Rhodopsin-mediated autosomal dominant retinitis pigmentosa (RHO-adRP) is a progressive inherited retinal degenerative disorder currently lacking effective treatments. A recurrent 3-base pair deletion in the RHO gene, resulting in the loss of isoleucine at codon 255 or 256 (RHO∆I255 or RHO∆I256), has been identified in patients from the United Kingdom, Germany, Belgium, China, and Korea, suggesting a broad geographic distribution. This mutation leads to rhodopsin (RHO) misfolding, its retention in the endoplasmic reticulum (ER), and aggregation with wild-type (WT) RHO, ultimately triggering ER stress and photoreceptor degeneration. These aggregates are primarily cleared via the ER-associated degradation (ERAD) pathway, with valosin-containing protein (VCP) playing a key role in their retrotranslocation and proteasomal degradation. Pharmacological or genetic inhibition of VCP has shown neuroprotective effects in other models of adRP, but the poor aqueous solubility of VCP inhibitors and challenges in retinal drug delivery hinder clinical translation. To overcome these limitations, we evaluated and compared three VCP-targeted therapeutic strategies in Rho∆I255 knock-in mouse retinae: (1) small-molecule inhibitors (ML240, NMS-873) solubilized in DMSO, (2) ML240 encapsulated in monomethoxy-polyethylene glycol (mPEG)-cholane nanoparticles, and (3) small interfering RNA (siRNA) targeting VCP, delivered via magnetic nanoparticles. Neuroprotective effects were assessed in vitro in retinal explants and in vivo following intravitreal injection. Our findings provide the first evidence that VCP inhibition restores RHO trafficking to the outer segments and prevents photoreceptor cell death in the Rho∆I255 model. Among the three approaches, nanocarrier-encapsulated ML240 exhibited superior efficacy, enabling sustained drug delivery and enhanced photoreceptor protection. These results establish a preclinical proof-of-concept for nanocarrier-mediated VCP inhibition as a promising therapeutic strategy for RHO-adRP and potentially other ER-stress-related retinal degenerations.

In this study, the suitability of organotypic cultures of neonatal rat retina as a controlled ex vivo platform for the evaluation of intraocular microparticulate drug delivery systems is assessed, while also exploring their ability to identify formulation-dependent differences.Poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with glial cell line-derived neurotrophic factor (GDNF) and/or α-tocopherol acetate (VitE) were evaluated in short-term (7 days) and long-term (20 days) retinal explant cultures. Retinas from wild-type rats were explanted at postnatal day 9 or 10 and cultured with GDNF/VitE-loaded microspheres with different VitE content (PLGA-E20-GDNF and PLGA-E40-GDNF), VitE-loaded microspheres (PLGA-E20 and PLGA-E40), blank microspheres (PLGA), or left untreated. After exposures, retinal sections were analyzed by immunohistochemistry and TUNEL assay.In short-term cultures, both GDNF/VitE-loaded formulations significantly reduced photoreceptor cell death, whereas in long-term cultures a significant reduction of TUNEL-positive cells was only observed for the PLGA-E40-GDNF formulation. The formulation containing higher VitE content showed a greater neuroprotective effect despite lower GDNF loading, suggesting a possible contribution of VitE to the overall effect. No signs of cytotoxicity or structural damage were observed.The concordance between the present ex vivo findings and previously reported in vivo data in terms of neuroprotective efficacy and biocompatibility supports the use of organotypic retinal cultures as an intermediate ex vivo model to evaluate ocular drug delivery systems.

PURPOSE:PDE6A-associated retinitis pigmentosa (RP) is a rare inherited retinal disease leading to severe vision loss and blindness, with no available treatment. This study assessed the safety and vision outcomes of a gene therapy using an adeno-associated virus (AAV) vector encoding PDE6A (AAV8.hPDE6A). METHODS:In an open-label, non-randomised controlled phase I/IIa trial, nine patients with biallelic PDE6A variants received a single subretinal injection of AAV8.hPDE6A. Doses were either 1.0×10¹⁰ (n=6) or 5.0×10¹⁰ (n=3) total vector genomes. Safety was the primary endpoint, assessed via clinical examinations, laboratory analyses and optical coherence tomography imaging. Secondary outcomes included changes in visual function, such as best corrected visual acuity (BCVA), contrast sensitivity, colour perception, dark adaptation thresholds, visual fields, patient-reported outcomes and chromatic pupil campimetry over 1 year. RESULTS:The mean patient age was 40.1 years, with baseline BCVA ranging from 40 to 82 letters (0.9-0.1 logMAR). No systemic adverse events occurred, and most ocular events resolved without treatment. Persistent adverse events included small peripheral atrophic areas (n=2), disturbed colour discrimination (n=3), cataract (n=1), slight central retinal thinning (n=5) and moderate visual acuity loss (n=2, 1 in each dose group). BCVA, full-field stimulus thresholds and other visual function measures showed statistically non-significant changes, with a trend towards worsening of retinal sensitivity in the treated eyes. CONCLUSION:Subretinal gene therapy with AAV8.hPDE6A did not improve visual function over 1 year and posed risks, including central retinal thinning and visual acuity decline. This is in contrast to the safety and efficacy profile established in preclinical models.

BACKGROUND:For many patients with age-related macular degeneration, diabetic retinopathy and other partially monogenetic retinal diseases as well as for tumors of the eye that are relatively rare but are usually associated with profound consequences for affected patients, there is still no effective treatment available. Metastatic melanoma, for example, remains poorly predictable with respect to disease progression, response to treatment and outcome. This illustrates the urgent need for a deeper molecular understanding of the disease with the goal to develop novel therapeutic strategies. Liquid biopsies of the aqueous humor represent a promising possibility for molecular analyses in the eyes of patients. OBJECTIVE:A clinical and scientific perspective with respect to potential fields of applications of liquid biopsy proteomics in ophthalmology is presented. MATERIAL AND METHODS:A systematic literature search was carried out in PubMed and the personal experiences of the authors are presented. RESULTS AND CONCLUSION:Aqueous humor proteomics offer a plethora of potential applications in ophthalmology and could become a key factor in personalized ophthalmology. Potential areas of application include the selection of treatment based on the activated biological signalling pathways, the selection of patients for clinical trials as well as the diagnostics, prognosis estimation and monitoring of the response to treatment. In addition, it can be a valuable component of multimodal diagnostics and enable insights into neurodegenerative diseases, such as Alzheimer's or Parkinson's disease.

Overexpression of complement genes in the tumor microenvironment, including Factor H (FH), is a strong predictor of poor prognosis in multiple cancers. Its canonical functions in the bloodborne complement cascade, though, cannot explain this prognostic impact. Here, we demonstrate that FH operates within the intracellular space in fibroblasts and tumor cells. By transcriptomics approach in patient tumors, cellular and biochemical assays we revealed that the prognostic impact of FH overexpression is mediated mainly by its cell-intrinsic functions in tumor-promoting fibroblasts and malignant cells. Intranuclear FH interacts with the cell cycle-transcription factor E2F3. FH also promotes proliferation by lowering the nuclear p53 pool. Moreover, in ccRCC cancer cells, FH also regulates cytoskeleton organization and cell morphology, potentially, via interaction with the actin capping CapZ complex. Therefore, complement FH acts as a multitasking effector, regulating cell cycle and actin polymerization, challenging the paradigm of extracellular space-restricted functioning, considered for many complement proteins.

Leucine-rich repeat kinase 2 (LRRK2) not only plays a vital role in familial forms of Parkinson's disease (PD) but also represents a risk factor for idiopathic PD. Its multi-domain architecture enables fine-tuned regulation of its biological function by orchestrating intra- and inter-molecular interactions. Here, we present BioID proximity proteomes of LRRK2 that reveal new interactors, which we further characterize using a novel evolutionary and structural bioinformatics pipeline. Co-evolutionary analysis of the protein-protein interaction network identifies a structural and functional module enriched in cytoskeletal components associated with the centrosome and microtubules. In addition, structural modeling of binary interactions using AlphaFold-Multimer reveals distinct groups of interactors that engage LRRK2 in a manner dependent on specific conformations and epitopes. Furthermore, we identify distinct changes in the LRRK2 proximity proteome that are induced by the type I kinase inhibitor MLi-2 or by co-expression of the LRRK2 upstream effector RAB29. Depending on its activity state and conformation, these protein-protein interactions link LRRK2 to defined cellular sub-compartments, including centriolar satellites and vesicular sub-compartments.

Abstract Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss in older adults. It is characterised by early retinal pigment epithelium (RPE) dysfunction followed by progressive photoreceptor degeneration. Cigarette smoking is a major environmental risk factor for AMD, and hydroquinone (HQ), a redox-active cigarette smoke component, induces oxidative stress and apoptosis in RPE cells. To analyse how RPE stress contributes to photoreceptor degeneration, we employed a retinal co-culture model composed of human induced pluripotent stem cell-derived RPE (iPSC-RPE) cells in conjunction with porcine neuroretina explants. Exposure to HQ induced oxidative stress in iPSC-RPE cells as well as retinal photoreceptors (RPR), resulting in apoptosis, executed at least in part by caspase activation. Concomitantly, HQ caused endoplasmic reticulum (ER) stress (ERAD) in RPR followed by their degeneration, evidenced by reduced outer nuclear layer (ONL) rows and shortened RPR outer segments (OS). Based on earlier results, which suggest a perturbation of proteostasis due to HQ, we tested whether ML240, a bona fide inhibitor of valosin-containing protein (VCP), would influence the degree of degenerative activities. ML240 did not prevent HQ-induced apoptosis in iPSC-RPE cells. However, it significantly preserved photoreceptor integrity, retaining OS length and cone density in HQ-stressed co-cultures. Proteomic analysis suggested that ML240 reshapes stress response patterns of the HQ-exposed neuroretina, as evidenced by a reduction in ERAD-associated markers, increased levels of antioxidant response proteins, and the preservation of cytochrome c enrichment in photoreceptor inner segments, which indicates improved mitochondrial integrity consistent with the observed preservation of photoreceptor structure. Together, these findings establish the iPSC-RPE/neuroretina co-culture as a platform to analyse pathophysiological features of AMD, dissect cell type-specific retinal responses to environmental stress and test neuroprotective pharmacological approaches to protect photoreceptors in oxidative stress-associated retinal degeneration.

The deposition of circulating complement factor H-related (FHR) proteins in tissues around the body has been implicated in a series of complement-mediated diseases. However, the array of blood-borne binding partners with which they interact remains unclear. Here, we identify novel blood-borne binding partners of FHR proteins, firstly through preliminary untargeted immunoprecipitation and mass spectrometry, and subsequently validating direct interactions through solid-phase binding assays. We uncover direct interactions between FHRs and soluble immune mediators including complement C4 (C4), cathepsin G (CTSG), mannose-binding lectin 2 (MBL2), and platelet basic protein (PPBP). Functional assays show that FHR-1 and FHR-2 attenuate CTSG-mediated C3b degradation, while FHR-5 and FHL-1 appear to affect lectin pathway activation via MBL2 binding. These interactions suggest that FHRs perhaps confer activity not only through surface competition with factor H, but also via selective engagement with circulating ligands. Our findings expand the known FHR interactome and reveal potential new avenues for understanding FHR biology and targeting complement dysregulation in disease.

Objective: To describe the ophthalmic examination protocol within the German National Cohort (NAKO) / NAKO Gesundheitsstudie, to report the baseline profile of participants undergoing ophthalmological assessment, and to illustrate the potential of these data as a population-based open resource for artificial intelligence (AI) research in eye health. Design: Baseline analysis of ophthalmic data within the nationwide, population-based multicenter prospective NAKO study. Participants: 48,460 adults in the ophthalmological level 2 module of 205,053 adults enrolled in NAKO, aged 19-74 years, with mean age 48.9 ± 12.5 years and 52.7% male. Methods: All participants underwent standardized assessments of a wide range of biomedical examinations and detailed questionnaire-based data collection, including non-dilated color fundus imaging, visual acuity testing, recording of a brief ocular history. Ocular and systemic health measures were summarized using descriptive statistics. Fundus image quality and morphological features (e.g. cup-to-disc ratio, ateriole-to-venule-ratio) were assessed using open-source deep learning models. Standard deep learning architectures were trained on the fundus images to predict age, sex and blood pressure. Main Outcome Measures: Percentage of fundus images graded as good quality; mean absolute error for age and blood pressure prediction; accuracy for sex prediction. Results: The analysis includes 48,460 participants who successfully completed the level 2 ophthalmological baseline examination across 18 study sites in Germany. Mean visual acuity (logMAR) was 0.01 ± 0.20 (left eye) and 0.03 ± 0.21 (right eye). Self-reported ocular disease prevalence was 4.2% for cataract, 2.0% for glaucoma, and 0.9% for macular degeneration. 68.2% of fundus images were classified as gradable as a consensus of four deep learning-based quality grading models Morphological features such as cup-to-disc ratio and arteriole-to-venule-ratio showed systematic differences across age groups. Standard deep learning architectures showed comparative performance to the state-of-the-art for age, sex and blood pressure prediction (2.96 MAE for age prediction, 0.84 accuracy for sex prediction, 10.78 and 7.01 MAE for systolic and diastolic blood pressure prediction). Conclusions: NAKO provides a large-scale, nationwide population-based resource with visual acuity measurements and systemic health indicators, as well as color fundus images in about 50,000 NAKO participants. The data sets the ground for studying eye health in the general adult population in Germany and can serve as a strong foundation for developing and validating AI tools in eye health research. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This project was conducted with data from the German National Cohort (NAKO; www.nako.de; Application No. NAKO-590 and NAKO-810). The NAKO is funded by the Federal Ministry of Research, Technology and Space (BMFTR; project funding reference numbers: 01ER1301A/B/C, 01ER1511D, 01ER1801A/B/C/D and 01ER2301A/B/C), federal states of Germany and the Helmholtz Association, the participating universities and the institutes of the Leibniz Association. We thank all participants who took part in the NAKO study and the staff of this research initiative. The project was additionally funded by the Hertie Foundation. PB is a member of the Excellence Cluster 2064 "Machine Learning - New Perspectives for Science" and the NAKO AI expert group. AS and MU are members of the NAKO retina expert group and the NAKO ophthalmic competence unit. AB is a member of the Digital Clinician Scientist Program at University Medical Center Hamburg-Eppendorf (UKE). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This project was conducted with data from the German National Cohort (NAKO; www.nako.de; Application No. NAKO-590 and NAKO-810). The study protocol of NAKO was approved by the ethics committees of all participating institutions, and all participants provided written informed consent in accordance with German legal and data protection requirements. It is conducted in accordance with the Declaration of Helsinki and national standards for good clinical and epidemiological practice. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data is accessible to researchers through the official NAKO Transfer Hub (https://transfer.nako.de), subject to application and approval by the NAKO Use and Access Committee, under established governance and data protection frameworks.

Retinal degenerative diseases represent a complex global health problem due to their significant impact on patients’ daily lives and their highly heterogeneous pathogenesis, which challenges therapeutic development. Despite this complexity, many diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), share common features, including disrupted proteostasis, oxidative stress, and inflammatory responses, eventually leading to photoreceptor (PR) degeneration and vision loss. The inhibition of valosin-containing protein (VCP) has emerged as a promising mutation-independent therapeutic strategy for RP. However, clinical translation requires rigorous validation in models that closely reflect human retinal physiology. Organotypic retinal explants from porcine, macaque, and human donors were placed in an in vitro culture setup and treated with ML240, a selective VCP inhibitor, delivered either as a free compound or encapsulated in mPEG5kDa-cholane. Photoreceptor survival was assessed via TUNEL assay, outer nuclear layer (ONL) row quantification, and immunostaining. Retinal inflammation was evaluated by microglial staining. A dose–response study was performed to determine safety margins across species, and additional retinal markers were used to assess the preservation of non-photoreceptor retinal cell populations. Porcine retinal explants exhibited progressive photoreceptor degeneration under ex vivo conditions. Treatment with ML240, particularly when formulated with mPEG5kDa-cholane, significantly reduced photoreceptor cell death and microglial activation. Macaque and human explants exhibited minimal to no signs of degeneration. Treatment did not affect morphological or histological features of the explant, demonstrating the safety of ML240 in the primate retina. VCP inhibition via ML240 demonstrates an uncompromised safety profile in porcine, macaque, and human retinal explants. In addition, the neuroprotective activity of ML240 was evident in porcine tissue. Formulation with mPEG5kDa-cholane enhances the overall performance of the compound, supporting its use for future clinical application as a mutation-independent therapeutic approach for retinal degenerative diseases.

Disruption of proteostasis is a defining feature of cancer and other chronic diseases. The AAA+ ATPase VCP/p97 (valosin containing protein) is a key regulator of proteostasis by disassembling ubiquitinated substrates for degradation. VCP overexpression supports cancer cell survival and correlates with poor prognosis, promoting the development of VCP inhibitors as anti-cancer agents. However, the molecular basis for cancer-selective vulnerability of VCP inhibition remains unclear. Here, we demonstrate that allosteric VCP inhibition triggers cell-type specific macroautophagy/autophagy through dynamic reorganization of organelle contact sites. In human umbilical vein endothelial cells (HUVECs), VCP inhibition induces adaptive autophagy through coordinated reorganization of plasma membrane (PM)-ER-mitochondria contacts. Controlled opening of the mitochondrial permeability transition pore (mPTP) releases calcium into the cytosol, activating AMP-activated protein kinase (AMPK) and TFEB pathways, collectively enhancing autophagic flux and sustaining endothelial survival. Critically, calcium-activated kinase inhibitor or calcium chelators blocked VCP inhibitor-induced autophagy in HUVECs, confirming calcium signaling as the central mediator of adaptive autophagy. In contrast, HCT116 colon cancer cells fail to maintain calcium homeostasis under VCP inhibition, leading to mitochondrial calcium overload, defective autophagy, and cell death. Together, our findings identify organelle contact reorganization and calcium homeostasis as key determinants of cell fate under conditions of proteotoxic stress, revealing how VCP inhibition selectively suppresses tumor progression while preserving vascular integrity that could enhance drug delivery and reduce tumor hypoxia.Abbreviations: ATF4: activating transcription factor 4; ATG3: autophagy related 3; ATG7: autophagy related 7; ATP: adenosine triphosphate; CAMKK2: calcium/calmodulin dependent protein kinase 2; CETSA: cellular thermal shift assay; CQ: chloroquine; CTS: cryptotanshinone; DDIT3/CHOP: DNA damage inducible transcript 3; DQ-BSA: dye quenched-bovine serum albumin; EIF2A: eukaryotic initiation factor 2A; ER: endoplasmic reticulum; ERAD: endoplasmic reticulum-associated protein degradation; HCT116: human colon carcinoma cell line; HSPA5: heat shock protein family A (Hsp70) member 5; HUVECs: human umbilical vein endothelial cells; ITPR1/IP3R1: inositol 1,4,5-trisphosphate receptor type 1; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; mPTP: mitochondrial permeability transition pore; MT-ND1: mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1; MTOR: mechanistic target of rapamycin kinase; ORAI1: ORAI calcium release-activated calcium modulator 1; PLA: proximity ligation assay; PM: plasma membrane; PRKAA/AMPK: protein kinase AMP-activated catalytic subunit alpha; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; STIM1: stromal interaction molecule 1; TFEB: transcription factor EB; U87MG: human glioblastoma astrocytoma cell line; FAF2/UBXD8: Fas associated factor family member 2; UPR: unfolded protein response; VCP/p97: valosin containing protein; VDAC1: voltage dependent anion channel 1.

Background:Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by biallelic ARSA variants. Deficient arylsulfatase A (ARSA) leads to accumulation of sulfatides, major constituents of myelin, resulting in progressive central and peripheral demyelination. Although sulfatide excretion in urine is a recognized hallmark, an extended urinary lipidomic signature of MLD and its clinical utility across subtypes and treatment contexts has not been systematically characterized in a large cohort.Objective:To determine whether targeted urinary lipidomics can (1) distinguish MLD from controls, (2) differentiate clinical subtypes, (3) assess whether urinary lipid profiles relate to hematopoietic stem cell transplantation (HSCT) outcomes, and (4) characterize MLD in relation to mimicking disorders and conditions.Methods:We performed high-resolution LC–MS/MS lipidomics on 155 spontaneous urine samples from 85 individuals, including 129 samples from 61 patients with confirmed MLD. Lipid panels included sulfatides, sphingomyelin (SM) species, and globotriaosylceramide (Gb3).Results:Urinary sulfatides were markedly elevated in all MLD samples and provided clear discrimination from controls, with very-long-chain and 2-hydroxy sulfatides (C22:0, C24:0, C24:0-OH, C24:1-OH) being most informative. At the group level, distinct lipid patterns associated with MLD subtypes were identified, with disease severity correlating with higher sulfatide levels. In HSCT-treated MLD patients, stabilized patients showed a trend toward lower post-HSCT sulfatide levels compared to progressive patients, although substantial inter-patient variability precluded individual-level prediction. Lipid profiling reliably distinguished MLD from both heterozygous carriers of disease-causing ARSA variants and individuals with ARSA pseudodeficiency but did not differentiate MLD from biochemically similar disorders (multiple sulfatase deficiency, saposin B deficiency). Gb3, particularly the C18:0 species, was consistently elevated, most likely a secondary signature of lysosomal dysfunction in sulfatide-burdened renal cells rather than primary Gb3 storage.Conclusions:Targeted urinary lipidomics provides a robust, non-invasive biochemical tool for diagnosing MLD and distinguishing it from pseudodeficiency and carrier states. Sulfatide profiles show associations with clinical subtype but do not always permit reliable individual classification. Sulfatides remain the primary disease storage marker; the Gb3 C18:0 elevation may reflect autophagic and/or lysosomal stress and warrants further investigation. The use of spontaneous urine samples demonstrates the feasibility of implementing this approach in real-world pediatric settings.

Organelles such as mitochondria, lysosomes, peroxisomes, and the endoplasmic reticulum form highly dynamic cellular networks and exchange information through sites of physical contact. While each organelle performs unique functions, this inter-organelle crosstalk helps maintain cell homeostasis. Age-related macular degeneration (AMD) is a devastating blinding disease strongly associated with mitochondrial dysfunction, oxidative stress, and decreased clearance of cellular debris in the retinal pigment epithelium (RPE). However, how these occur, and how they relate to organelle function both with the RPE and potentially the photoreceptors are fundamental, unresolved questions in AMD biology. Here, we report the discussions of the “Mitochondria, Lysosomes, and other Organelle Interactions” task group of the 2024 Ryan Initiative for Macular Research (RIMR). Our group focused on understanding the interplay between cellular organelles in maintaining homeostasis in the RPE and photoreceptors, how this could be derailed to promote AMD, and identifying where these pathways could potentially be targeted therapeutically.

Rhodopsin-mediated autosomal dominant retinitis pigmentosa ( RHO -adRP) is a progressive inherited retinal degenerative disorder currently lacking effective treatments. A recurrent 3-base pair deletion in the RHO gene, resulting in the loss of isoleucine at codon 255 or 256 ( RHO ΔI255 or RHO ΔI256), has been identified in patients from the United Kingdom, Germany, Belgium, China, and Korea, suggesting a broad geographic distribution. This mutation leads to rhodopsin (RHO) misfolding, its retention in the endoplasmic reticulum (ER), and aggregation with wild-type (WT) RHO, ultimately triggering ER stress and photoreceptor degeneration. These aggregates are primarily cleared via the ER-associated degradation (ERAD) pathway, with valosin-containing protein (VCP) playing a key role in their retrotranslocation and proteasomal degradation. Pharmacological or genetic inhibition of VCP has shown neuroprotective effects in other models of adRP, but the poor aqueous solubility of VCP inhibitors and challenges in retinal drug delivery hinders clinical translation. To overcome these limitations, we evaluated and compared three VCP-targeted therapeutic strategies in Rho ΔI255 knock-in mouse retinae: (1) small-molecule inhibitors (ML240, NMS-873) solubilized in DMSO, (2) ML240 encapsulated in monomethoxy-polyethylene glycol (mPEG)-cholane nanoparticles, and (3) small interfering RNA (siRNA) targeting VCP, delivered via magnetic nanoparticles. Neuroprotective effects were assessed in vitro in retinal explants and in vivo following intravitreal injection. Our findings provide the first evidence that VCP inhibition restores RHO trafficking to the outer segments and prevents photoreceptor cell death in the Rho ΔI255 model. Among the three approaches, nanocarrier-encapsulated ML240 exhibited superior efficacy, enabling sustained drug delivery and enhanced photoreceptor protection. These results establish a preclinical proof-of-concept for nanocarrier-mediated VCP inhibition as a promising therapeutic strategy for RHO -adRP and potentially other ER-stress-related retinal degenerations. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. Tistou and Charlotte Kerstan Foundation Fighting Blindness Canada ProRetina foundation Zinke Heritage foundation [1]: pending:yes

Age-related macular degeneration (AMD), affecting circa 200 million people worldwide, is a debilitating disease leading to visual loss in the elderly population. The main risk for AMD is advanced age. Genetic predisposition as well as modern lifestyle habits, such as smoking or unhealthy diets, increase this risk. On the molecular level, these risks convert into complex systemic changes at the interface of the choriocapillaris, Bruch's membrane, RPE, and neuroretina, affecting the functional integrity and survival of RPE and photoreceptors cells. To date, therapeutic options for AMD patients are limited. Pathway identification and a detailed understanding of the molecular mechanisms driving AMD are prerequisites to defining potential novel druggable targets. This review aims to give a short overview of the known cell signaling pathways focusing on RPE cells in response to stress conditions occurring in AMD.

The classical approach of using adjacent pieces of fresh-frozen tissue for various omics analysis from the same sample possesses a risk of biological mismatch between arising from intrinsic tissue heterogeneity. We propose an alternative approach of tissue cryogenic pulverization and lyophilization before distribution for omics studies for a more reliable analysis. Here, we compare individual omics layer readouts from fresh-frozen adjacent tissue pieces and homogenized powder in mouse brain, kidney, and liver. Genomics, transcriptomics, proteomics, and metabolomics analyses showed comparable RNA integrity, DNA methylation, and coverage of transcripts, proteins, and metabolites across both methods. Moreover, the homogenized-lyophilized powder usage led to reduced heterogeneity between biological replicates. We conclude that the cryogenically pulverized-lyophilized tissue approach not only maintains a critical molecular feature coverage and quality but also provides a homogenous basis for various omics analysis enhancing reproducibility, sample transport, storage and enabling multi omics base on one and the same tissue aliquot.

Genotype-phenotype correlations of rare diseases are complicated by low patient number, high phenotype variability, and compound heterozygosity. Mutations may cause instability of single proteins, and affect protein complex formation or overall robustness of a specific process in a given cell. Ciliopathies offer an interesting case for studying genotype-phenotype correlations as they have a spectrum of severity and include diverse phenotypes depending on different mutations in the same protein. For instance, mutations in the intraflagellar transport protein IFT140 cause a vast spectrum of ciliopathies ranging from isolated retinal dystrophy to severe skeletal abnormalities and multi-organ diseases such as Mainzer-Saldino and Jeune syndrome. Here, the quantitative effects of 23 missense mutations in IFT140, which forms part of the crucial IFT-A complex of the ciliary machinery, were analyzed using affinity purification coupled with mass spectrometry (AP-MS). A subset of 10 mutations led to a significant and domain-specific reduction in IFT140-IFT-A complex interaction indicating complex formation issues and potentially hampering its molecular function. Knockout of IFT140 led to loss of cilia, as shown before. However, phenotypically only mild effects concerning cilia assembly were observed for two out of four tested IFT140 missense mutations. Therefore, our results demonstrate the utility of AP-MS in discerning pathogenic MMs from polymorphisms, and we postulate that reduced function is tolerated by the evolutionarily highly conserved IFT-A system.

Introduction: Nephronophthisis (NPHP) is an autosomal recessive kidney disease resulting mainly from primary cilium defects, with unspecific and variable symptoms that can progress to kidney failure needing replacement therapy at a young age. Currently, up to 64% of likely NPHP cases can be diagnosed by assessing known genes. Therefore, there is a need to gain more insight in what genes can cause this disease. METHODS:In a diagnostic setting, we performed broad genetic testing in patients with advanced kidney disease. We carried out in silico and in vitro analyses for TMEM72, including immunohistochemistry and affinity proteomics, and in vivo experiments to further interpret our findings. RESULTS:We identified biallelic TMEM72 variants in 9 patients from six families with a phenotype suggestive for NPHP. Five families presented with kidney failure at a (young) adult age. One family had a different phenotype with prenatal onset of kidney failure and neurological symptoms. The phenotypes of the patients correspond to TMEM72 expression mainly in the kidney. In silico analyses indicate that homozygous loss-of-function variants are likely not tolerated in TMEM72. Immunohistochemistry staining of kidney biopsies revealed altered localization and expression of TMEM72 in cases compared to controls. In human-derived tubuloids, we showed that TMEM72 localizes to the cilium. Furthermore, using an affinity proteomics approach, we found an association of TMEM72 and ciliary function, more specifically in selective ciliary cholesterol transport. CONCLUSION:We present the first genetic evidence, underlined by immunohistochemistry and protein binding assays, linking TMEM72 variants to kidney disease and ciliary function. We conclude that TMEM72 is a candidate gene for NPHP. Future work is needed to further characterize TMEM72 variants and unravel its disease mechanism. .

Background: Metastasis and resistance to therapy are major drivers of cancer-related mortality. Complement component C1R is overexpressed in multiple tumor types and correlates with poor prognosis and lack of response to immunotherapy, particularly in clear cell renal cell carcinoma (ccRCC), the most common renal cancer. In several models, C1R knockout reduces proliferation, viability, and migration of cancer cells, suggesting critical cell-intrinsic functions. However, the underlying mechanisms remain poorly understood. We aimed to investigate whether and how C1r contributes to the hallmarks of cancer Methods: We used gene silencing, phenotype profiling, RNA-seq, and metabolomics in C1r-expressing ccRCC cell lines (A498, Caki-1) and fibroblasts (BJ) to study C1r function. Findings were corroborated using snRNA-seq data ccRCC patients, including cancer cells and cancer-associated fibroblasts (CAFs). Subcellular fractionation and multiplexed immunofluorescence were used to localize C1r, and co-immunoprecipitation with mass spectrometry identified candidate interactors. A 40-marker immunofluorescence panel was applied to ccRCC tumor sections (n = 6), and a larger patient cohort was stained with an 8-plex panel targeting C1r, stromal and immune cell markers. Results: C1R expression was primarily detected in tumor cells and CAFs in ccRCC. Silencing C1R in vitro impaired proliferation, migration, viability, and sphere formation in both cancer cells and fibroblasts. This phenotype could not be rescued by adding purified C1r, indicating a non-canonical, intracellular role. C1r localized to organelles and nuclei in vitro and in situ. Proteomic analysis identified ~30 candidate interactors related to proliferation and migration. Transcriptomic and metabolic analyses of siC1R cells confirmed altered pathways involved in cell cycle, migration, energy and aminoacid metabolism. C1r levels also influenced immune signaling: C1R knockdown reduced inflammatory signatures in vitro, while its overexpression in tumors correlated with altered T cell infiltration in both RNA-seq and hyperplex imaging data. The relationship between C1r expression and immune cell phenotype is currently under investigation in a patient cohort Conclusion: Our findings reveal novel intracellular functions of C1r in tumor cells and CAFs, independent of the classical complement pathway. These results highlight C1r as a multifunctional, cell-autonomous regulator of tumor progression and immune contexture, and support its potential as a therapeutic target in cancers expressing C1r.