly, this AC activity helped synchronise GC activity. Conclusion: Our µERG protocol simultaneously records the light-dependent activities of the first-, second-, and third-order neurons within the native neuronal circuitry, providing unprecedented insights into retinal physiology and pathophysiology. This method now also enables complete in vitro retinal function testing of therapeutic interventions, providing critical guidance for later in vivo investigations.

Abstract Cilia are protrusions of the cell surface and composed of hundreds of proteins many of which are evolutionary and functionally well conserved. In cells assembling motile cilia the expression of numerous ciliary components is under the control of the transcription factor FOXJ1. Here, we analyse the evolutionary conserved FOXJ1 target CFAP161 in Xenopus and mouse. In both species Cfap161 expression correlates with the presence of motile cilia and depends on FOXJ1. Tagged CFAP161 localises to the basal bodies of multiciliated cells of the Xenopus larval epidermis, and in mice CFAP161 protein localises to the axoneme. Surprisingly, disruption of the Cfap161 gene in both species did not lead to motile cilia-related phenotypes, which contrasts with the conserved expression in cells carrying motile cilia and high sequence conservation. In mice mutation of Cfap161 stabilised the mutant mRNA making genetic compensation triggered by mRNA decay unlikely. However, genes related to microtubules and cilia, microtubule motor activity and inner dyneins were dysregulated, which might buffer the Cfap161 mutation.

Therapy development for neurodegenerative diseases of the retina constitutes a major unmet medical need, and this may be particularly relevant for inherited diseases of the retina, which are largely untreatable to this day. Therapy development necessitates appropriate models to improve the understanding of the underlying degenerative mechanisms, as well as for the testing and evaluation of novel treatment approaches. This review provides an overview of various in vitro model systems used to study retinal neuroprotection. The in vitro methods and technologies discussed range from primary retinal cell cultures and cell lines, to retinal organoids and organotypic retinal explants, to the cultivation of whole eyeballs. The advantages and disadvantages of these methods are compared and evaluated, also in view of the 3R principles (i.e., the refinement, reduction, and replacement of live animal testing), to identify suitable in vitro alternatives for in vivo experimentation. The article further expands on the use of in vitro models to test and evaluate neuroprotective treatments and to aid the development of retinal drug delivery systems. Among the pharmacological agents tested and characterized in vitro are such that interfere with aberrant cyclic guanosine monophosphate (cGMP) -signaling or such that inhibit the activities of poly (ADP-ribose) polymerase (PARP), histone deacetylases (HDAC), calpain-type proteases, as well as unfolded protein response-related stress. We then introduce nanoparticle-based drug delivery systems and discuss how different in vitro systems may be used to assess their efficacy in the treatment of retinal diseases. The summary provides a brief comparison of available in vitro models and relates their advantages and limitations to the various experimental requirements, for instance, for studies into disease mechanisms, novel treatments, or retinal toxicity. In many cases, combinations of different in vitro models may be required to obtain a comprehensive view of the efficacy of a given retinal neuroprotection approach.

Insights into the pathogenesis of age-related macular degeneration (AMD), a leading cause of blindness, point towards a complex interplay of genetic and lifestyle factors triggering various systemic pathways. This study aimed to characterize metabolomic profiles for AMD and to evaluate their position in the trias with genetics and lifestyle. This study included 5923 individuals from five European studies. Blood metabolomics were assessed using a nuclear magnetic resonance platform of 146 metabolites. Associations were studied using regression analyses. A genetic risk score (GRS) was calculated using β-values of 49 AMD variants, a lifestyle risk score (LRS) using smoking and diet data, and a metabolite risk score (MRS) using metabolite values. We identified 61 metabolites associated with early-intermediate AMD, of which 94% were lipid-related, with higher levels of HDL-subparticles and apolipoprotein-A1, and lower levels of VLDL-subparticles, triglycerides, and fatty acids (false discovery rate (FDR) p-value < 1.4 × 10−2). Late AMD was associated with lower levels of the amino acids histidine, leucine, valine, tyrosine, and phenylalanine, and higher levels of the ketone bodies acetoacetate and 3-hydroxybutyrate (FDR p-value < 1.5 × 10−3). A favorable lifestyle characterized by a healthy diet was associated with higher levels of amino acids and lower levels of ketone bodies, while an unfavorable lifestyle, including smoking, showed opposite effects (FDR p-value < 2.7 × 10−2). The MRS mediated 5% of the effect of the GRS and 20% of that of the LRS on late AMD. Our findings show that metabolomic profiles differ between AMD stages and show that blood metabolites mostly reflect lifestyle. The severity-specific profiles spur further interest into the systemic effects related to disease conversion.

The mechanism by which adenosine monophosphate modulates dynein ATPase-mediated ciliary and flagellar beating remains obscure. Here the authors identify an axonemal module including cilia and flagella associated protein 45 that supports adenine nucleotide homeostasis and underlies a human ciliopathy

In yeast, the phosphatase Cdc14 controls mitotic exit. Here the authors show that mammalian CDC14B antagonizes CDK1, to keep the deubiquitinase USP9X unphosphorylated and inactive, and that Wilms’ tumor protein 1 is a substrate for active USP9X that directs mitosis-specific transcription to regulate mitotic survival.

Kras is often mutated in colorectal cancer but how this oncogenic mutation alters signalling pathways globally is undetermined. Here, the authors analyse how this mutation affects protein interaction networks and signal flow showing an extensive re‐wiring of signalling in response to KRas mutation

Primary ciliary defects cause a group of developmental conditions known as ciliopathies. Here, we provide mechanistic insight into ciliary ubiquitin processing in cells and for mouse model lacking the ciliary protein Mks1. In vivo loss of Mks1 sensitises cells to proteasomal disruption, leading to abnormal accumulation of ubiquitinated proteins. We identified UBE2E1, an E2 ubiquitin-conjugating enzyme that polyubiquitinates β-catenin, and RNF34, an E3 ligase, as novel interactants of MKS1. UBE2E1 and MKS1 colocalised, and loss of UBE2E1 recapitulates the ciliary and Wnt signalling phenotypes observed during loss of MKS1. Levels of UBE2E1 and MKS1 are co-dependent and UBE2E1 mediates both regulatory and degradative ubiquitination of MKS1. We demonstrate that processing of phosphorylated β-catenin occurs at the ciliary base through the functional interaction between UBE2E1 and MKS1. These observations suggest that correct β-catenin levels are tightly regulated at the primary cilium by a ciliary-specific E2 (UBE2E1) and a regulatory substrate-adaptor (MKS1).

Mutations in PDE6D impair the function of its cognate protein, phosphodiesterase 6D (PDE6D), in prenylated protein trafficking towards the ciliary membrane, causing the human ciliopathy Joubert Syndrome (JBTS22) and retinal degeneration in mice. In this study, we purified the prenylated cargo of PDE6D by affinity proteomics to gain insight into PDE6D-associated disease mechanisms. By this approach, we have identified a specific set of PDE6D-interacting proteins that are involved in photoreceptor integrity, GTPase activity, nuclear import, or ubiquitination. Among these interacting proteins, we identified novel ciliary cargo proteins of PDE6D, including FAM219A, serine/threonine-protein kinase NIM1 (NIM1K), and ubiquitin-like protein 3 (UBL3). We show that NIM1K and UBL3 localize inside the cilium in a prenylation-dependent manner. Furthermore, UBL3 also localizes in vesicle-like structures around the base of the cilium. Through affinity proteomics of UBL3, we confirmed its strong interaction with PDE6D and its association with proteins that regulate small extracellular vesicles (sEVs) and ciliogenesis. Moreover, we show that UBL3 localizes in specific photoreceptor cilium compartments in a prenylation-dependent manner. Therefore, we propose that UBL3 may play a role in the sorting of proteins towards the photoreceptor outer segment, further explaining the development of PDE6D-associated retinal degeneration.

Introduction The actual prevalence of a SARS-CoV-2 infection and the individual assessment of being or having been infected may differ. Facing the great uncertainty—especially at the beginning of the pandemic—and the possibility of asymptomatic or mildly symptomatic, subclinical infections, we evaluate the experience of SARS-CoV-2 antibody screening at a tertiary clinical setting.Methods and analysis All employees of a tertiary eye centre and a research institute of ophthalmology were offered antibody testing in May 2020, using a sequential combination of different validated assays/antigens and point-of-care (POC) testing for a subset (NCT04446338). Before taking blood, a systematic inquiry into past symptoms, known contacts and a subjective self-assessment was documented. The correlations between serostatus, patient contacts and demographic characteristics were analysed. Different tests were compared by Kappa statistics.Results Among 318 participants, SARS-CoV-2 antibodies were detected in 9 employees. Chemiluminescence assays (chemiluminescence immunoassay and electrochemiluminescence) showed superior specificity and high reproducibility, compared with ELISA and POC results.In contrast to the low seropositivity (2.8%) of healthcare workers, higher than that of the other departments of the hospital, a large proportion mistakenly assumed that they might have already been infected. Antiviral antibody titres increased and remained on a plateau for at least 3 months.Conclusions The great demand and acceptance confirmed the benefit of highly sensitive testing methods in the early phase of the pandemic. The coincidence of low seroprevalence and anxious employees may have contributed to internalising the need of hygiene measures.

The very large G protein-coupled receptor (VLGR1, ADGRV1) is the largest member of the adhesion GPCR family. Mutations in VLGR1 have been associated with the human Usher syndrome (USH), the most common form of inherited deaf-blindness as well as childhood absence epilepsy. VLGR1 was previously found as membrane–membrane adhesion complexes and focal adhesions. Affinity proteomics revealed that in the interactome of VLGR1, molecules are enriched that are associated with both the ER and mitochondria, as well as mitochondria-associated ER membranes (MAMs), a compartment at the contact sites of both organelles. We confirmed the interaction of VLGR1 with key proteins of MAMs by pull-down assays in vitro complemented by in situ proximity ligation assays in cells. Immunocytochemistry by light and electron microscopy demonstrated the localization of VLGR1 in MAMs. The absence of VLGR1 in tissues and cells derived from VLGR1-deficient mouse models resulted in alterations in the MAM architecture and in the dysregulation of the Ca2+ transient from ER to mitochondria. Our data demonstrate the molecular and functional interaction of VLGR1 with components in MAMs and point to an essential role of VLGR1 in the regulation of Ca2+ homeostasis, one of the key functions of MAMs.

No effective therapies exist for dry age-related macular degeneration. In this perspective, the authors propose that research should emphasize system biology approaches that integrate various ‘omics’ data into mathematical models to establish pathogenic mechanisms on which to design novel treatments, and identify biomarkers that predict disease progression and therapeutic response.

Summary: VLGR1 (very large G protein-coupled receptor-1) is by far the largest adhesion G protein-coupled receptor in humans. Homozygous pathologic variants of VLGR1 cause hereditary deaf blindness in Usher syndrome 2C and haploinsufficiency of VLGR1 is associated with epilepsy. However, its molecular function remains elusive. Herein, we used affinity proteomics to identify many components of focal adhesions (FAs) in the VLGR1 interactome. VLGR1 is localized in FAs and assembles in FA protein complexes in situ. Depletion or loss of VLGR1 decreases the number and length of FAs in hTERT-RPE1 cells and in astrocytes of Vlgr1 mutant mice. VLGR1 depletion reduces cell spread and migration kinetics as well as the response to mechanical stretch characterizing VLGR1 as a metabotropic mechanosensor in FAs. Our data reveal a critical role of VLGR1 in the FA function and enlighten potential pathomechanisms in diseases related to VLGR1.

Primary cilia are sensory organelles vital for developmental and physiological processes. Their dysfunction causes a range of phenotypes including retinopathies. Although primary cilia have been described in the retinal pigment epithelium (RPE), little is known about their contribution to biological processes within this tissue. Ciliary proteins are increasingly being identified in non-ciliary locations and might carry out additional functions, disruption of which possibly contributes to pathology. The RPE is essential for maintaining photoreceptor cells and visual function. We demonstrate that upon loss of Bbs8, predominantly thought to be a ciliary gene, the RPE shows changes in gene and protein expression initially involved in signaling pathways and developmental processes, and at a later time point RPE homeostasis and function. Differentially regulated molecules affecting the cytoskeleton and cellular adhesion, led to defective cellular polarization and morphology associated with a possible epithelial-to-mesenchymal transition (EMT)-like phenotype. Our data highlights the benefit of combinatorial “omics” approaches with in vivo data for investigating the function of ciliopathy proteins. It also emphasizes the importance of ciliary proteins in the RPE and their contribution to visual disorders, which must be considered when designing treatment strategies for retinal degeneration.

Background/objectivesThe systemic organ involvement of SARS-CoV-2 needs to be thoroughly investigated including the possibility of an ocular reservoir in humans. To examine retinal tissues and vitreous for histopathology and SARS-CoV-2 presence with regard to possible effects on the human retina and/ or vitreous. We performed histopathological analyses and quantitative (q)RT-PCR-testing for SARS-CoV-2 RNA on retinal tissues and vitreous of COVID-19 postmortem donors.Subjects/methodsIncluded in this study were 10 eyes of 5 deceased COVID-19 patients. The diagnosis of SARS-CoV-2 infection was confirmed via pharyngeal swabs and broncho-alveolar fluids. The highest level of personal protective equipment (PPE) and measures was employed during fluid-tissue procurement and preparation. Histopathological examinations and qRT-PCR-testing were carried out for all retinal tissues and vitreous fluids.ResultsThe histopathological examinations revealed no signs of morphologically identifiable retinal inflammation or vessel occlusions based on hematoxylin and eosin stains. By qRT-PCRs, we detected no significant level of viral RNA in human retina and vitreous.ConclusionsIn this study, no significant level of SARS-CoV-2-RNA was detected in the human retinal and vitreous fluid samples of deceased COVID-19 patients. Histopathological examinations confirmed no morphological sign of damage to retinal vasculature or tissues. Further studies are needed to confirm or refute the results.

Summary: PINK1 loss-of-function mutations cause early onset Parkinson disease. PINK1-Parkin mediated mitophagy has been well studied, but the relevance of the endogenous process in the brain is debated.Here, the absence of PINK1 in human dopaminergic neurons inhibits ionophore-induced mitophagy and reduces mitochondrial membrane potential. Compensatory, mitochondrial renewal maintains mitochondrial morphology and protects the respiratory chain. This is paralleled by metabolic changes, including inhibition of the TCA cycle enzyme mAconitase, accumulation of NAD+, and metabolite depletion. Loss of PINK1 disrupts dopamine metabolism by critically affecting its synthesis and uptake. The mechanism involves steering of key amino acids toward energy production rather than neurotransmitter metabolism and involves cofactors related to the vitamin B6 salvage pathway identified using unbiased multi-omics approaches.We propose that reduction of mitochondrial membrane potential that cannot be controlled by PINK1 signaling initiates metabolic compensation that has neurometabolic consequences relevant to Parkinson disease.

The very large G-protein-coupled receptor 1 (VLGR1/ADGRV1) is the largest member of the adhesion G-protein-coupled receptor (ADGR) family. Mutations in VLGR1/ADGRV1 cause human Usher syndrome (USH), a form of hereditary deaf-blindness, and have been additionally linked to epilepsy. In the absence of tangible knowledge of the molecular function and signaling of VLGR1, the pathomechanisms underlying the development of these diseases are still unknown. Our study aimed to identify novel, previously unknown protein networks associated with VLGR1 in order to describe new functional cellular modules of this receptor. Using affinity proteomics, we have identified numerous new potential binding partners and ligands of VLGR1. Tandem affinity purification hits were functionally grouped based on their Gene Ontology terms and associated with functional cellular modules indicative of functions of VLGR1 in transcriptional regulation, splicing, cell cycle regulation, ciliogenesis, cell adhesion, neuronal development, and retinal maintenance. In addition, we validated the identified protein interactions and pathways in vitro and in situ. Our data provided new insights into possible functions of VLGR1, related to the development of USH and epilepsy, and also suggest a possible role in the development of other neuronal diseases such as Alzheimer’s disease.

Purpose: To evaluate the status of ocular donor tissues of a COVID-19 postmortem donor. Methods: SARS-CoV-2 was detected via a pharyngeal swab and broncho-alveolar lavage in the COVID-19 suspect. Postmortem tissue procurement and preparation were performed with personal protective equipment (PPE) and the necessary protective measures. qRT-PCR-testing was performed for the following ocular tissues and fluids: conjunctival fluid swabs, bulbar conjunctiva, corneal epithelium, corneal stroma, corneal endothelium, anterior chamber fluid, lens, iris, vitreous, retina, uvea, sclera, and optic nerve. Informed consent and Institutional Review Board approval was obtained prior to this study (196/2020BO2; Date of approval: 03/26/2020; Ethics Committee of the University of Tuebingen). Results: In all ocular tissue and fluid samples no SARS-CoV-2 RNA was detected via qRT-PCR of the confirmed COVID-19 postmortem donor. Conclusions and Importance: Late-stage COVID-19 patients might not harbor an ocular reservoir of SARS-CoV-2. The risk of transmitting SARS-CoV-2 via ocular tissues and fluids might be low. This may bear future implications for patient management in ophthalmological practice, surgery and transplantation.

Abstract The transcription factor FOXJ1 is essential for the formation of motile cilia throughout the animal kingdom. Target genes therefore likely constitute an important part of the motile cilia program. Here, we report on the analysis of one of these targets, Fam183b, in Xenopus and mice. Fam183b encodes a protein with unknown function which is conserved from the green algae Chlamydomonas to humans. Fam183b is expressed in tissues harbouring motile cilia in both mouse and frog embryos. FAM183b protein localises to basal bodies of cilia in mIMCD3 cells and of multiciliated cells of the frog larval epidermis. In addition, FAM183b interacts with NUP93, which also localises to basal bodies. During frog embryogenesis, Fam183b was dispensable for laterality specification and brain development, but required for ciliogenesis and motility of epidermal multiciliated cells and nephrostomes, i.e. the embryonic kidney. Surprisingly, mice homozygous for a null allele did not display any defects indicative of disrupted motile ciliary function. The lack of a cilia phenotype in mouse and the limited requirements in frog contrast with high sequence conservation and the correlation of gene expression with the presence of motile cilia. This finding may be explained through compensatory mechanisms at sites where no defects were observed in our FAM183b-loss-of-function studies.

Age-related macular degeneration (AMD) is a complex degenerative disease of the retina with multiple risk-modifying factors, including aging, genetics, and lifestyle choices. The combination of these factors leads to oxidative stress, inflammation, and metabolic failure in the retinal pigment epithelium (RPE) with subsequent degeneration of photoreceptors in the retina. The alternative complement pathway is tightly linked to AMD. In particular, the genetic variant in the complement factor H gene (CFH), which leads to the Y402H polymorphism in the factor H protein (FH), confers the second highest risk for the development and progression of AMD. Although the association between the FH Y402H variant and increased complement system activation is known, recent studies have uncovered novel FH functions not tied to this activity and highlighted functional relevance for intracellular FH. In our previous studies, we show that loss of CFH expression in RPE cells causes profound disturbances in cellular metabolism, increases the vulnerability towards oxidative stress, and modulates the activation of pro-inflammatory signaling pathways, most importantly the NF-kB pathway. Here, we silenced CFH in hTERT-RPE1 cells to investigate the mechanism by which intracellular FH regulates RPE cell homeostasis. We found that silencing of CFH results in hyperactivation of mTOR signaling along with decreased mitochondrial respiration and that mTOR inhibition via rapamycin can partially rescue these metabolic defects. To obtain mechanistic insight into the function of intracellular FH in hTERT-RPE1 cells, we analyzed the interactome of FH via immunoprecipitation followed by mass spectrometry-based analysis. We found that FH interacts with essential components of the ubiquitin-proteasomal pathway (UPS) as well as with factors associated with RB1/E2F signalling in a complement-pathway independent manner. Moreover, we found that FH silencing affects mRNA levels of the E3 Ubiquitin-Protein Ligase Parkin and PTEN induced putative kinase (Pink1), both of which are associated with UPS. As inhibition of mTORC1 was previously shown to result in increased overall protein degradation via UPS and as FH mRNA and protein levels were shown to be affected by inhibition of UPS, our data stress a potential regulatory link between endogenous FH activity and the UPS.

The treatment of retinal diseases by intravitreal injections requires frequent administration unless drug delivery systems with long retention and controlled release are used. In this work, we focused on pullulan (≈67 kDa) conjugates of dexamethasone as therapeutic systems for intravitreal administration. The pullulan–dexamethasone conjugates self-assemble into negatively charged nanoparticles (average size 326 ± 29 nm). Intravitreal injections of pullulan and pullulan–dexamethasone were safe in mouse, rat and rabbit eyes. Fluorescently labeled pullulan particles showed prolonged retention in the vitreous and they were almost completely eliminated via aqueous humor outflow. Pullulan conjugates also distributed to the retina via Müller glial cells when tested in ex vivo retina explants and in vivo. Pharmacokinetic simulations showed that pullulan–dexamethasone conjugates may release free and active dexamethasone in the vitreous humor for over 16 days, even though a large fraction of dexamethasone may be eliminated from the eye as bound pullulan–dexamethasone. We conclude that pullulan based drug conjugates are promising intravitreal drug delivery systems as they may reduce injection frequency and deliver drugs into the retinal cells.

Age-related Macular degeneration (AMD) is a degenerative disease of the macula affecting the elderly population. Treatment options are limited, partly due to the lack of understanding of AMD pathology and the lack of suitable research models that replicate the complexity of the human macula and the intricate interplay of the genetic, aging and lifestyle risk factors contributing to AMD. One of the main genetic risks associated with AMD is located on the Complement Factor H (CFH) gene, leading to an amino acid substitution in the Factor H (FH) protein (Y402H). However, the mechanism of how this FH variant promotes the onset of AMD remains unclear. Previously, we have shown that FH deprivation in RPE cells, via CFH silencing, leads to increased inflammation, metabolic impairment and vulnerability toward oxidative stress. In this study, we established a novel co-culture model comprising CFH silenced RPE cells and porcine retinal explants derived from the visual streak of porcine eyes, which closely resemble the human macula. We show that retinae exposed to FH-deprived RPE cells show signs of retinal degeneration, with rod cells being the first cells to undergo degeneration. Moreover, via Raman analyses, we observed changes involving the mitochondria and lipid composition of the co-cultured retinae upon FH loss. Interestingly, the detrimental effects of FH loss in RPE cells on the neuroretina were independent of glial cell activation and external complement sources. Moreover, we show that the co-culture model is also suitable for human retinal explants, and we observed a similar trend when RPE cells deprived of FH were co-cultured with human retinal explants from a single donor eye. Our findings highlight the importance of RPE-derived FH for retinal homeostasis and provide a valuable model for AMD research.

Rhodopsin (RHO) misfolding mutations are a common cause of the blinding disease autosomal dominant retinitis pigmentosa (adRP). The most prevalent mutation, RHOP23H, results in its misfolding and retention in the endoplasmic reticulum (ER). Under homeostatic conditions, misfolded proteins are selectively identified, retained at the ER, and cleared via ER-associated degradation (ERAD). Overload of these degradation processes for a prolonged period leads to imbalanced proteostasis and may eventually result in cell death. ERAD of misfolded proteins, such as RHOP23H, includes the subsequent steps of protein recognition, targeting for ERAD, retrotranslocation, and proteasomal degradation. In the present study, we investigated and compared pharmacological modulation of ERAD at these four different major steps. We show that inhibition of the VCP/proteasome activity favors cell survival and suppresses P23H-mediated retinal degeneration in RHOP23H rat retinal explants. We suggest targeting this activity as a therapeutic approach for patients with currently untreatable adRP.

Age-related macular degeneration (AMD), the leading cause of vision loss in the elderly, is a degenerative disease of the macula, where retinal pigment epithelium (RPE) cells are damaged in the early stages of the disease, and chronic inflammatory processes may be involved. Besides aging and lifestyle factors as drivers of AMD, a strong genetic association to AMD is found in genes of the complement system, with a single polymorphism in the complement factor H gene (CFH), accounting for the majority of AMD risk. However, the exact mechanism of CFH dysregulation confers such a great risk for AMD and its role in RPE cell homeostasis is unclear. To explore the role of endogenous CFH locally in RPE cells, we silenced CFH in human hTERT-RPE1 cells. We demonstrate that endogenously expressed CFH in RPE cells modulates inflammatory cytokine production and complement regulation, independent of external complement sources, or stressors. We show that loss of the factor H protein (FH) results in increased levels of inflammatory mediators (e.g., IL-6, IL-8, GM-CSF) and altered levels of complement proteins (e.g., C3, CFB upregulation, and C5 downregulation) that are known to play a role in AMD. Moreover, our results identify the NF-κB pathway as the major pathway involved in regulating these inflammatory and complement factors. Our findings suggest that in RPE cells, FH and the NF-κB pathway work in synergy to maintain inflammatory and complement balance, and in case either one of them is dysregulated, the RPE microenvironment changes towards a proinflammatory AMD-like phenotype.

Rod photoreceptors are thought to be saturated under bright light. Here, the authors describe the physiological parameters that mediate response saturation of rod photoreceptors in mouse retina, and show that rods can drive visual responses in photopic conditions.

Intracellular trafficking depends on the function of Rab GTPases, whose activation is regulated by guanine exchange factors (GEFs). The Rab5 GEF, Rabex5, was previously proposed to be auto-inhibited by its C-terminus. Here, we studied full-length Rabex5 and Rabaptin5 proteins as well as domain deletion Rabex5 mutants using hydrogen deuterium exchange mass spectrometry. We generated a structural model of Rabex5, using chemical cross-linking mass spectrometry and integrative modeling techniques. By correlating structural changes with nucleotide exchange activity for each construct, we uncovered new auto-regulatory roles for the ubiquitin binding domains and the Linker connecting those domains to the catalytic core of Rabex5. We further provide evidence that enhanced dynamics in the catalytic core are linked to catalysis. Our results suggest a more complex auto-regulation mechanism than previously thought and imply that ubiquitin binding serves not only to position Rabex5 but to also control its Rab5 GEF activity through allosteric structural alterations.

The devastating effects and incurable nature of hereditary and sporadic retinal diseases such as Stargardt disease, age-related macular degeneration or retinitis pigmentosa urgently require the development of new therapeutic strategies. Additionally, a high prevalence of retinal toxicities is becoming more and more an issue of novel targeted therapeutic agents. Ophthalmologic drug development, to date, largely relies on animal models, which often do not provide results that are translatable to human patients. Hence, the establishment of sophisticated human tissue-based in vitro models is of upmost importance. The discovery of self-forming retinal organoids (ROs) derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) is a promising approach to model the complex stratified retinal tissue. Yet, ROs lack vascularization and cannot recapitulate the important physiological interactions of matured photoreceptors and the retinal pigment epithelium (RPE). In this study, we present the retina-on-a-chip (RoC), a novel microphysiological model of the human retina integrating more than seven different essential retinal cell types derived from hiPSCs. It provides vasculature-like perfusion and enables, for the first time, the recapitulation of the interaction of mature photoreceptor segments with RPE in vitro. We show that this interaction enhances the formation of outer segment-like structures and the establishment of in vivo-like physiological processes such as outer segment phagocytosis and calcium dynamics. In addition, we demonstrate the applicability of the RoC for drug testing, by reproducing the retinopathic side-effects of the anti-malaria drug chloroquine and the antibiotic gentamicin. The developed hiPSC-based RoC has the potential to promote drug development and provide new insights into the underlying pathology of retinal diseases.

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.

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/.

The use of synthetic RNA for research purposes as well as RNA-based therapy and vaccination has gained increasing importance. Given the anatomical seclusion of the eye, small interfering RNA (siRNA)-induced gene silencing bears great potential for targeted reduction of pathological gene expression that may allow rational treatment of chronic eye diseases in the future. However, there is yet an unmet need for techniques providing safe and efficient siRNA delivery to the retina. We used magnetic nanoparticles (MNPs) and magnetic force (Reverse Magnetofection) to deliver siRNA/MNP complexes into retinal explant tissue, targeting valosin-containing protein (VCP) previously established as a potential therapeutic target for autosomal dominant retinitis pigmentosa (adRP). Safe and efficient delivery of VCP siRNA was achieved into all retinal cell layers of retinal explants from the RHO P23H rat, a rodent model for adRP. No toxicity or microglial activation was observed. VCP silencing led to a significant decrease of retinal degeneration. Reverse Magnetofection thus offers an effective method to deliver siRNA into retinal tissue. Used in combination with retinal organotypic explants, it can provide an efficient and reliable preclinical test platform of RNA-based therapy approaches for ocular diseases.

Abstract The mitotic spindle assembly checkpoint (SAC) maintains genome stability and marks an important target for antineoplastic therapies. However, it has remained unclear how cells execute cell fate decisions under conditions of SAC‐induced mitotic arrest. Here, we identify USP9X as the mitotic deubiquitinase of the X‐linked inhibitor of apoptosis protein (XIAP) and demonstrate that deubiquitylation and stabilization of XIAP by USP9X lead to increased resistance toward mitotic spindle poisons. We find that primary human aggressive B‐cell lymphoma samples exhibit high USP9X expression that correlate with XIAP overexpression. We show that high USP9X/XIAP expression is associated with shorter event‐free survival in patients treated with spindle poison‐containing chemotherapy. Accordingly, aggressive B‐cell lymphoma lines with USP9X and associated XIAP overexpression exhibit increased chemoresistance, reversed by specific inhibition of either USP9X or XIAP. Moreover, knockdown of USP9X or XIAP significantly delays lymphoma development and increases sensitivity to spindle poisons in a murine Eμ‐Myc lymphoma model. Together, we specify the USP9X–XIAP axis as a regulator of the mitotic cell fate decision and propose that USP9X and XIAP are potential prognostic biomarkers and therapeutic targets in aggressive B‐cell lymphoma.

Mutations in proteins that localize to primary cilia cause devastating diseases, yet the primary cilium is a poorly understood organelle. Here the authors use interaction proteomics to identify a network of human ciliary proteins that provides new insights into several biological processes and diseases.

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.

Ciliary neurotrophic factor (CNTF) is one of the most studied neuroprotective agents with acknowledged potential in treating diseases of the posterior eye segment. Although its efficacy and mechanisms of action in the retina have been studied extensively, it is still not comprehensively understood which retinal cells mediate the therapeutic effects of CNTF. As with therapeutic proteins in general, it is poorly elucidated whether exogenous CNTF administered into the vitreous can enter and distribute into the retina and hence reach potentially responsive target cells. Here, we have characterized our purified recombinant human CNTF (rhCNTF), studied the protein’s in vitro bioactivity in a cell-based assay, and evaluated the thermodynamic and oligomeric status of the protein during storage. Biological activity of rhCNTF was further evaluated in vivo in an animal model of retinal degeneration. The retinal penetration and distribution of rhCNTF after 24 h was studied utilizing two ex vivo retina models. Based on our characterization findings, our rhCNTF is correctly folded and biologically active. Moreover, based on initial screening and subsequent follow-up, we identified two buffers in which rhCNTF retains its stability during storage. Whereas rhCNTF did not show photoreceptor preservative effect or improve the function of photoreceptors in vivo, this could possibly be due to the used disease model or the short duration of action with a single intravitreal injection of rhCNTF. On the other hand, the lack of in vivo efficacy was shown to not be due to distribution limitations; permeation into the retina was observed in both retinal explant models as in 24 h rhCNTF penetrated the inner limiting membrane, and being mostly observed in the ganglion cell layer, distributed to different layers of the neural retina. As rhCNTF can reach deeper retinal layers, in general, having direct effects on resident CNTF-responsive target cells is plausible.

Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases. Genome editing represents a promising emerging field in the treatment of monogenic disorders, as it aims to correct disease-causing mutations within the genome. Genome editing relies on highly specific endonucleases and the capacity of the cells to repair double-strand breaks (DSBs). As DSB pathways are cell-cycle dependent, their activity in postmitotic retinal neurons, with a focus on photoreceptors, needs to be assessed in order to develop therapeutic in vivo genome editing. Three DSB-repair pathways are found in mammalian cells: Non-homologous end joining (NHEJ); microhomology-mediated end joining (MMEJ); and homology-directed repair (HDR). While NHEJ can be used to knock out mutant alleles in dominant disorders, HDR and MMEJ are better suited for precise genome editing, or for replacing entire mutation hotspots in genomic regions. Here, we analyzed transcriptomic in vivo and in vitro data and revealed that HDR is indeed downregulated in postmitotic neurons, whereas MMEJ and NHEJ are active. Using single-cell RNA sequencing analysis, we characterized the dynamics of DSB repair pathways in the transition from dividing cells to postmitotic retinal cells. Time-course bulk RNA-seq data confirmed DSB repair gene expression in both in vivo and in vitro samples. Transcriptomic DSB repair pathway profiles are very similar in adult human, macaque, and mouse retinas, but not in ground squirrel retinas. Moreover, human-induced pluripotent stem-cell-derived neurons and retinal organoids can serve as well suited in vitro testbeds for developing genomic engineering approaches in photoreceptors. Our study provides additional support for designing precise in vivo genome-editing approaches via MMEJ, which is active in mature photoreceptors.

Human induced pluripotent stem cell (hiPSC)-derived organoids mimicking tissues and organs in vitro have advanced medical research, as they opened up new possibilities for in-depth basic research on human organ development as well as providing a human in vitro model for personalized therapeutic approaches. hiPSC-derived retinal organoids have proven to be of great value for modeling the human retina featuring a very similar cellular composition, layering, and functionality. The technically challenging imaging of three-dimensional structures such as retinal organoids has, however, raised the need for robust whole-organoid imaging techniques. To improve imaging of retinal organoids we optimized a passive clearing technique (PACT), which enables high-resolution visualization of fragile intra-tissue structures. Using cleared retinal organoids, we could greatly enhance the antibody labeling efficiency and depth of imaging at high resolution, thereby improving the three-dimensional microscopy output. In that course, we were able to identify the spatial morphological shape and organization of, e.g., photoreceptor cells and bipolar cell layers. Moreover, we used the synaptic protein CtBP2/Ribeye to visualize the interconnection points of photoreceptor and bipolar cells forming the retinal-specific ribbon synapses.

The seven-transmembrane receptor Smoothened (Smo) activates all Hedgehog (Hh) signaling by translocation into the primary cilia (PC), but how this is regulated is not well understood. Here we show that Pitchfork (Pifo) and the G protein-coupled receptor associated sorting protein 2 (Gprasp2) are essential components of an Hh induced ciliary targeting complex able to regulate Smo translocation to the PC. Depletion of Pifo or Gprasp2 leads to failure of Smo translocation to the PC and lack of Hh target gene activation. Together, our results identify a novel protein complex that is regulated by Hh signaling and required for Smo ciliary trafficking and Hh pathway activation.

PURPOSE:Hypothermia has been shown to be neuroprotective in the therapy of ischemic stroke in the brain. To date no studies exist on the level of the inner retina and it is unclear if hypothermia would prolong the ischemic tolerance time of retinal ganglion cells, which are decisive in many ischemic retinopathies. METHODS:Bovine eyes were enucleated and stored either at 21°C or 37°C for 100 or 340 minutes, respectively. Afterwards the globes were dissected, the retina was prepared and either the spontaneous ganglion cell responses were measured or the retina was incubated as an organotypic culture for additional 24 hours. After incubation the retina was either processed for histology (H&E and DAPI staining) or real-time PCR (Thy-1 expression) was performed. RESULTS:Hypothermia prolonged ganglion cell survival up to 340 minutes under ischemic conditions. In contrast to eyes kept at 37°C the eyes stored at 21°C still showed spontaneous ganglion cell spiking (56.8% versus 0%), a 5.8 fold higher Thy-1 mRNA expression (not significant, but a trend) and a preserved retinal structure after 340 minutes of ischemia. CONCLUSION:Hypothermia protects retinal ganglion cells against ischemia and prolongs their ischemic tolerance time.