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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.

Protein-protein interaction analysis is an important tool to elucidate the function of proteins and protein complexes as well as their dynamic behavior. To date, the analysis of tissue- or even cell- or compartment-specific protein interactions is still relying on the availability of specific antibodies suited for immunoprecipitation. Here, we aimed at establishing a method that allows identification of protein interactions and complexes from intact tissues independent of specific, high affinity antibodies used for protein pull-down and isolation. Tagged bait proteins were expressed in human HEK293T cells and residual interactors removed by SDS. The resulting tag-fusion protein was then used as bait to pull proteins from tissue samples. Tissue-specific interactions were reproducibly identified from porcine retina as well as from retinal pigment epithelium using the ciliary protein lebercilin as bait. Further, murine heart-specific interactors of two gene products of the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type 1 (cGK1) were investigated. Here, specific interactions were associated with the cGK1α and β gene products, that differ only in their unique amino-terminal region comprising about 100 aa. As such, the new protocol provides a fast and reliable method for tissue-specific protein complex analysis which is independent of the availability or suitability of antibodies for immunoprecipitation. SIGNIFICANCE: Protein-protein interaction in the functional relevant tissue is still difficult due to the dependence on specific antibodies or bait production in bacteria or insect cells. Here, the tagged protein of interest is produced in a human cell line and bound proteins are gently removed using SDS. Because applying the suitable SDS concentration is a critical step, different SDS solutions were tested to demonstrate their influence on interactions and the clean-up process. The established protocol enabled a tissue-specific analysis of the ciliary proteins lebercilin and TMEM107 using pig eyes. In addition, two gene products of the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type 1 showed distinct protein interactions in mouse heart tissue. With the easy, fast and cheap protocol presented here, deep insights in tissue-specific and functional relevant protein complex formation is possible., (Copyright © 2020. Published by Elsevier B.V.)

Nephrotic syndrome is characterized by urinary excretion of plasma proteases or proteasuria. There is a lack of data on the quantity, activity status and identity of these aberrantly filtered proteases. We established a fluorescence-based substrate assay to quantify protease activity in urine samples from healthy and nephrotic humans and mice. Protease class activity was determined after addition of specific inhibitors. Individual proteases were identified by tandem mass spectrometry (MS/MS). In spot urine samples from 10 patients with acute nephrotic syndrome of various etiology, urinary protease activity was significantly increased compared to that of healthy persons (753 ± 178 vs. 244 ± 65 relative units, p < 0.05). The corresponding proteases were highly sensitive to inhibition by the serine protease inhibitors AEBSF (reduction by 85 ± 6% and 72 ± 8%, respectively) and aprotinin (83 ± 9% vs. 25 ± 6%, p < 0.05). MS/MS of all urinary proteins or after AEBSF purification showed that most of them were active serine proteases from the coagulation and complement cascade. These findings were recapitulated in mice, pointing to a similar pathophysiology. In conclusion, nephrotic syndrome leads to increased urinary excretion of active plasma proteases which can be termed proteasuria. Serine proteases account for the vast majority of urinary protease activity in health and nephrotic syndrome. SIGNIFICANCE STATEMENT: In this study, we found that nephrotic urine samples of humans and mice have a significantly increased protease activity compared to healthy urine samples, using a universal pentapeptide substrate library. This was driven by increased excretion of aprotinin-sensitive serine proteases. With tandem mass spectrometry, we provide a comprehensive and systematic overview of all urinary proteases or the "urine proteasome". We identified renally expressed proteases in health and addition of proteases from the coagulation and complement cascade in the nephrotic state. These results set the basis to study the role of urinary proteases at both health and nephrotic syndrome to find diagnostic markers of renal disease as well as possible therapeutic targets., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Preliminary investigations of human corneal tissues from coronavirus disease 2019 (COVID-19) cadaveric donors indicated that no severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is present. Current eye banking guidelines do not recommend any type of routine testing for SARS-CoV‑2 RNA in post-mortem donor tissue. This is partly based on factors that can influence the test results of the reverse transcription polymerase chain reaction (RT-PCR).

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 m Aconitase, 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., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors.)

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.

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.

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.

RING finger protein 11 (RNF11) is an evolutionary conserved Really Interesting New Gene E3 ligase that is overexpressed in several human tumours. Although several reports have highlighted its involvement in crucial cellular processes, the mechanistic details underlying its function are still poorly understood. Utilizing stable isotope labelling by amino acids in culture (SILAC)-based proteomics analysis, we identified 51 proteins that co-immunoprecipitate with wild-type RNF11 and/or with its catalytically inactive mutant. We focused our attention on the interaction of RNF11 with Ankyrin repeat domain-containing protein 13 (ANKRD13)s family. Members of the ANKRD13 family contain ubiquitin-interacting motifs (UIM) that recognize the Lys-63-linked ubiquitin (Ub) chains appended to Epidermal growth factor receptor (EGFR) soon after ligand binding. We show that ANKRD13A, ANKRD13B and ANKRD13D form a complex with RNF11 in vivo and that the UIMs are required for complex formation. However, at odds with the conventional UIM binding mode, Ub modification of RNF11 is not required for the interaction with ANKRD13 proteins. We also show that the interaction between ANKRD13A and RNF11 is modulated by the EGF stimulus and that a complex formed by ANKRD13A, RNF11 and activated EGFR is transiently assembled in the early phases of receptor endocytosis. Moreover, loss of function of the E3 ligases Itchy E3 ubiquitin-protein ligase (ITCH) or RNF11, respectively, abrogates or increases the ubiquitination of endogenous ANKRD13A, affecting its ability to bind activated EGFR. We propose a model whereby the ANKRD13 proteins act as molecular scaffolds that promote the transient formation of a complex between the activated EGFR and the E3 ligases ITCH and RNF11. By regulating the ubiquitination status of ANKRD13A and consequently its endocytic adaptor function, RNF11 promotes sorting of the activated EGFR for lysosomal degradation., (© 2020 Federation of European Biochemical Societies.)

Preliminary investigations of human corneal tissues from coronavirus disease 2019 (COVID-19) cadaveric donors indicated that no severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is present. Current eye banking guidelines do not recommend any type of routine testing for SARS-CoV‑2 RNA in post-mortem donor tissue. This is partly based on factors that can influence the test results of the reverse transcription polymerase chain reaction (RT-PCR).

The SARS-CoV‑2 causes a disease spectrum that includes asymptomatic and mildly symptomatic infections with subclinical manifestations but which can nevertheless still be potentially contagious. Evidence from SARS-CoV‑2 infected macaque monkeys and from studies with seasonal coronaviruses suggests that the infection is likely to produce an immunity that is protective for a certain period of time. Available test methods enable a high degree of reliability, e.g. if high-quality serological methods are combined. Although individual test results have to be interpreted with caution, serosurveillance in a tertiary eye care center and large eye research institute can reduce anxiety and provide clarity regarding the actual number of (unreported) SARS-CoV‑2 infections.

Coronaviruses are a genetically highly variable family of viruses that infect vertebrates and have succeeded in infecting humans many times by overcoming the species barrier. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which initially appeared in China at the end of 2019, exhibits a high infectivity and pathogenicity compared to other coronaviruses. As the viral coat and other viral components are recognized as being foreign by the immune system, this can lead to initial symptoms, which are induced by the very efficiently working immune defense system via the respiratory epithelium. During severe courses a systemically expressed proinflammatory cytokine storm and subsequent changes in the coagulation and complement systems can occur. Virus-specific antibodies, the long-term expression of which is ensured by the formation of B memory cell clones, generate a specific immune response that is also detectable in blood (seroconversion). Specifically effective cytotoxic CD8+ T‑cell populations are also formed, which recognize viral epitopes as pathogen-specific patterns in combination with MHC presentation on the cell surface of virus-infected cells and destroy these cells. At the current point in time it is unclear how regular, robust and durable this immune status is constructed. Experiences with other coronavirus infections (SARS and Middle East respiratory syndrome, MERS) indicate that the immunity could persist for several years. Based on animal experiments, already acquired data on other coronavirus types and plausibility assumptions, it can be assumed that seroconverted patients have an immunity of limited duration and only a very low risk of reinfection. Knowledge of the molecular mechanisms of viral cycles and immunity is an important prerequisite for the development of vaccination strategies and development of effective drugs.

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.

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., Competing Interests: Tarek Bayyoud, MD: Conflicts of interest: none. Angelika Iftner: Conflicts of interest: none. Thomas Iftner, PhD: Conflicts of interest: none. Karl Ulrich Bartz-Schmidt, MD: Conflicts of interest: none. Marius Ueffing, PhD: Conflicts of interest: none. Michael Schindler, PhD: Conflicts of interest: none. Sebastian Thaler, MD: Conflicts of interest: none., (© 2020 The Authors.)

Polymorphisms in the Complement Factor H (CFH) gene, coding for the Factor H protein (FH), can increase the risk for age-related macular degeneration (AMD). AMD-associated CFH risk variants, Y402H in particular, impair FH function leading to complement overactivation. Whether this alone suffices to trigger AMD pathogenesis remains unclear. In AMD, retinal homeostasis is compromised due to the dysfunction of retinal pigment epithelium (RPE) cells. To investigate the impact of endogenous FH loss on RPE cell balance, we silenced CFH in human hTERT-RPE1 cells. FH reduction led to accumulation of C3, at both RNA and protein level and increased RPE vulnerability toward oxidative stress. Mild hydrogen-peroxide exposure in combination with CFH knock-down led to a reduction of glycolysis and mitochondrial respiration, paralleled by an increase in lipid peroxidation, which is a key aspect of AMD pathogenesis. In parallel, cell viability was decreased. The perturbations of energy metabolism were accompanied by transcriptional deregulation of several glucose metabolism genes as well as genes modulating mitochondrial stability. Our data suggest that endogenously produced FH contributes to transcriptional and metabolic homeostasis and protects RPE cells from oxidative stress, highlighting a novel role of FH in AMD pathogenesis.

Cilia are complex cellular protrusions consisting of hundreds of proteins. Defects in ciliary structure and function, many of which have not been characterised molecularly, cause ciliopathies: a heterogeneous group of human syndromes. Here, we report on the FOXJ1 target gene Cfap206 , orthologues of which so far have only been studied in Chlamydomonas and Tetrahymena In mouse and Xenopus , Cfap206 was co-expressed with and dependent on Foxj1 CFAP206 protein localised to the basal body and to the axoneme of motile cilia. In Xenopus crispant larvae, the ciliary beat frequency of skin multiciliated cells was enhanced and bead transport across the epidermal mucociliary epithelium was reduced. Likewise, Cfap206 knockout mice revealed ciliary phenotypes. Electron tomography of immotile knockout mouse sperm flagella indicated a role in radial spoke formation reminiscent of FAP206 function in Tetrahymena Male infertility, hydrocephalus and impaired mucociliary clearance of the airways in the absence of laterality defects in Cfap206 mutant mice suggests that Cfap206 may represent a candidate for the subgroup of human primary ciliary dyskinesias caused by radial spoke defects., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Importance: Achromatopsia linked to variations in the CNGA3 gene is associated with day blindness, poor visual acuity, photophobia, and involuntary eye movements owing to lack of cone photoreceptor function. No treatment is currently available., Objective: To assess safety and vision outcomes of supplemental gene therapy with adeno-associated virus (AAV) encoding CNGA3 (AAV8.CNGA3) in patients with CNGA3-linked achromatopsia., Design, Setting, and Participants: This open-label, exploratory nonrandomized controlled trial tested safety and vision outcomes of gene therapy vector AAV8.CNGA3 administered by subretinal injection at a single center. Nine patients (3 per dose group) with a clinical diagnosis of achromatopsia and confirmed biallelic disease-linked variants in CNGA3 were enrolled between November 5, 2015, and September 22, 2016. Data analysis was performed from June 6, 2017, to March 12, 2018., Intervention: Patients received a single unilateral injection of 1.0 × 1010, 5.0 × 1010, or 1.0 × 1011 total vector genomes of AAV8.CNGA3 and were followed up for a period of 12 months (November 11, 2015, to October 10, 2017)., Main Outcomes and Measures: Safety as the primary end point was assessed by clinical examination of ocular inflammation. Systemic safety was assessed by vital signs, routine clinical chemistry testing, and full and differential blood cell counts. Secondary outcomes were change in visual function from baseline in terms of spatial and temporal resolution and chromatic, luminance, and contrast sensitivity throughout a period of 12 months after treatment., Results: Nine patients (mean [SD] age, 39.6 [11.9] years; age range, 24-59 years; 8 [89%] male) were included in the study. Baseline visual acuity letter score (approximate Snellen equivalent) ranged from 34 (20/200) to 49 (20/100), whereas baseline contrast sensitivity log scores ranged from 0.1 to 0.9. All 9 patients underwent surgery and subretinal injection of AAV8.CNGA3 without complications. No substantial safety problems were observed during the 12-month follow-up period. Despite the congenital deprivation of cone photoreceptor-mediated vision in achromatopsia, all 9 treated eyes demonstrated some level of improvement in secondary end points regarding cone function, including mean change in visual acuity of 2.9 letters (95% CI, 1.65-4.13; P = .006, 2-sided t test paired samples). Contrast sensitivity improved by a mean of 0.33 log (95% CI, 0.14-0.51 log; P = .003, 2-sided t test paired samples)., Conclusions and Relevance: Subretinal gene therapy with AAV8.CNGA3 was not associated with substantial safety problems and was associated with cone photoreceptor activation in adult patients, as reflected by visual acuity and contrast sensitivity gains., Trial Registration: ClinicalTrials.gov Identifier: NCT02610582.

The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the protein encoded by the photoreceptor-specific gene C2orf71 , which is mutated in inherited retinal dystrophy (RP54). We demonstrate that C2orf71/PCARE (photoreceptor cilium actin regulator) can interact with the Arp2/3 complex activator WASF3, and efficiently recruits it to the primary cilium. Ectopic coexpression of PCARE and WASF3 in ciliated cells results in the remarkable expansion of the ciliary tip. This process was disrupted by small interfering RNA (siRNA)-based down-regulation of an actin regulator, by pharmacological inhibition of actin polymerization, and by the expression of PCARE harboring a retinal dystrophy-associated missense mutation. Using human retinal organoids and mouse retina, we observed that a similar actin dynamics-driven process is operational at the base of the photoreceptor OS where the PCARE module and actin colocalize, but which is abrogated in Pcare -/- mice. The observation that several proteins involved in retinal ciliopathies are translocated to these expansions renders it a potential common denominator in the pathomechanisms of these hereditary disorders. Together, our work suggests that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane at the initiation of new outer segment disk formation., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Malfunctions of motile cilia cause a variety of developmental defects and diseases in humans and animal model organisms. Defects include impaired mucociliary clearance of the airways, sperm immotility, hydrocephalus and organ laterality. Here, we characterize the evolutionary conserved Cfap43 gene by loss-of-function experiments in the mouse and the frog Xenopus laevis. Cfap43 is expressed in tissues carrying motile cilia and acts as a target gene of the transcription factor FOXJ1, which is essential for the induction of motile ciliogenesis. We show that CFAP43, a protein of unknown biochemical function, localizes to the ciliary axoneme. CFAP43 is involved in the regulation of the beating frequency of tracheal cilia and loss of CFAP43 causes severe mucus accumulation in the nasal cavity. Likewise, morphant and crispant frog embryos revealed impaired function of motile cilia of the larval epidermis, a model for airway mucociliary epithelia. CFAP43 participates in the formation of flagellar axonemes during spermatogenesis as mice mutant for Cfap43 display male infertility, consistent with observations in male sterile patients. In addition, mice mutant for Cfap43 display early onset hydrocephalus. Together, these results confirm the role of CFAP43 in the male reproductive tract and pinpoint additional functions in airway epithelia mucus clearance and brain development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Regulation of mitosis secures cellular integrity and its failure critically contributes to the development, maintenance, and treatment resistance of cancer. In yeast, the dual phosphatase Cdc14 controls mitotic progression by antagonizing Cdk1-mediated protein phosphorylation. By contrast, specific mitotic functions of the mammalian Cdc14 orthologue CDC14B have remained largely elusive. Here, we find that CDC14B antagonizes CDK1-mediated activating mitotic phosphorylation of the deubiquitinase USP9X at serine residue 2563, which we show to be essential for USP9X to mediate mitotic survival. Starting from an unbiased proteome-wide screening approach, we specify Wilms' tumor protein 1 (WT1) as the relevant substrate that becomes deubiquitylated and stabilized by serine 2563-phosphorylated USP9X in mitosis. We further demonstrate that WT1 functions as a mitotic transcription factor and specify CXCL8/IL-8 as a target gene of WT1 that conveys mitotic survival. Together, we describe a ubiquitin-dependent signaling pathway that directs a mitosis-specific transcription program to regulate mitotic survival.

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., Competing Interests: The authors declare no conflict of interest.

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 (KRAS G13D ) 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 KRAS G13D (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.

Purpose: Choroideremia (CHM) is a rare inherited retinal degeneration resulting from mutation of the CHM gene, which results in absence of functional Rab escort protein 1 (REP1). We evaluated retinal gene therapy with an adeno-associated virus vector that used to deliver a functional version of the CHM gene (AAV2-REP1)., Methods: THOR (NCT02671539) is a Phase 2, open-label, single-center, randomized study. Six male patients (51-60 years) with CHM received AAV2-REP1, by a single 0.1-mL subretinal injection of 10 genome particles during vitrectomy. Twelve-month data are reported., Results: In study eyes, 4 patients experienced minor changes in best-corrected visual acuity (-4 to +1 Early Treatment Diabetic Retinopathy Study [ETDRS] letters); one gained 17 letters and another lost 14 letters. Control eyes had changes of -2 to +4 letters. In 5/6 patients, improvements in mean (95% confidence intervals) retinal sensitivity (2.3 [4.0] dB), peak retinal sensitivity (2.8 [3.5] dB), and gaze fixation area (-36.1 [66.9] deg) were recorded. Changes in anatomical endpoints were similar between study and control eyes. Adverse events were consistent with the surgical procedure., Conclusion: Gene therapy with AAV2-REP1 can maintain, and in some cases, improve, visual acuity in CHM. Longer term follow-up is required to establish whether these benefits are maintained.

Purpose: Mutations in the CRB1 gene cause early-onset retinal degeneration (EORD). Clinical disease progression markers, such as visual fields or electrophysiology, are not reliably measurable in most patients to follow the retinal function in patients with CRB1 -mutations., Methods: Ten patients (five females, five males; age 22-56 years) with EORD caused by CRB1 mutations were examined in a cross-sectional manner using best corrected visual acuity (BCVA), perimetry, full-field and multifocal electroretinography, full-field stimulus threshold (FST), and pupillography to red and blue light. Disease duration was defined as the difference between the age at the first symptoms to the age at examination in years., Results: BCVA was quantifiable in six patients and ranged from light perception to 20/50. The visual field was measurable only in three patients who had the shortest disease duration. Full-field and multifocal electroretinography were not measurable in any patient. FST to blue and red light were measurable in all patients except the one with the longest disease duration; the thresholds ranged from -16.7 to 1.5 dB for red light and from -40.2 to 2.5 dB for blue light (0 dB = 0.01 cd.s/m 2 ) and showed correlations with disease duration ( r = 0.87 for blue, r = 0.65 for red, r = 0.8 for blue-red difference). The maximal relative pupil constriction amplitude (MRA) showed low or no correlations with disease duration ( r = -0.55 for blue, r = -0.3 for red light); the blue-red difference in the post-illumination pupil responses (PIPR) showed no correlation with disease duration ( r = -0.05). Compared to healthy eyes, the MRA to red and blue light was significantly decreased ( P < 0.001) and the blue-red PIPR difference was significantly increased ( P = 0.003)., Conclusions: FST features a valid clinical marker in late-stage early-onset retinitis pigmentosa caused by CRB1 mutations correlating with disease duration. This indicates the potential as a progression marker of disease. The pupil responses to full-field chromatic stimuli show significant differences from the normal population: the remaining responses, although reduced, indicate a partially preserved inner retinal function despite severe photoreceptor dysfunction., Translational Relevance: The functional measurements presented in this study present a valid clinical progression marker in late-stage early onset retinitis pigmentosa caused by biallelic CRB1 mutations. Additionally, they can be used as outcome measures for safety and efficacy in clinical therapy trials., (Copyright 2019 The Authors.)

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., Competing Interests: JL, SS, AC, GG, FR, MG, VA, MH, YK, MU, AL, CG, MZ No competing interests declared, (© 2019, Lauer et al.)

Importance: Choroideremia (CHM) is a rare, degenerative, genetic retinal disorder resulting from mutation of the CHM gene, leading to an absence of functional ras-associated binding escort protein 1 (REP1). There is currently no approved treatment for CHM., Objective: To assess the safety and efficacy of retinal gene therapy with an adeno-associated virus vector (AAV2) designed to deliver a functional version of the CHM gene (AAV2-REP1) for treatment of patients with choroideremia., Design, Setting, and Participants: Tübingen Choroideremia Gene Therapy (THOR) was a single-center, phase 2, open-label randomized clinical trial. Data were collected from January 11, 2016, to February 26, 2018. Twenty-four-month data are reported for 6 men with a molecularly confirmed diagnosis of CHM. Intention-to-treat analysis was used., Interventions: Patients received AAV2-REP1 by a single, 0.1-mL subretinal injection of 1011 genome particles during vitrectomy into 1 eye randomly assigned to receive treatment., Main Outcomes and Measures: Primary end point was change in best-corrected visual acuity (BCVA) on the Early Treatment Diabetic Retinopathy Study chart from baseline to month 24 in the treated eye vs the control eye. Secondary end points included microperimetry variables, change in fundus autofluorescence, and spectral-domain optical coherence tomographic evaluations from baseline to month 24 in the treated eye vs the control eye., Results: On enrollment, the mean (SD) age of the 6 men included in the study was 54.9 (4.1) years. The mean (SD) BCVA score was 60.3 (13.4) (approximately 20/63 Snellen equivalent) in the study eyes and 69.3 (20.6) (approximately 20/40 Snellen equivalent) in the control eyes. At 24 months, the BCVA change was 3.7 (7.5) in the treated eyes and 0.0 (5.1) in the control eyes (difference, 3.7; 95% CI, -7.2 to 14.5; P = .43). Mean change in retinal sensitivity was 10.3 (5.5) dB in the treated eyes and 9.7 (4.9) dB in the control eyes (difference, 0.6; 95% CI, -10.2 to 11.4; P = .74). A total of 28 adverse events were reported; all were consistent with the surgical procedure (eg, conjunctival hyperemia, foreign body sensation), and none were regarded as severe., Conclusions and Relevance: Among 6 participants, gene therapy with AAV2-REP1 was associated with maintenance or improvement of visual acuity, although no significant difference was found from control eyes. All safety issues were associated with the surgical procedure and none were judged severe. Continued investigations could more precisely define the efficacy and safety of gene therapy with AAV2-REP1 in CHM., Trial Registration: ClinicalTrials.gov identifier: NCT02671539.

Adhesion G protein-coupled receptors (ADGRs) have recently become a target of intense research. Their unique protein structure, which consists of a G protein-coupled receptor combined with long adhesive extracellular domains, suggests a dual role in cell signaling and adhesion. Despite considerable progress in the understanding of ADGR signaling over the past years, the knowledge about ADGR protein networks is still limited. For most receptors, only a few interaction partners are known thus far. We aimed to identify novel ADGR-interacting partners to shed light on cellular protein networks that rely on ADGR function. For this, we applied affinity proteomics, utilizing tandem affinity purifications combined with mass spectrometry. Analysis of the acquired proteomics data provides evidence that ADGRs not only have functional roles at synapses but also at intracellular membranes, namely at the endoplasmic reticulum, the Golgi apparatus, mitochondria, and mitochondria-associated membranes (MAMs). Specifically, we found an association of ADGRs with several scaffold proteins of the membrane-associated guanylate kinases family, elementary units of the γ-secretase complex, the outer/inner mitochondrial membrane, MAMs, and regulators of the Wnt signaling pathways. Furthermore, the nuclear localization of ADGR domains together with their physical interaction with nuclear proteins and several transcription factors suggests a role of ADGRs in gene regulation., (© 2019 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals, Inc. on behalf of New York Academy of Sciences.)

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., Competing Interests: KA, CP, JH, SB, JR, JC, MN, VC, LA, WH, NS, KS, MU, SL, PL No competing interests declared, (© 2019, Achberger et al.)

Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly in the developed world. While treatment is effective for the neovascular or "wet" form of AMD, no therapy is successful for the non-neovascular or "dry" form. Here we discuss the current knowledge on dry AMD pathobiology and propose future research directions that would expedite the development of new treatments. In our view, these should emphasize system biology approaches that integrate omic, pharmacological, and clinical data into mathematical models that can predict disease onset and progression, identify biomarkers, establish disease causing mechanisms, and monitor response to therapy.

Background: Activating mutations in KRAS frequently occur in colorectal cancer (CRC) patients, leading to resistance to EGFR-targeted therapies., Methods: To better understand the cellular reprogramming which occurs in mutant KRAS cells, we have undertaken a systems-level analysis of four CRC cell lines which express either wild type (wt) KRAS or the oncogenic KRAS G13D allele (mtKRAS)., Results: RNAseq revealed that genes involved in ribosome biogenesis, mRNA translation and metabolism were significantly upregulated in mtKRAS cells. Consistent with the transcriptional data, protein synthesis and cell proliferation were significantly higher in the mtKRAS cells. Targeted metabolomics analysis also confirmed the metabolic reprogramming in mtKRAS cells. Interestingly, mtKRAS cells were highly transcriptionally responsive to EGFR activation by TGFα stimulation, which was associated with an unexpected downregulation of genes involved in a range of anabolic processes. While TGFα treatment strongly activated protein synthesis in wtKRAS cells, protein synthesis was not activated above basal levels in the TGFα-treated mtKRAS cells. This was likely due to the defective activation of the mTORC1 and other pathways by TGFα in mtKRAS cells, which was associated with impaired activation of PKB signalling and a transient induction of AMPK signalling., Conclusions: We have found that mtKRAS cells are substantially rewired at the transcriptional, translational and metabolic levels and that this rewiring may reveal new vulnerabilities in oncogenic KRAS CRC cells that could be exploited in future.

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., Competing Interests: The commercial affiliation of S.F. to La Roche AG does not alter our adherence to PLOS ONE policies on sharing data and materials.

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/., Competing Interests: The authors have declared that no competing interests exist.

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.

Cone photoreceptor loss is the main cause of color blindness and loss of visual acuity in patients suffering from inherited cone dystrophies. Despite the crucial role of cones in everyday life, knowledge on mechanisms of cone cell death and the identification of potential targets for the preservation or delay of cone loss are scarce. Recent findings have shown that excessive histone deacetylase (HDAC) activity is associated with both primary rod and primary cone degeneration. Importantly, pharmacological inhibition of HDAC activity in vivo at the onset of cone degeneration offers a prolonged protection of cones in a mouse model of inherited cone degeneration (cpfl1). In this study, we evaluated the potential of trichostatin A (TSA), a pan-HDAC inhibitor, to prevent cone cell death at a later stage of degeneration in the cpfl1 model. We demonstrate that a single intravitreal TSA injection protected the cpfl1 cones even when administered after the onset of degeneration. In addition, the TSA treatment significantly improved aberrant cone nucleokinesis present in the cpfl1 retina. These results highlight the feasibility of targeted cone neuroprotection in vivo even at later disease stages of inherited cone dystrophies.

Purpose: To establish a robust workflow for combined mass spectrometry-based analysis of metabolites and proteins in tear fluid with regard to clinical applicability., Methods: Tear fluid was taken from 12 healthy volunteers at different time points using specially designed Schirmer strips. Following the liquid extraction of metabolites from standardized punches, the remaining material was processed for bottom-up proteomics. Targeted metabolite profiling was performed adapting a metabolomics kit, which targets 188 metabolites from four different analyte classes. Proteomics was performed of the identical samples targeting 15 tear proteins relevant to ocular health., Results: Sixty metabolites could be consistently determined in all tear samples (98 metabolites were detectable in average) covering acylcarnitines, amino acids, biogenic amines, and glycerophospholipids. Following normalization, the majority of metabolites exhibited intraindividual variances of less than 20%, both regarding different times of sampling, and the individual eye. The targeted analysis of tear proteins revealed a mean intraindividual variation of 23% for the three most abundant proteins. Even extreme differences in tear secretion rates resulted in interindividual variability below 30% for 65 metabolites and two proteins., Conclusions: The newly established workflow can be used for combined targeted detection of metabolites and proteins in one punch of a Schirmer strip in a clinical setting., Translational Relevance: Our data about intra- and interindividual as well as intereye variation provide a valuable basis for the design of clinical studies, and for the applicability of multiplexed "omics" to well accessible tear fluid with regard to future routine use.

Nonlinear compression of laser pulses with tens of millijoule energy in a gas-filled multipass cell is a promising approach to realize a new generation of high average power femtosecond sources. For the first time, to the best of our knowledge, we demonstrate nonlinear broadening of pulses with about 18 mJ of energy at a 5 kHz repetition rate in an argon-filled Herriott cell and show compressibility from 1.3 ps to 41 fs. In addition to the large compression factor, the output beam has an outstanding quality and excellent spectral homogeneity. Furthermore, we discuss prospects to scale the energy to the 100 mJ level in the near future.

RAB28, a member of the RAS oncogene family, is a ubiquitous, farnesylated, small GTPase of unknown function present in photoreceptors and the retinal pigmented epithelium (RPE). Nonsense mutations of the human RAB28 gene cause recessive cone-rod dystrophy 18 (CRD18), characterized by macular hyperpigmentation, progressive loss of visual acuity, RPE atrophy, and severely attenuated cone and rod electroretinography (ERG) responses. In an attempt to elucidate the disease-causing mechanism, we generated Rab28 -/- mice by deleting exon 3 and truncating RAB28 after exon 2. We found that Rab28 -/- mice recapitulate features of the human dystrophy ( i.e. they exhibited reduced cone and rod ERG responses and progressive retina degeneration). Cones of Rab28 -/- mice extended their outer segments (OSs) to the RPE apical processes and formed enlarged, balloon-like distal tips before undergoing degeneration. The visual pigment content of WT and Rab28 -/- cones was comparable before the onset of degeneration. Cone phagosomes were almost absent in Rab28 -/- mice, whereas rod phagosomes displayed normal levels. A protein-protein interaction screen identified several RAB28-interacting proteins, including the prenyl-binding protein phosphodiesterase 6 δ-subunit (PDE6D) and voltage-gated potassium channel subfamily J member 13 (KCNJ13) present in the RPE apical processes. Of note, the loss of PDE6D prevented delivery of RAB28 to OSs. Taken together, these findings reveal that RAB28 is required for shedding and phagocytosis of cone OS discs., (© 2018 Ying et al.)

The intellectual disability gene, Sox11, encodes for a critical neurodevelopmental transcription factor with functions in precursor survival, neuronal fate determination, migration and morphogenesis. The mechanisms regulating SOX11's activity remain largely unknown. Mass spectrometric analysis uncovered that SOX11 can be post-translationally modified by phosphorylation. Here, we report that phosphorylatable serines surrounding the high-mobility group box modulate SOX11's transcriptional activity. Through Mass Spectrometry (MS), co-immunoprecipitation assays and in vitro phosphorylation assays followed by MS we verified that protein kinase A (PKA) interacts with SOX11 and phosphorylates it on S133. In vivo replacement of SoxC factors in developing adult-generated hippocampal neurons with SOX11 S133 phospho-mutants indicated that phosphorylation on S133 modulates dendrite development of adult-born dentate granule neurons, while reporter assays suggested that S133 phosphorylation fine-tunes the activation of select target genes. These data provide novel insight into the control of the critical neurodevelopmental regulator SOX11 and imply SOX11 as a mediator of PKA-regulated neuronal development.

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., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

Achromatopsia is an autosomal recessively inherited congenital defect characterized by a lack of cone photoreceptor function, leading to severely impaired vision. In this clinical study, achromatopsia patients were treated with a single subretinal injection of rAAV.hCNGA3 to restore cone function. The focus of this trial was on the safety of the treatment. After surgery, patients were monitored in eight extensive visits during the first year, followed by a 4-year follow-up period with annual visits. For essential complementation of the standard ophthalmological and systemic examinations, disease-specific methods were developed to assess the safety, efficacy, and patient-reported outcomes in this trial.

Pericentrin (Pcnt) is a multifunctional scaffold protein and mutations in the human PCNT gene are associated with several diseases, including ciliopathies. Pcnt plays a crucial role in ciliary development in olfactory receptor neurons, but its function in the photoreceptor-connecting cilium is unknown. We downregulated Pcnt in the retina ex vivo and in vivo via a virus-based RNA interference approach to study Pcnt function in photoreceptors. ShRNA-mediated knockdown of Pcnt impaired the development of the connecting cilium and the outer segment of photoreceptors, and caused a nuclear migration defect. In protein interaction screens, we found that the outer nuclear membrane protein Syne-2 (also known as Nesprin-2) is an interaction partner of Pcnt in photoreceptors. Syne-2 is important for positioning murine photoreceptor cell nuclei and for centrosomal migration during early ciliogenesis. CRISPR/Cas9-mediated knockout of Syne-2 in cell culture led to an overexpression and mislocalization of Pcnt and to ciliogenesis defects. Our findings suggest that the Pcnt-Syne-2 complex is important for ciliogenesis and outer segment formation during retinal development and plays a role in nuclear migration., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

CRISPR/Cas9-mediated gene editing allows manipulation of a gene of interest in its own chromosomal context. When applied to the analysis of protein interactions and in contrast to exogenous expression of a protein, this can be studied maintaining physiological stoichiometry, topology, and context. We have used CRISPR/Cas9-mediated genomic editing to investigate Cluap1/IFT38, a component of the intraflagellar transport complex B (IFT-B). Cluap1 has been implicated in human development as well as in cancer progression. Cluap1 loss of function results in early developmental defects with neural tube closure, sonic hedgehog signaling and left-right defects. Herein, we generated an endogenously tagged Cluap1 for protein complex analysis, which was then correlated to the corresponding interactome determined by ectopic expression. Besides IFT-B complex components, new interacting proteins like Ephrin-B1 and TRIP6, which are known to be involved in cytoskeletal arrangement and protein transport, were identified. With the identification of platelet-derived growth factor A (PDGFA) and coiled-coil domain-containing protein 6 (CCDC6) two new interactions were discovered, which link Cluap1 to ciliogenesis and cancer development. The CRISPR/Cas9-mediated knockout of Cluap1 revealed a new phenotype affecting the actin cytoskeleton. Together, these data provide first evidence for a role of Cluap1 not only for cilia assembly and maintenance but also for cytoskeletal rearrangement and intracellular transport processes., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

We present an efficient method for compressing sub-picosecond pulses at 200 W average power with 2 mJ pulse energy in a multipass cell filled with different gases. We demonstrate spectral broadening by more than a factor of five using neon, argon, and nitrogen as nonlinear media. The 210 fs input pulses are compressed down to 37 fs and 35 GW peak power with a beam quality factor of 1.3×1.5 at a power throughput of >93%. This concept represents an excellent alternative to hollow-core fiber-based compression schemes and optical parametric amplifiers (OPAs).

Purpose: To study longitudinal changes of anti-drug antibody (ADA) titers to recombinant adeno-associated virus serotype 8 (rAAV8) capsid epitopes in nonhuman primates (NHP) and patients., Methods: Three groups of six NHP each received subretinal injections (high dose: 1 × 1012 vector genomes [vg], low dose: 1 × 1011 vg, or vehicle only). Four additional animals received intravitreal injections of the high dose (1 × 1012 vg). Three patients received 1 × 1010 vg as subretinal injections. ELISA quantified ADA levels at baseline and 1, 2, 3, 7, 28, and 90 days after surgery in NHP and at baseline and 1, 3, and 6 months after surgery in patients., Results: Two out of 22 animals lacked ADA titers at baseline and developed low ADA titers toward the end of the study. Titers in the low-dose group stayed constant, while two of six animals from the high-dose group developed titers that rose beyond the range of the assay. All animals from the intravitreal control group showed a rise in ADA titer by day 7 that peaked at day 28. Preliminary data from the clinical trial (NCT02610582) show no humoral immune response in patients following subretinal delivery of 1 × 1010 vg., Conclusions: No significant induction of ADA occurred in NHP when mimicking the clinical scenario of subretinal delivery with a clinical-grade rAAV8 and concomitant immunosuppression. Likewise, clinical data showed no humoral immune response in patients. In contrast, intravitreal delivery was associated with a substantial humoral immune response. Subretinal delivery might be superior to an intravitreal application regarding immunologic aspects.

Inherited retinal degeneration (RD) is a devastating and currently untreatable neurodegenerative condition that leads to loss of photoreceptor cells and blindness. The vast genetic heterogeneity of RD, the lack of "druggable" targets, and the access-limiting blood-retinal barrier (BRB) present major hurdles toward effective therapy development. Here, we address these challenges ( i ) by targeting cGMP (cyclic guanosine- 3',5'-monophosphate) signaling, a disease driver common to different types of RD, and ( ii ) by combining inhibitory cGMP analogs with a nanosized liposomal drug delivery system designed to facilitate transport across the BRB. Based on a screen of several cGMP analogs we identified an inhibitory cGMP analog that interferes with activation of photoreceptor cell death pathways. Moreover, we found liposomal encapsulation of the analog to achieve efficient drug targeting to the neuroretina. This pharmacological treatment markedly preserved in vivo retinal function and counteracted photoreceptor degeneration in three different in vivo RD models. Taken together, we show that a defined class of compounds for RD treatment in combination with an innovative drug delivery method may enable a single type of treatment to address genetically divergent RD-type diseases., Competing Interests: Conflict of interest statement: A. Rentsch, H.-G.G., P.G., V.M., P.E., and F.P.-D. have filed for three patents on the synthesis and use of cGMP analogues (PCTWO2016/146669A1, PCT/EP2017/066113, and PCT/EP2017/071859) and have obtained a European Medicine Agency orphan drug designation for the use of CN03 for the treatment of retinitis pigmentosa (EU/3/15/1462). H.-G.G., P.G., V.M., P.E., and F.P.-D. are shareholders of, or have other financial interest in, the company Mireca Medicines, which intends to forward clinical testing of CN03., (Copyright © 2018 the Author(s). Published by PNAS.)

DNAAF1 (LRRC50) is a cytoplasmic protein required for dynein heavy chain assembly and cilia motility, and DNAAF1 mutations cause primary ciliary dyskinesia (PCD; MIM 613193). We describe four families with DNAAF1 mutations and complex congenital heart disease (CHD). In three families, all affected individuals have typical PCD phenotypes. However, an additional family demonstrates isolated CHD (heterotaxy) in two affected siblings, but no clinical evidence of PCD. We identified a homozygous DNAAF1 missense mutation, p.Leu191Phe, as causative for heterotaxy in this family. Genetic complementation in dnaaf1-null zebrafish embryos demonstrated the rescue of normal heart looping with wild-type human DNAAF1, but not the p.Leu191Phe variant, supporting the conserved pathogenicity of this DNAAF1 missense mutation. This observation points to a phenotypic continuum between CHD and PCD, providing new insights into the pathogenesis of isolated CHD. In further investigations of the function of DNAAF1 in dynein arm assembly, we identified interactions with members of a putative dynein arm assembly complex. These include the ciliary intraflagellar transport protein IFT88 and the AAA+ (ATPases Associated with various cellular Activities) family proteins RUVBL1 (Pontin) and RUVBL2 (Reptin). Co-localization studies support these findings, with the loss of RUVBL1 perturbing the co-localization of DNAAF1 with IFT88. We show that RUVBL1 orthologues have an asymmetric left-sided distribution at both the mouse embryonic node and the Kupffer's vesicle in zebrafish embryos, with the latter asymmetry dependent on DNAAF1. These results suggest that DNAAF1-RUVBL1 biochemical and genetic interactions have a novel functional role in symmetry breaking and cardiac development., (© The Author(s) 2017. Published by Oxford University Press.)

Photoreceptor cell death in inherited retinal degeneration is accompanied by over-activation of histone deacetylases (HDAC). Excessive HDAC activity is found both in primary rod degeneration (such as in the rd10 mouse) and in primary cone death, including the cone photoreceptor function loss 1 (cpfl1) mouse. We evaluated the potential of pharmacological HDAC inhibition to prevent photoreceptor degeneration in primary rod and cone degeneration. We show that a single in vivo treatment of cpfl1 mice with the HDAC inhibitor trichostatin A (TSA) resulted in a significant protection of cpfl1 mutant cones. Similarly, HDAC inhibition with the clinically approved HDAC inhibitor vorinostat (SAHA) resulted in a significant improvement of rod survival in rd10 retinal explant cultures. Altogether, these results highlight the feasibility of targeted neuroprotection in vivo and create hope to maintain vision in patients suffering from both rod and cone dystrophies.

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., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Ocular gene therapy has evolved rapidly into the clinical realm due to promising pre-clinical proof-of-concept studies, recognition of the high unmet medical need of blinding disorders, and the excellent safety profile of the most commonly used vector system, the adeno-associated virus (AAV). With several trials exposing subjects to AAV, investigators independently report about cases with clinically evident inflammation in treated eyes despite the concept of ocular immune privilege. Here, we provide a detailed analysis of innate and adaptive immune response to clinical-grade AAV8 in non-human primates and compare this to preliminary clinical data from a retinal gene therapy trial for CNGA3-based achromatopsia (ClinicalTrials.gov: 02610582)., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)