Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease

Pathogenic mutations in the ABCA4 gene cause several inherited retinal diseases, particularly Stargardt disease (STGD1). However, many missense variants remain classified as variants of uncertain significance (VUS) due to inconclusive evidence regarding their pathogenic impact. The missense VUS span across all the domains of ABCA4, with the majority found in the larger extracellular domains (ECDs). The largest uncharacterized region of ABCA4 is located in ECD1, where limited structural information and inconsistent computational predictions hinder clinical interpretation of missense VUS in this region. Here, we integrated in silico analysis with in vitro functional assays to evaluate the pathogenicity of VUS in this region and improve their diagnostic classification. Missense VUS in the ECD1 uncharacterized region were curated from ClinVar. Six multiallelic sites were identified in the uncharacterized region and 13 missense VUS on these multiallelic sites were characterized using the integrated analysis. In the in silico platform, the pathogenicity of the VUS were predicted using multiple algorithms, and the structural effects of the variants were analyzed compared to the wild type. Recombinant variants were expressed in virus-like particles (VLPs), and protein expression, membrane localization, and ATPase activity were quantified relative to wild type to identify potential disease-causing variants. From the integrated analysis, variants with pronounced structural destabilization, impaired membrane trafficking, and reduced or absent N-retinylidene-phosphatidylethanolamine (NRPE) substrate stimulated ATPase activities were identified as potentially deleterious. Notably, VUS at p.H193P and p.I214N showed loss of function, with p.I214N reflecting selectively impaired membrane targeting and p.H193P reflecting combined expression and trafficking defects. Additionally, NRPE-stimulated ATPase activities were impaired in VUS, p.V195L, p.V195I, p.D197H, p.I214F and p.N269S. Overall structural destabilization interfered with the NRPE-stimulated ATPase activities of p.N269S, while the lack of NRPE-stimulated ATPase activities of p.D197H, p.V195L, p.V195I and p.I214F are thought to be due to impaired NRPE interactions with ABCA4. All the VUS at p.R140, p.H193Y, p.D197N and p.N269H showed both the basal and NRPE-stimulated ATPase activities but less than that of the wild type, displaying a mild functional deficit. Together, these findings demonstrated that certain VUS within the unresolved ECD1 region disrupt ABCA4 stability and function, supporting their contribution to disease pathogenesis. This integrative approach highlights key residues likely to be pathogenic and advances the interpretation of VUS in inherited retinal disorders.

Objectives; The role of nuclear receptor subfamily 4, group A, member 3 (NR4A3) in hepatic steatosis, inflammation, and insulin resistance (IR) within the context of metabolic dysfunction-associated steatotic liver disease (MASLD) remains largely underexplored. Consequently, this study aimed to examine NR4A3's impact on MASLD and the potential underlying mechanisms. Methods; We aimed to elucidate the functional role of NR4A3 in MASLD through its knockdown in cell culture and animal models. To establish the cell culture model of MASLD, LO2 cells were treated with free fatty acids (FFAs), while male C57BL/6 mice were fed a high-fat diet (HFD) to create the animal model. NR4A3 knockdown was achieved using specific short hairpin RNA (NR4A3-shRNA) in the mice model and three small interfering RNAs (NR4A3-siRNAs) in the cell culture model. The lipids content, fatty acid synthesis, inflammatory factors, and IR were then assessed with and without NR4A3 knockdown. Furthermore, the underlying mechanism through which NR4A3 exerts its influence was explored by analyzing the interaction between NR4A3 and activating transcription factor 3 (ATF3). Results: In the cell culture experiments, the knockdown of NR4A3 significantly decreased the lipids content, fatty acid synthesis, and inflammatory factors in the LO2 cells treated with FFAs in the NR4A3-shRNA group compared with those in the NC-shRNA control group. In the animal model experiments, NR4A3 knockdown in the HFD male C57BL/6 mice significantly ameliorated HFD-induced hepatic steatosis, inflammation, and IR. Mechanistically, the knockdown of NR4A3 downregulated the expression and transcriptional activity of ATF3, resulting in an impaired ATF3 function. ATF3 overexpression significantly reversed lipid accumulation decline and reduced inflammation after NR4A3 knockdown. Conclusion: The downregulation of NR4A3 alleviates MASLD by modulating ATF3, suggesting this may be a promising therapeutic target.

Using a genetic screening approach based on an inducible gene-activating system and cell sorting, Down syndrome critical region 3 (DSCR3) was isolated as a gene whose overexpression increased cell size. Fibroblasts derived from individuals with Down syndrome (DS) exhibit elevated DSCR3 expression at both the mRNA and protein levels, correlating with increased cell volume compared to fibroblasts from healthy donors. Despite a slower proliferation rate, DS fibroblasts demonstrate higher basal and maximal mitochondrial respiration, suggesting enhanced metabolic activity associated with increased cell size. siRNA-mediated knockdown of DSCR3 reduces cell size in both DS and normal fibroblasts, indicating its general role in cell size regulation. As DSCR3 is a component of the retriever complex involved in endosomal cargo recycling, these findings position membrane protein trafficking as a novel module for cell size control.

Alpha-1 antitrypsin deficiency (AATD) caused by the Z variant leads to hepatic accumulation of misfolded AAT polymers and liver disease. Although proteotoxic stress is well established, its impact on lipid metabolism, mitochondrial function, and organelle homeostasis remains incompletely understood. The effects of Z-AAT accumulation were investigated in Z-HepG2 cells and 3D patient-derived ZZ hepatic organoids through protein aggregation, lipid storage, mitochondrial structure and function, peroxisomal dynamics, and comprehensive transcriptomic and proteomic analyses. Z-AAT expression led to intracellular polymer accumulation and reduced secretion, together with lipid accumulation, mitochondrial structural abnormalities, increased mitochondrial number but impaired respiratory capacity. Metabolic profiling revealed reduced oxidative phosphorylation and partial reliance on glucose metabolism. Peroxisomes displayed increased mass, consistent with altered lipid handling. Multi-omics analysis demonstrated widespread transcriptional and proteomic reprogramming related to protein synthesis, lipid metabolism, and mitochondrial function. Proteomic analysis confirmed proteotoxic stress-induced mitochondrial dysfunction, impaired lipid handling, and activation of stress response, inflammatory and vesicular trafficking pathways. Importantly, lipid supplementation elicited adaptive mitochondrial transcriptional responses in control cells, whereas Z-HepG2 cells showed a blunted response to lipid challenge. In conclusion, Z-AAT accumulation disrupts hepatic lipid processing and impaired mitochondrial and peroxisomal homeostasis, producing diminished metabolic flexibility likely contributing to AATD-associated liver disease.

Laminin-2 (gene: LAMA2) is a key protein in the basement membrane of muscle and Schwann cells. A complete lack of this protein results in LAMA2-related congenital muscular dystrophy (LAMA2-RD), a severe muscle disease characterized by progressive muscle weakness, respiratory insufficiency, failure to thrive and shortened life span. One key signature of this disease is early onset of fibrosis coupled with poor muscle growth. We previously showed that TGF-{beta} and its activator, integrin-V, are elevated in dystrophic fibers of DyW mice, a mouse model of LAMA2- RD. Other than activating TGF-{beta}, integrin-V is also known to facilitate the transdifferentiation of various cell types to myofibroblasts. In this study we present evidence for transcriptional dysregulation of genes driving myofibroblast transdifferentiation and extracellular matrix (ECM) remodelling during the early development of DyW mice that is also reflected in muscle biopsies from young LAMA2-RD patients. We hypothesize that the early ECM remodelling, seen in both DyW mice and LAMA2-RD children, may explain the congenital onset of fibrosis with poor muscle growth seen in the disease.

Significance Missense mutations can lead to pathological effects in human cells. Predictive methods that account for structural context, such as AlphaMissense, can provide pathogenicity scores. The accumulation of pathogenicity hotspots can reveal important structural features within individual proteins of protein families, such as GLUT transporters. Mapping pathogenicity scores onto the structure can thus provide a mechanistic explanation of the protein function necessary for its role in the cell. Abstract Non-synonymous amino acid substitutions (missense mutations) are common in the general population; some are causative of serious disease. Depending on their structural context, they can disrupt protein function, folding, or dynamics. Computational predictive methods developed in recent years, such as AlphaMissense, provide new insights into how missense mutations affect protein structure by predicting and mapping their pathogenicity across each amino acid in the human proteome. In this study, we identify recurring patterns of pathogenicity prediction across the GLUT family membrane transporters encoded by genes slc2a1-14. Within the GLUT transporter family, we observe higher pathogenicity profiles in the transmembrane domains, particularly in pore-lining and binding-site residues. Predicted missense pathogenicity is elevated throughout residues assigned to the central cavity, suggesting sensitivity of the transport pathway. Another finding shows higher pathogenicity in specific transmembrane helices of the protein, with the same pattern across all proteins. On the other hand, we observed lower pathogenicity values in some representatives of the GLUT family. These findings show that the pathogenicity of glucose transport within the GLUT family may be shaped by functional redundancy and physiological essentiality across GLUT groups.

Phenylketonuria (PKU) is a genetic metabolic disorder caused by the lack of functional phenylalanine hydroxylase (PAH). Elevated levels of phenylalanine (Phe) are known to be neurotoxic; however, the molecular mechanisms underlying Phe's effects remain elusive. This study investigates the impact of PKU on proteostasis, redox balance, and metabolism in BTBR PAHenu2 mice, a severe disease animal model. Combined proteomics and metabolomics revealed impaired redox homeostasis in the brain and disrupted mitochondrial energy metabolism (ATP and TCA intermediates). The dysregulation was further supported by decreased levels of ATP, reduced glutathione (GSH), cysteine, and reduced catalase activity. Western blot analyses revealed substantial remodeling of protein degradation systems: the 19S regulatory (Rpt1) subunit and 26S proteasome content and activity were significantly increased, and ubiquitinated protein levels were elevated, indicating protein turnover and activation of the ubiquitin-proteasome system. Autophagy was also activated, as evidenced by a reduced LC3-II/LC3-I ratio, decreased p62 levels, unchanged ATG5 levels, and increased HSPA8 protein expression. By contrast, UPR markers remained stable despite an increase in the oxidized-to-reduced PDI ratio, suggesting a localized shift without activation of a full ER stress response. In parallel, systemic alterations were assessed in whole blood. Indeed, GSH, cysteine, ATP and ADP were decreased in PKU, whereas NADPH increased. These changes were accompanied by reduced activities of GSH reductase and GSH peroxidase, thereby confirming metabolic and redox disruption. Collectively, these findings indicate that PKU is associated with activation of protein degradation pathways as an adaptive response to cellular stress combined with redox imbalance and energy dysregulation.

Erythrocytosis, a disorder with increased erythrocyte production, has a heterogeneous aetiology, including rare congenital types linked to dysregulation of the oxygen-sensing pathway. Variants in the EGLN1 gene, encoding the prolyl hydroxylase that regulates hypoxia-inducible factor (HIF) stability, are associated with familial erythrocytosis type 3 (ECYT3). In patients with idiopathic erythrocytosis we previously identified two novel EGLN1 variants, c.1072C>T (p.(Pro358Ser)) and c.1124A>G (p.(Glu375Gly)), classified as variants of uncertain significance. Herein, we performed in silico and in vitro analyses to assess their structural and functional effects, using the known pathogenic variant p.(His374Arg) as a positive control. AlphaFold3 predictions revealed minimal conformational changes in the protein core for all variants, while stability predictions suggested reduced protein stability. Functional assays in HEK293 cells demonstrated significantly decreased protein levels and stability for p.(Pro358Ser) and p.(Glu375Gly), comparable to p.(His374Arg). However, luciferase reporter assays showed that, unlike p.(His374Arg), the novel variants did not substantially impair EGLN1 enzymatic activity or activate HIF signalling. Our results suggest that the novel variants may contribute to erythrocytosis through destabilization of EGLN1, supporting further studies to elucidate their precise impact on hypoxia regulation. This study highlights the complexity of studying EGLN1 variants and the importance of functional evaluation for clinical interpretation.

BackgroundThe etiology and molecular mechanisms of Moyamoya disease (MMD) remain unclear. Exosomes, as carriers of bioactive molecules, may reflect disease-specific alterations and serve as potential biomarkers. This study aimed to investigate disease mechanisms using proteomic profiling of serum-derived exosomes (SDEs) in MMD. Materials and MethodsPeripheral blood each from 15 MMD patients and 15 healthy-controls were used to isolate SDEs via ultracentrifugation. Proteins from pooled SDEs were extracted, digested, and analyzed by LC-MS/MS. Differentially expressed proteins were examined using MetaboAnalyst, DAVID, Enrichr, STRING, and Cytoscape. Key targets were validated at transcript and protein levels using RT-qPCR and ELISA in independent cohorts. ResultsA total of 2,554 proteins were identified, with 213 showing differential expression (118 upregulated, 95 downregulated; p [≤] 0.05). Functional and pathway analyses revealed enrichment in angiogenesis, cytoskeletal remodeling, and endothelial signaling. PRKG2 and MYC were upregulated, while RHOA was downregulated, highlighting their involvement in focal adhesion and PI3K-AKT pathways. Validation confirmed these findings. ConclusionDysregulated proteins were linked to RHOA-ROCK and PI3K-Akt signaling, suggesting their role in driving VSMC phenotypic switching, contributing to vascular occlusion. These findings indicate that altered exosomal-proteins may participate in maladaptive vascular remodeling, although the initial trigger for VSMC transition remains unknown.

Apolipoprotein E (ApoE) is the primary genetic risk modifier of late-onset Alzheimers disease, with the {varepsilon}4 allele increasing risk up to 15-fold relative to {varepsilon}3. The structural differences between isoforms are thought to underlie their distinct effects on lipid transport, receptor binding, and disease risk. ApoE4 exhibits reduced thermodynamic stability compared to ApoE3, but prior characterisation has relied on purified recombinant protein, leaving open whether these differences are preserved in native cellular environments and how they relate to rare disease-associated variants. Here, we employed the cellular thermal shift assay (CETSA) and a bioluminescence-based thermal stability assay (BiTSA) to systematically characterise ApoE thermal stability across isoforms and variants. Using CETSA on brain tissue from humanised APOE knock-in mice and post-mortem human brain, we confirm that ApoE4 exhibits significantly reduced thermal stability compared to ApoE3 in native tissue, with this difference conserved across species despite variation in absolute melting temperatures. We developed BiTSA, which leverages a split-luciferase HiBiT tag to quantify soluble ApoE across a thermal gradient in living cells, providing a higher-throughput platform that faithfully recapitulates isoform stability differences. Applying BiTSA to rare AD-associated variants, we found that L28P exerts divergent, isoform-dependent effects, destabilising ApoE3 while paradoxically stabilising ApoE4--a finding supported by AlphaFold modelling revealing isoform-specific differences in helix 1 architecture. These results establish BiTSA as a robust cellular tool for ApoE variant characterisation and demonstrate that isoform background critically modulates the structural consequences of rare mutations.

Amyotrophic lateral sclerosis (ALS) is categorized by TDP-43 proteinopathy, however, the nuclear pathological events remain poorly defined. While cytoplasmic TDP-43 inclusions dominate the late disease stages, accumulating evidence indicates that nuclear TDP-43 assemblies arise earlier and impair RNA splicing. Here, we characterized a single RRM-proximal TDP-43 variant, G148V, designed to disrupt nucleic-acid engagement without altering canonical RNA-binding residues. Structural and biophysical analyses revealed conformational changes and loss of DNA/RNA binding. In mammalian cells, TDP-43 G148V robustly formed nuclear puncta with high penetrance, exhibiting solid-like properties, pathological phosphorylation, splicing dysfunction, and toxicity. Furthermore, we identified molecular chaperone HSC70 as an important regulator of the nuclear puncta assembly. HSC70 redistributed into G148V nuclear puncta to modulate their material state, whereas HSC70 depletion significantly promoted puncta solidification, increased insoluble TDP-43 accumulation, and enhanced cytotoxicity. Disease-associated K181E and K263E mutants also formed nuclear puncta and induced HSC70 nuclear redistribution. These findings establish G148V as a model of early nuclear TDP-43 pathology and highlight HSC70-mediated regulation as a key factor of TDP-43 nuclear assembly. HighlightsO_LIA single TDP-43 mutation, G148V, in RRM1 domain robustly induces nuclear puncta without exogenous stress. C_LIO_LIG148V disrupts nucleic-acid binding, driving solid-like nuclear assemblies with hyperphosphorylation. C_LIO_LINuclear G148V puncta impair splicing regulation and reduce cell viability, recapitulating early ALS pathology. C_LIO_LIThe molecular chaperone HSC70 modulates puncta material states and mitigates G148V-associated cytotoxicity. C_LI Graphical abstrac O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/739729v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@d019c8org.highwire.dtl.DTLVardef@4ca632org.highwire.dtl.DTLVardef@331fd9org.highwire.dtl.DTLVardef@6fe8e4_HPS_FORMAT_FIGEXP M_FIG C_FIG eTOC blurbA structure-guided TDP-43 G148V mutation reveals how loss of nucleic-acid engagement promotes early nuclear condensation, splicing dysfunction, and toxicity, while uncovering a protective role for HSC70 in regulating condensate properties during ALS pathogenesis.

Pigmentation is a critical protective mechanism that safeguards the skin against UV-induced damage, whereas dysregulated pigmentation predisposes to pigmentary disorders and skin malignancies. Although calcium signaling has emerged as an important regulator of melanogenesis, the identity of the calcium-handling proteins and the molecular mechanisms linking calcium dynamics to pigmentation remain poorly understood. Here, we identify the ER calcium pump SERCA2b as a negative regulator of pigmentation through modulation of ER stress and mitochondrial calcium uptake. We demonstrate that SERCA2b expression inversely correlates with pigmentation levels, and gain- and loss-of-function studies establish SERCA2b as a suppressor of melanogenesis. Mechanistically, SERCA2b depletion induces adaptive ER stress, enhances ER-mitochondrial proximity, and promotes mitochondrial calcium uptake. Notably, mutations in SERCA2b are associated with Darier disease, a condition characterized by hyperpigmented skin lesions, although the underlying mechanism remains unknown. To address this, we generated SERCA2b mutants corresponding to variants identified in Indian Dariers disease patients and examined their effects on pigmentation, ER stress, and mitochondrial calcium dynamics. The mutant phenotypes closely recapitulated SERCA2b loss-of-function effects, demonstrating that adaptive ER stress and enhanced mitochondrial calcium signaling underlie hyperpigmentation associated with Dariers disease. Importantly, treatment with 4-phenylbutyrate (4-PBA), an FDA-approved ER stress alleviator, rescued mutant-induced hyperpigmentation, reduced ER stress, and normalized mitochondrial calcium uptake. Collectively, our findings uncover a previously unrecognized role of SERCA2b in skin pigmentation, establish a mechanistic link between SERCA2b mutations and hyperpigmentation, and identify adaptive ER stress pathways as potential therapeutic target for pigmentary disorders.

Mitochondrial dysfunction is implicated in a variety of complex neurological disorders. Primary mitochondrial diseases are caused directly by mutations in genes encoding mitochondrial proteins, leading to mitochondrial dysfunction and disease. Mitochondrial dysfunction is also a key contributor to pathogenesis in multiple neurodegenerative diseases. Rescue of mitochondrial function is therefore an attractive therapeutic target in both groups of diseases. In this study, we used primary fibroblasts derived from patients with the primary mitochondrial disease Leigh syndrome (LS) and the neurodegenerative disease Huntingtons disease (HD) to investigate mitochondrial phenotypes in these patients. We used these to identify modifiable measures of mitochondrial phenotype using a high content imaging screen. Despite having distinct underlying disease causes, different mitochondrial phenotypes in LS and HD patient derived cells converged on an imbalance between functional and dysfunctional mitochondria. Through multi-parameter screening of the mitochondrial phenotype we identified the AMPK activator A769662 as a small molecule able to rescue this imbalance in both LS and HD patient derived fibroblasts via different pathways. Our findings indicate that high throughput screening for mitochondrial phenotypes could identify novel therapeutic agents to rescue mitochondrial dysfunction in complex neurological disorders. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSMitochondrial dysfunction is the primary driver of mitochondrial disease and key contributing factor to neurodegenerative disease pathogenesis. Rescuing mitochondrial function is a promising therapeutic strategy, yet strategies to identify mitochondrial modulators in patient cells are limited. Added value of this studyOur in-depth mitochondrial characterisation of Leigh syndrome and Huntingtons disease patient fibroblasts shows the potential for using this methodology to identify mitochondrial therapeutics. We identify a mitochondrial phenotype common across diseases and an AMPK activator capable of rescuing this phenotype. Implications of all the available evidenceOur work extends our understanding of the mitochondrial dysfunction associated with Leigh syndrome and Huntingtons disease and expands the tool set available for identifying modulators of mitochondrial health as potential therapeutics for complex neurological disorders.

Pompe disease (glycogen storage disease type II) is an autosomal recessive lysosomal storage disorder characterized by progressive glycogen accumulation within lysosomes. It leads to their enlargement, autophagosome build-up and defective autophagic flux. Among the pathophysiological features, mitochondrial abnormalities have long been regarded as secondary consequences of lysosomal dysfunction. Typically, they have been described in electron microscopy, revealing paracrystalline inclusions, cristae lost, swollen mitochondria, and glycogen-filled structures. However, the spatial organization and interplay between mitochondria and lysosomes in skeletal muscle remain poorly understood, as does the progression of these alterations with respect to muscle metabolic profile. Here, we present a novel approach combining super-resolution imaging with a deep learning- based image analysis workflow to quantitatively assess mitochondrial and lysosomal remodeling as well as their interactions in skeletal muscle of the main murine model of the Pompe disease. Organelles were analyzed at two specific stages of the disease, according to muscle type, fiber type and subcellular location of the mitochondria. We show that the overall structure of the mitochondrial network is affected as early as the pre-symptomatic stage (1 month), while changes in mitochondrial density are more restricted at this stage and become more widespread as disease progresses (4 months). Importantly, these pathophysiological modifications are highly dependent on the muscle, fiber type and subcellular location. Alongside a rapid and widespread increase in lysosomal size, and a subsequent shift toward tighter lysosomal clustering at the later stage, we observe a progressive, region-specific increase in mitochondria-lysosome interactions that is most pronounced in the intermyofibrillar region. Our findings establish that this original imaging approach provides a relevant and powerful framework for quantitatively analyzing interactions between organelles within skeletal muscle fibers, thus offering new opportunities to explore the subcellular changes underlying disease progression. As such, it represents an interesting tool for monitoring pathophysiology and evaluating the effectiveness of therapeutic interventions.

Neurodegenerative diseases are complex disorders characterised by progressive neuronal loss and widespread transcriptomic dysregulation; however, the coordinated interactions among coding and non-coding RNAs that contribute to disease progression remain incompletely understood. In this study, RNA-seq datasets from disease-relevant neuronal populations and brain regions representing Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS) were analysed using an integrative network-based framework. Differential expression analysis coupled with weighted gene co-expression network analysis identified modules significantly correlated with disease and prioritised highly connected hub genes. Integration of these hub genes with curated RNA interaction database enabled the construction of candidate lncRNA-miRNA-mRNA regulatory networks. Functional enrichment analysis revealed Gene Ontology biological processes associated with synaptic signalling, mitochondrial function, RNA metabolism and neuroinflammatory responses across neurodegenerative conditions. The inferred regulatory networks suggested both disease-specific and shared post-transcriptional regulatory modules involving key hub genes and non-coding RNAs. Additionally, putative sequence variants were identified within untranslated regions of selected hub genes, suggesting potential alterations in miRNA-mediated regulations. Therefore, this study provides a systems-level view of transcriptomic dysregulation across major neurodegenerative diseases and identifies candidate regulatory interactions and molecular targets for future functional investigation

The multiligand endocytic receptor, megalin (LRP2), is expressed in the retinal pigment epithelium (RPE) and patients lacking the receptor develop high myopia. Despite its established role in retinal development, the contribution of megalin to retinal homeostasis in the normally developed/mature eye remains poorly understood. Here, we investigated megalin function using an inducible knockout mouse (KO) model and human iPSC-derived RPE with megalin knockdown (KD) to distinguish post-developmental homeostatic functions from developmental effects. In vivo, megalin ablation caused progressive retinal degeneration and visual impairment, with morphological abnormalities in the RPE but no changes in myopia-associated ocular phenotypes including axial length and intraocular pressure. Proteomic profiling of megalin-KO RPE revealed reduction of autophagy-related proteins. In line with this, megalin deficiency was associated with accumulation of pro-cathepsin D, and perturbed rhodopsin turnover. This was supported in vitro, where trafficking of photoreceptor outer segment (POS) containing phagosomes to lysosomes was reduced, suggesting disturbed phagosome maturation. Megalin KD did not measurably impair initial uptake of POS discs, but delayed rhodopsin degradation, indicating defective post-ingestion processing. Together, these findings establish megalin as a key regulator of retinal homeostasis in the mature eye by controlling phagosome-lysosome fusion in the RPE and suggest that megalin dysfunction contributes to slowly progressive retinal degeneration. This positions megalin as a potential therapeutic target in lysosomal degenerative diseases in the retina.

The aim of this study was to characterize the BXD51 mouse strain as a reproducible model of chronic progressive glaucoma. Unlike the highly susceptible DBA/2J (D2) mutant strain, BXD51 is a genetically stable recombinant inbred line derived from C57BL/6J (B6) and D2 parental lines. Longitudinal assessments of intraocular pressure (IOP), visual acuity (VA), contrast sensitivity (CS), and pattern electroretinogram (pERG) demonstrated that BXD51 mice undergo a delayed decline in visual and retinal ganglion cell (RGC) function. Their decline is biphasic, with a period of initial ocular stress followed by a late-onset, accelerated structural and functional deterioration of RGCs. Anterior segment structural analysis by optical coherence tomography (OCT) and histology demonstrated increasing pigment dispersion and subsequent iridocorneal angle closure. Immunofluorescence analysis of structural neuronal markers (TUBB3 and MAP1A/2) exhibited thinning of the ganglion cell layer (GCL) and inner plexiform layer (IPL) together with axonal degeneration, mirroring the laminar degeneration seen in human glaucoma patients. BXD51 also revealed marked spatial heterogeneity between peripheral and central retina. Multivariate analysis confirmed that BXD51 follows a distinct clinical trajectory that separates it from both wild-type (B6) and a severe glaucoma model (D2). By spanning the range between resistance and extreme susceptibility to glaucomatous neurodegeneration, this study establishes the BXD51 mouse as a translational platform for mechanistic studies and for evaluating long-term neuroprotective strategies.

Purpose: Oxidative stress is thought to contribute to the development of age-related cataracts (ARCs), but the mechanisms by which oxidative damage leads to the opacification of the lens remain unclear. Previous studies suggest that oxidative stress can disrupt lens proteostasis. Therefore, it was hypothesized that ARCs arise from proteomic changes driven by an age-associated decline in oxidative stress defenses that interact with the lens proteostatic state. To test this hypothesis, proteomic analyses of lenses exposed to oxidative stress were performed to examine oxidative and proteostatic stress responses in vivo. Methods: Cataract formation was induced by injecting hydrogen peroxide into the aqueous humor of adult zebrafish. nrf2fh318/fh318 zebrafish were used to model the reduced oxidative stress protection observed in aged human lenses, while cryaba-/- zebrafish were used to model impaired lens proteostasis. Resulting opacities in WT, cryaba-/-, nrf2fh318/fh318, and cryaba-/-; nrf2fh318/fh318 lenses were quantified and proteomic changes in the cortex were analyzed using data independent acquisition Parallel Accumulation Serial Fragmentation mass spectrometry. Results: Hydrogen peroxide treatment induced the formation of cortical cataracts. Proteomic results showed that, dependent on genotype and day, oxidative stress activates the unfolded and mitochondrial unfolded protein responses. Additional changes were also observed in energy metabolism, Ca2+ homeostasis, protein degradation, and cytoskeletal and extracellular matrix remodeling pathways. Conclusions: Treated zebrafish lenses successfully model ARC and mass spectrometry proteomics identified the unfolded and mitochondrial unfolded protein responses as potential therapeutic targets for ARC.

IntroductionsAortic dissection (AD) is a life-threatening vascular disease with limited therapeutic targets. Apolipoprotein H (APOH), a circulating glycoprotein implicated in lipid metabolism, has not been studied in AD. MethodsPlasma APOH levels and aortic deposition were examined in AD patients. A {beta}-aminopropionitrile (BAPN) and angiotensin II (Ang-II)-induced mouse AD model with AAV-mediated Apoh knockdown was used to evaluate survival, aortic dilation, and extracellular matrix remodeling. Transcriptomic profiling, chromatin immunoprecipitation, and gene silencing in human aortic vascular smooth muscle cells (HAVSMC) were performed to dissect the mechanism. PPAR{gamma} agonist rescue was conducted in vivo. ResultsAPOH was elevated in plasma and deposited in AD aortas. Apoh knockdown improved survival, reduced AD incidence and ascending aortic dilation, and attenuated elastic fiber disruption and collagen deposition. Transcriptomics revealed enrichment of the PPAR pathway. APOH promoted HAVSMC phenotypic switching from a contractile to a synthetic state, decreasing ACTA2/TAGLN and increasing OPN/MMP9. Mechanistically, APOH upregulated NR5A1, which directly bound the PPAR{gamma} promoter to enhance PPAR{gamma} and FABP4 expression. Silencing NR5A1 or PPAR{gamma} reversed APOH-induced phenotypic switching and inflammation. In vivo, PPAR{gamma} agonist diminished the protective effects of Apoh silencing. ConclusionAPOH promotes AD progression through the NR5A1-PPAR{gamma} axis, driving vascular smooth muscle cell phenotypic switching and inflammation, and represents a potential therapeutic target. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/739043v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@323110org.highwire.dtl.DTLVardef@133260dorg.highwire.dtl.DTLVardef@1075c9eorg.highwire.dtl.DTLVardef@51a965_HPS_FORMAT_FIGEXP M_FIG C_FIG Circulating APOH promotes aortic dissection through the NR5A1-PPAR{gamma} axis in human aortic vascular smooth muscle cells. Clinical observations showed that plasma APOH levels were elevated in patients with aortic dissection. Circulating APOH acts on human aortic vascular smooth muscle cells (HAVSMC) and upregulates NR5A1, which binds to the PPARG promoter and enhances PPAR{gamma} transcription. Activation of the NR5A1-PPAR{gamma} signaling axis promotes the phenotypic transition of HAVSMCs from a contractile phenotype to a synthetic phenotype, as indicated by decreased ACTA2 and TAGLN expression and increased OPN and MMP9 expression, accompanied by enhanced production of the inflammatory mediators IL-6, MCP-1, and TNF-. Silencing NR5A1 or PPAR{gamma} reverses APOH-induced phenotypic switching and inflammatory responses, supporting the critical role of the NR5A1-PPAR{gamma} axis in APOH-mediated vascular injury.

PACS1 syndrome is a rare neurodevelopmental disorder caused by a recurrent de novo missense variant (p.R203W) in the PACS1 protein. However, it remains unclear whether the p.R203W variant acts through a loss-of-function or alternative mechanism. Here, we used isogenic iPSC-derived neurons (iNs) to directly compare the effects of PACS1 p.R203W to complete loss of PACS1 function. Using a combination of proteomic, biochemical and electrophysiological approaches, we identified molecular and functional phenotypes associated with each genotype. While PACS1(+/R203W) and PACS1(-/-) iNs shared phenotypic abnormalities, the overall molecular and functional consequences of the p.R203W variant were distinct from those caused by PACS1 deficiency. Notably, PACS1(+/R203W) presented with unique proteomic and kinase signaling signatures and a shift in stimulus dependent excitability. These findings demonstrate that PACS1 syndrome is not caused by a simple loss of function and instead support a non-loss-of-function mechanism. Lastly, our interactome analysis suggests that the p.R203W variant retains aspects of canonical PACS1 function while acquiring novel molecular interactions that could contribute to PACS1 syndrome pathogenesis. Altogether, these findings provide a framework for future mechanistic studies and therapeutic development in PACS1 syndrome. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/747101v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@d1522corg.highwire.dtl.DTLVardef@69e4dforg.highwire.dtl.DTLVardef@30eebcorg.highwire.dtl.DTLVardef@899b9d_HPS_FORMAT_FIGEXP M_FIG C_FIG