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The mechanisms that connect complement system activation and basal deposit formation in early stages of age-related macular degeneration (AMD) are insufficiently understood, which complicates the design of efficient therapies to prevent disease progression. Using human fetal (hf) retinal pigment epithelial (RPE) cells, we have established an in vitro model to investigate the effect of complement C3a on RPE cells and its role in the formation of sub-RPE deposits. The results of these studies revealed that C3a produced after C3 activation is sufficient to induce the formation of sub-RPE deposits via complement-driven proteasome inhibition. C3a binds the C3a receptor (C3aR), stimulates deposition of collagens IV and VI underneath the RPE, and impairs the extracellular matrix (ECM) turnover by increased MMP-2 activity, all mediated by downregulation of the ubiquitin proteasome pathway (UPP). The formation of basal deposits can be prevented by the addition of a C3aR antagonist, which restores the UPP activity and ECM turnover. These findings indicate that the cell-based model can be used to test potential therapeutic agents in vitro . The data suggest that modulation of C3aR-mediated events could be a therapeutic approach for treatment of early AMD.

This protocol describes the isolation and culture of primary mouse RPE cells from various mouse models to produce viable RPE cells in vitro that mimic in vivo characteristics This protocol describes the isolation and culture of primary mouse RPE cells from various mouse models to produce viable RPE cells in vitro that mimic in vivo characteristics Mouse models are powerful tools for the study of ocular diseases. Alterations in the morphology and function of the retinal pigment epithelium (RPE) are common features shared by many ocular disorders. We report a detailed protocol to collect, seed, culture and characterize RPE cells from mice. We describe a reproducible method that we previously developed to collect and culture murine RPE cells on Transwells as functional polarized monolayers. The collection of RPE cells takes ∼3 h, and the cultures mimic in vivo RPE cell features within 1 week. This protocol also describes methods to characterize the cells on Transwells within 1–2 weeks by transmission and scanning electron microscopy (TEM and SEM, respectively), immunostaining of vibratome sections and flat mounts, and measurement of transepithelial electrical resistance. The RPE cell cultures are suitable to study the biology of the RPE from wild-type and genetically modified strains of mice between the ages of 10 d and 12 months. The RPE cells can also be manipulated to investigate molecular mechanisms underlying the RPE pathology in the numerous mouse models of ocular disorders. Furthermore, modeling the RPE pathology in vitro represents a new approach to testing drugs that will help accelerate the development of therapies for vision-threatening disorders such as macular degeneration (MD).

A study assessed the use of gene editing to treat CEP290 -associated inherited retinal degeneration, a disease that causes severe early-onset vision loss in childhood, and showed safety and early evidence of efficacy.

Unconsciousness is a fundamental component of general anesthesia (GA), but anesthesiologists have no reliable ways to be certain that a patient is unconscious. To develop EEG signatures that track loss and recovery of consciousness under GA, we recorded high-density EEGs in humans during gradual induction of and emergence from unconsciousness with propofol. The subjects executed an auditory task at 4-s intervals consisting of interleaved verbal and click stimuli to identify loss and recovery of consciousness. During induction, subjects lost responsiveness to the less salient clicks before losing responsiveness to the more salient verbal stimuli; during emergence they recovered responsiveness to the verbal stimuli before recovering responsiveness to the clicks. The median frequency and bandwidth of the frontal EEG power tracked the probability of response to the verbal stimuli during the transitions in consciousness. Loss of consciousness was marked simultaneously by an increase in low-frequency EEG power (<1 Hz), the loss of spatially coherent occipital alpha oscillations (8–12 Hz), and the appearance of spatially coherent frontal alpha oscillations. These dynamics reversed with recovery of consciousness. The low-frequency phase modulated alpha amplitude in two distinct patterns. During profound unconsciousness, alpha amplitudes were maximal at low-frequency peaks, whereas during the transition into and out of unconsciousness, alpha amplitudes were maximal at low-frequency nadirs. This latter phase–amplitude relationship predicted recovery of consciousness. Our results provide insights into the mechanisms of propofol-induced unconsciousness, establish EEG signatures of this brain state that track transitions in consciousness precisely, and suggest strategies for monitoring the brain activity of patients receiving GA.

Purpose Current sequencing strategies can genetically solve 55–60% of inherited retinal degeneration (IRD) cases, despite recent progress in sequencing. This can partially be attributed to elusive pathogenic variants (PVs) in known IRD genes, including copy-number variations (CNVs), which have been shown as major contributors to unsolved IRD cases. Methods Five hundred IRD patients were analyzed with targeted next-generation sequencing (NGS). The NGS data were used to detect CNVs with ExomeDepth and gCNV and the results were compared with CNV detection with a single-nucleotide polymorphism (SNP) array. Likely causal CNV predictions were validated by quantitative polymerase chain reaction (qPCR). Results Likely disease-causing single-nucleotide variants (SNVs) and small indels were found in 55.6% of subjects. PVs in USH2A (11.6%), RPGR (4%), and EYS (4%) were the most common. Likely causal CNVs were found in an additional 8.8% of patients. Of the three CNV detection methods, gCNV showed the highest accuracy. Approximately 30% of unsolved subjects had a single likely PV in a recessive IRD gene. Conclusion CNV detection using NGS-based algorithms is a reliable method that greatly increases the genetic diagnostic rate of IRDs. Experimentally validating CNVs helps estimate the rate at which IRDs might be solved by a CNV plus a more elusive variant.

Early-onset inherited retinal degenerations (IRDs), such as Leber congenital amaurosis (LCA) caused by pathogenic variants in the NMNAT1 gene, lead to severe vision loss in children. Despite its ubiquitous expression, reduced NMNAT1 function primarily affects photoreceptor cells (PRs) of the retina, yet the mechanisms underlying their vulnerability remain incompletely understood. Here, we demonstrate that reduced NMNAT1 enzyme function due to the p.V9M mutation leads to DNA damage in PRs, characterized by the progressive accumulation of the oxidative DNA adduct 8-oxo-dG in Nmnat1 V9M/V9M mutant mice. Cells with oxidative DNA damage also demonstrate DNA double-strand breaks, as evidenced by co-staining with antibodies to phosphorylated H2AX (γH2A.X). This DNA damage correlates with apoptosis-driven PR degeneration, as evidenced by caspase-9 activation and TUNEL staining in the PRs of the Nmnat1 V9M/V9M mutant mice, while alternative cell death pathways such as necroptosis and parthanatos were not significantly activated. Treatment with the antioxidant N-acetylcysteine (NAC) reduced oxidative DNA damage and retinal immune responses, mitigated apoptosis, and preserved cone PRs. Longitudinal assessment via optical coherence tomography (OCT) and electroretinography (ERG) revealed sustained structural and functional protection of the retina in NAC-treated mice. These findings establish oxidative DNA damage as a key driver of PR degeneration in the Nmnat1 V9M/V9M model and highlight NAC’s potential as a therapeutic strategy for NMNAT1 -associated IRD and potentially other IRDs in which oxidative DNA damage contributes to disease pathogenesis.

Reliable projections of wildfire and associated socioeconomic risks are crucial for the development of efficient and effective adaptation and mitigation strategies. The lack of or limited observational constraints for modeling outputs impairs the credibility of wildfire projections. Here, we present a machine learning framework to constrain the future fire carbon emissions simulated by 13 Earth system models from the Coupled Model Intercomparison Project phase 6 (CMIP6), using historical, observed joint states of fire-relevant variables. During the twenty-first century, the observation-constrained ensemble indicates a weaker increase in global fire carbon emissions but higher increase in global wildfire exposure in population, gross domestic production, and agricultural area, compared with the default ensemble. Such elevated socioeconomic risks are primarily caused by the compound regional enhancement of future wildfire activity and socioeconomic development in the western and central African countries, necessitating an emergent strategic preparedness to wildfires in these countries. A new study develops a machine learning framework to observationally constrain CMIP6-simulated fire carbon emissions, finding a weaker increase in 21st-century global fires but higher increase in their socioeconomic risks than previously thought.

Electroretinography (ERG) provides a noninvasive functional measure of multiple cell types of the outer retina. We conducted an ERG-based screen of 530 single-gene knockout mouse strains generated as part of the International Mouse Phenotyping Consortium, representing 2.5% of all protein-coding genes, to identify genetic variants affecting retinal function. We identified 30 strains with significantly altered ERG amplitudes. Two of the genes identified, Cfap418 and Syne2 , have been previously reported with outer retinal dysfunction, thereby serving as internal controls that validate our screening protocol. Of the remaining 28 genes newly associated with altered retinal function, the majority lacked a contemporaneous histopathology correlate, highlighting the importance of ERG in early detection of functional abnormalities. A rare homozygous missense variant in FCHSD2 , the human orthologue of one of the 28 genes identified, was found in a patient presenting with retinal degeneration that lacked a molecular diagnosis. This report represents a useful resource for future investigations into the molecular mechanisms driving inherited retinal diseases and demonstrates the power of large-scale ERG screening in identifying novel genetic determinants of retinal function.

The complement system plays a role in the formation of sub-retinal pigment epithelial (RPE) deposits in early stages of age-related macular degeneration (AMD). But the specific mechanisms that connect complement activation and deposit formation in AMD patients are unknown, which limits the development of efficient therapies to reduce or stop disease progression. We have previously demonstrated that C3 blockage prevents the formation of sub-RPE deposits in a mouse model of EFEMP1 -associated macular degeneration. In this study, we have used double mutant Efemp1 R345W/R345W : C5 -/- mice to investigate the role of C5 in the formation of sub-RPE deposits in vivo and in vitro. The data revealed that the genetic ablation of C5 does not eliminate the formation of sub-RPE deposits. Contrarily, the absence of C5 in RPE cultures promotes complement dysregulation that results in increased activation of C3, which likely contributes to deposit formation even in the absence of EFEMP1-R345W mutant protein. The results also suggest that genetic ablation of C5 alters the extracellular matrix turnover through an effect on matrix metalloproteinases in RPE cell cultures. These results confirm that C3 rather than C5 could be an effective therapeutic target to treat early AMD.

Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 ( PRPF31 -mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31 +/− mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31 +/− mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical – basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof of concept for future therapeutic strategies. Mutations in pre-mRNA processing factors cause autosomal dominant retinitis pigmentosa. Here the authors provide insights into the pathophysiological mechanisms underlying non-syndromic retinal disease caused by heterozygous mutations in genes encoding ubiquitously expressed splicing factors.