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

The ultimate cause of blindness in glaucoma is the death of retinal ganglion cells, and understanding the mechanism behind retinal ganglion cell loss during glaucoma could lead to the development of novel treatments for glaucoma. Endothelin-1 has been shown to mediate retinal ganglion cell death during glaucoma through impairment of mitochondrial function. Retinal ganglion cells are highly metabolically active, and susceptible to oxidative damage and decreased respiratory capacity. Mitophagy is the process whereby damaged mitochondria are degraded to prevent further propagation of oxidative damage. The current study evaluates the effect of endothelin-1 on mitophagy in retinal ganglion cells. Electron microscopy revealed endothelin-1 administration lead to a decrease in healthy mitochondria in the optic nerve. The MitoQC mouse was used to evalute mitophagy in response to endothelin-1, along with immunohistochemical analysis of mitophagy proteins. Mitophagy follows different trends in the optic nerve and retinal ganglion cell bodies following endothelin-1 administration, mitophagy was increased in the optic nerve but decreased in the retina following endothelin administration. With elevation of intraocular pressure, mitophagy was increased in the retina but decreased in the optic nerve. In retinal ganglion cells, parkin expression and activation was unchanged 24 hours after endothelin-1 administration, but was decreased 72 hours following endothelin-1 administration. Taken together, these results suggest that endothelin-1 impacts mitophagy through parkin-independent mechanisms in retinal ganglion cell bodies, and the ganglion cell bodies and optic nerve appear to have different responses to endothelin-1.

Myotonic Dystrophy type 1 (DM1) is an autosomal multisystem disorder manifested due to unstable CTG nucleotide repeat expansion within the 3'-untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Although progress towards understanding of molecular pathogenesis in muscle and heart has been made, the pathways that affect the brain in DM1 is fundamentally unknown. In addition, the congenital DM1 manifest even more complicated brain abnormalities. Despite the wealth of existing cellular and animal models, iPSCs based studies are being fostered as they replicate the human model more closely to the disease. In view of this context, we set out to characterize the differentiation potential of congenital DM1 patient derived iPSC lines towards neuronal cells. Using neurogenin2 (NGN2) induced direct reprogramming of iPSCs into neurons and chemically defined media-induced neural induction protocol, we find that congenital DM1 mutant iPSC derived neurons exhibited precocious differentiation, as evidenced by their expression of pan-neuronal markers TUJ1 and Map2, along with increased processes extension and neurite length. Moreover, unbiased RNA sequencing analyses and qPCR validation revealed precocious and enhanced expression of several neurogenic transcription factors including, Ascl1, NeuroG2, and NeuroD1. Furthermore, immunofluorescence imaging of MBNL1 and MBNL2, RNA-splicing factors, displayed enhanced nuclear aggregations, a hallmark of the DM1 disease, in the mutant lines. Moreover, investigation of RNA splicing events identified mis-splicing in many important genes/transcripts including RMST, ANK3 and MBD1 during the neural conversion of congenital DM1 lines. These studies reveal novel paradigms that may contribute to neurological pathogenesis in CDM1 patients. These studies also provide a strong foundation for future mechanistic investigation aimed at understanding CDM1 pathology and may open new avenues for the development of gene therapy approaches for individuals with DM1.

BackgroundKATNIP (Katanin-interacting protein), also known as KIAA0556, is one of the human genes with pathogenic variants linked to Joubert syndrome, an archetypal neurodevelopmental ciliopathy. KATNIP is a scaffolding protein with a critical role in ciliogenesis. In this study, we characterized the ciliopathy phenotypes due to KATNIP gene deletion. ResultsWe produced a Katnip null mouse model using CRISPR-Cas12a (Cpf1). The null heterozygotes appeared normal while the homozygotes died around postnatal day 9, showing severe hydrocephalus and deficiency in neuroprogenitor cell proliferation. Katnip-deficient cells in the brain have a higher rate of cilia formation and longer cilia than wild type cells. ConclusionKATNIP loss of function gives rise to hydrocephalus found in Joubert syndrome. The results indicate that KATNIP restricts ciliogenesis and cilia extension and supports proliferation of neuroprogenitor cells in the brain.

Jordans Syndrome (JS) is a rare, neurodevelopmental disorder caused by de novo missense mutations in protein phosphatase 2 regulatory subunit Bdelta (PPP2R5D). JS is characterized by severe neurological impairments starting in early life. PPP2R5D encodes for B56{delta}, one of the regulatory subunits of protein phosphatase 2A (PP2A). PP2A is a heterotrimeric protein serine/threonine phosphatase that is highly expressed in the brain and the liver. Past studies have focused on PP2As role in liver and little is known about the holoenzymes behavior in neuronal cells. Although B56{delta} is known to play an important role in the substrate specificity of PP2A, the identification of validated downstream substrates in JS remains unclear. To better understand how the mutations affect neuronal cells, we developed cerebral cortical-like organoids from an engineered allele series of the most common JS mutations to characterize the physiological changes throughout different stages of neurodevelopment. Organoids were assessed for transcriptomic, protein, and electrophysiological changes utilizing bulk RNA sequencing, immunocytochemistry, Western Blot, and high-density MicroElectrode Array. The results identify differentially expressed genes and translated proteins, potential neuronal substrates, and significant electrophysiological signatures that suggest mutations in B56{delta} lead to variant-specific dysfunction of PP2A. Overexpression of PPP2R5D through AAV transduction of organoids rescued several phenotypes in the variants, suggesting different pathogenetic etiology underneath. Our findings successfully characterized cerebral cortical-like organoids in JS cell lines and demonstrated its potential as a model for studying neurodevelopmental disorder and for screening therapeutic approaches.

The Microphthalmia-associated transcription factor (MITF) plays a critical role in retinal pigment epithelium (RPE) development and function. Dysfunctional autophagy and lysosomal degradation in the RPE have been implicated in age-related retinal degeneration, yet the contribution of MITF to these pathways remains incompletely understood. Here, we show that reduced Mitf expression impairs autophagy in mouse and human RPE cells. Primary RPE cells from Mitfmi-vga9/+ heterozygotes mice displayed altered autophagic flux characterized by accumulation of LC3B-II and p62, while MITF knockdown in human ARPE-19 cells promoted autophagosome accumulation. Ultrastructural analysis further revealed age-dependent accumulation of autolysosomes and lipofuscin-like granules in mutant RPE cells. In addition, expression of autophagy-related genes was altered in mutant RPE tissue, supporting disrupted lysosomal-autophagic homeostasis. Together, our findings identify MITF as an important regulator of autophagy in the RPE and suggest that impaired MITF-dependent homeostasis may contribute to retinal degeneration.

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.

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.

S-acylation is a reversible posttranslational lipid modification important in the nervous system that dynamically regulates protein localization and function. Aberrant S-acylation has been implicated in several neurological conditions. While several de-S-acylases (deacylases hereafter) have been identified, little is known regarding their expression and localization in the brain and in neurons. Here, we characterized the expression, localization, and S-acylation of cytosolic deacylases, including acyl-protein thioesterases APT, APT2, and APT1L and /{beta} hydrolase domain-containing proteins ABHD7, ABHD10, ABHD13, ABHD16A, and ABHD17A-C. Mouse brain RNA sequencing data revealed high expression of Lypla1/APT1, Lypla2/APT2, Ephx4/ABHD7, Abhd16a, and Abhd17A-C in the brain, whereas Lyplal1/APT1L, Abhd10, and Abhd13 were expressed at very low levels. Protein analysis demonstrated region-specific expression, with expression of APT1 and ABHD16A highest in the cerebellum and APT2 highest in the hippocampus, with all three highly expressed in cultured hippocampal neurons. Deacylases were observed distributed throughout neurons on punctate structures, with APT2 and ABHD17C to the Golgi by immunocytochemistry. Finally, all ten cytosolic deacylases are themselves S-acylated. These data characterizing deacylase expression, localization, and S-acylation in neural contexts, provides a foundation for future studies investigating deacylase neuronal functions and potential roles in neurological disease.

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.

The tight regulation of iron homeostasis is of great importance for cellular health. An increase in intracellular iron levels results in the formation of free radicals, which damages macromolecules and membranes, eventually resulting in cell death by Ferroptosis. Recently, we showed that patients with mutations in VPS41 display a severe neurodegenerative phenotype with iron deposition in the brain. VPS41 is well known as subunit of the HOPS complex required for fusion of late endosomes and autophagosomes with lysosomes. However, VPS41 has also been identified as inhibitor of Ferroptosis and regulator of redox homeostasis. How VPS41 exerts these functions and if these are dependent on the HOPS complex is unknown. Here we show that depletion of VPS41 results in increased intracellular iron levels, ROS formation and mitochondrial fission. Our findings indicate an important role for VPS41 in the regulation of iron homeostasis and mitochondrial fission and suggest Ferroptosis as a possible cause for neurodegeneration in VPS41 patients.

Age-related macular degeneration (AMD) is a progressive complex eye disease and one of the leading causes of blindness. AMD progression is marked by molecular changes in the retinal pigmented epithelium (RPE) which include increased reactive oxygen species (ROS) accumulation, mitochondrial dysfunction - eventually leading to dysfunctional RPE. Mitophagy regulator, Pink1, is reduced in the RPE of AMD patients and Pink1 loss leads to a shift from mitochondrial respiration to glycolysis. Serine is a non-essential amino acid which is de novo synthesized from glycolytic intermediate 3-PG via the rate limiting enzyme PHGDH. Serine is tightly integrated into anabolic processes like glutathione (GSH) cycling, maintaining NADH/NADPH pools leading to changes in AMPK signaling. Here, we show that Pink1 loss leads to a reduction in PHGDH and serine levels in the RPE leading to impaired mitochondrial structure and function, increased ROS mediated damage, increased inflammation, and hampered retinal function. Serine supplementation rescued ROS accumulation, balanced GSH abundance, and increased retinal function. Overall, our study highlights the potential of dietary serine in ROS management in AMD.

Defects in lysosomal lipid handling provoke fatal disorders presenting neurovisceral symptoms with variable onset and life spans. A prime example is Niemann-Pick type C disease (NPCD), where export of cholesterol and other lipids from the endosomal-lysosomal system is impaired due to variants of either NPC intracellular cholesterol transporter 1 (NPC1) or NPC intracellular cholesterol transporter 2 (NPC2). Therapeutic options for NPCD are limited to palliative care and disease-modifying drugs, and there is an unmet need for new treatments. Based on positive effects in patient-derived fibroblasts in vitro, we explored how inhibition of bromodomain and extra-terminal domain (BET) proteins affects a well-established mouse model bearing the frequent I1061T variant of NPC1. Treatment with JQ1, a hydrophobic prototype BET protein inhibitor, induced beneficial but sex-dependent molecular and behavioral changes in mice. Our results indicate bromodomain proteins as therapeutic drug target for NPCD and reveal sex-dependent BET protein signaling in mice.

Oxidative stress is proposed to be a driver of age-related diseases. Age-related macular degeneration is one such disease, where the retinal pigment epithelium (RPE) is affected early in the disease. Vasculature damage also occurs, sometimes preceding RPE damage. To model some aspects of dry AMD, we used the NaIO3 mouse model of oxidative damage. Disruption of the deep retinal vascular plexus, disorganization and death of capillaries within the choriocapillaris, and marked electroretinographic decline were observed. AAV overexpressing the transcription factor, NRF2, which induces anti-oxidation enzymes and represses inflammation, was tested for protection of damage. The BEST1 promoter limited expression to the RPE. The RPE, photoreceptors, and vascular architecture in both retinal and choroidal compartments were protected. Conditioned medium from RPE-choroid explants, infected by AAV8/BEST1-NRF2, was sufficient to transfer partial protection in vivo, indicating that NRF2 induces a protective secreted factor(s). Analysis of RNA-seq data identified growth differentiation factor 15 (GDF15) as a candidate downstream mediator. Injection of recombinant GDF15 reproduced key protective phenotypes in vivo, whereas Gdf15-deficiency attenuated NRF2-mediated rescue. Pharmacologic inhibition of TGF-{beta} receptor signaling diminished NRF2 associated protection, supporting involvement of this signaling pathway. In a laser-induced choroidal neovascularization model, intravitreal GDF15 injection reduced fluorescein leakage and lesion size. These findings support a model in which NRF2 activation in the RPE induces expression of GDF15, which is capable of protecting the RPE, photoreceptors, and the retinal and choroidal vasculature. NRF2 and GDF15 have therapeutic potential for ocular diseases, as well as for other diseases with vascular pathology.

Pathogenic variants in IFT140 are associated with a spectrum of syndromic and non-syndromic ciliopathies, with retinal degeneration as a common feature. Despite advances in understanding IFT140 function across various tissues, human retina-specific models are lacking. Here, we show that knock-in mice homozygous for the IFT140 patient variant c.932A>G (p.Y311C) did not develop retinal degeneration, while mice with the homozygous variant c.1451C>T (p.T484M), associated with non-syndromic retinal dystrophy, were embryonic lethal. Therefore, to understand the effect of these variants on retinal homeostasis, we generated novel human in vitro models of IFT140-associated retinal dystrophy, including CRISPR/Cas9 IFT140 knock-out (IFT140KO) induced pluripotent stem cells (iPSC) and patient-derived iPSC retinal pigment epithelium (iPSC-RPE) and retinal organoids (iPSC-ROs). IFT140KO iPSC-RPE cells display stubby cilia compared to isogenic controls, while IFT140T484M/T484Mpatient-derived iPSC-RPE cells exhibit slightly shorter cilia and cilia tip protein accumulation. Both IFT140KO and IFT140T484M/T484M iPSC-ROs show accumulation of cilia proteins at the connecting cilium and outer segment of photoreceptors, and mislocalization of rhodopsin to the inner segments and outer nuclear layer. Pharmacological screening of compounds previously reported to improve cilia structure identified the flavonoid eupatilin as the most effective molecule. Treatment with eupatilin improved cilium length and IFT traffic in iPSC-RPE, and IFT traffic and rhodopsin localization in iPSC-ROs. These findings emphasize the importance of human stem cell derived models to investigate tissue specific disease mechanisms and highlight the therapeutic potential of eupatilin to ameliorate cilia defects in retinal tissue.

Thyroid eye disease (TED) is a debilitating condition characterized by orbital fibroblast (OF) activation and excessive hyaluronic acid (HA) accumulation within the retro-ocular space. While IGF-1R blockade with teprotumumab has significantly advanced TED management, incomplete clinical responses and disease relapse underscore the need to identify alternative targets. In this study, we used high-throughput RNA sequencing to map the transcriptomic landscape in TED OFs compared with non-TED OF controls. Our analysis identified robust enrichment of pathways critical to the TED phenotype, including PI3K-AKT signaling, the platelet-derived growth factor (PDGF) pathway, and extracellular matrix remodeling. We validated several key upregulated mediators that may contribute to orbital remodeling, including FOXC2, HGF, MET, and HMGA2, alongside the downregulation of the Wnt antagonist SFRP2. By employing a computational drug-repositioning approach, we identified the multi-kinase inhibitor sorafenib, which targets VEGFR, PDGFR, and RAF, as a potent candidate to neutralize the TED-specific gene signature. Functional assays demonstrated that sorafenib dose-dependently inhibited PDGF-induced AKT phosphorylation and significantly attenuated HA synthesis in primary TED OFs. These results define a persistent, receptor tyrosine kinase-driven program in the TED orbit and suggest that multi-kinase inhibition represents a viable therapeutic strategy for refractory TED. HighlightsO_LIThyroid eye disease (TED) orbital fibroblasts exhibit a transcriptomic signature characterized by elevated PI3K/AKT, angiogenic, and growth factor signaling. C_LIO_LIComputational drug prediction identifies sorafenib as a candidate to reverse the TED gene signature. C_LIO_LISorafenib dose-dependently inhibits AKT activation and hyaluronic acid production in TED orbital fibroblasts. C_LI

BackgroundDecreased hepatic removal of low density lipoproteins (LDL) and increased apolipoprotein B (apoB) production cause hypercholesterolemia, a major causal risk factor of atherosclerotic cardiovascular disease (ASCVD). By a genome-wide siRNA screen, we previously identified subunits of the Coat protein I (COPI) complex to limit LDL uptake into Huh-7 hepatocarcinoma cells. MethodsThese findings were validated by targeted in vitro experiments as well as genetic association studies in humans and three mouse models with mutated or disrupted COPI genes. ResultsSilencing of COPA, COPB1, COPB2, ARCN1, COPG1, and COPZ1 in Huh-7 cells resulted in decreased uptake of LDL and aberrant glycosylation and altered cell surface abundance of the LDL receptor (LDLR) as well as increased apoB secretion and cellular lipid storage. Single nucleotide polymorphisms of ARCN1 were associated with lower ARCN1 expression and higher levels of LDL-cholesterol (LDL-C). Rare variants of COPA and COPG1 were enriched among patients with LDL-C > 5 mmol/L. Patients and mice carrying other rare immunopathogenic missense variants of COPA and COPG1 did not present with elevated plasma levels of LDL-C, while hepatic knockdown of murine Copg1 increased the concentrations of non-HDL-cholesterol in plasma and triglycerides in the liver. ConclusionsThe COPI coatomer regulates LDLR activity and apoB secretion as well as lipid content of liver cells. Loss of function of some variants of COPI genes are associated with higher LDL-C levels.

BackgroundDuchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, which encodes dystrophin in skeletal muscle cells. Although the role of dystrophin as a structural protein is well known, the cellular processes underlying myofiber degeneration are still not fully understood. Despite advances from studies in murine models, these models do not fully replicate the human pathology. MethodsWe investigated sarcolemmal integrity, membrane repair capacity, and annexin protein expression in DMD patient muscle biopsies and human skeletal muscle cell lines using immunohistochemistry, both shear stress-based and laser irradiation injury assays, western blotting, and live-cell imaging of GFP-tagged annexins. ResultsWe identified defective membrane repair in DMD skeletal muscle cells, independent of increased membrane fragility, by evaluating resealing capacity in control and DMD derived-patient cell lines using both a shear stress assay (N = l2, p < 0.000l) and a laser irradiation assay (N = 3, p < 0.000l). Analyses performed on human DMD muscle biopsies (N = l0) further confirmed this defect, demonstrating massive intracellular IgG uptake (p < 0.000l) together with altered annexin expression profiles. While mechanical stress induces the upregulation of annexin A5 (ANXA5, p < 0.0l) and A6 (ANXA6, p < 0.05) in healthy skeletal muscle cells - suggesting an adaptive response to membrane damage, given the annexin familys central role in membrane repair - we observed dysregulated expression patterns of these proteins in DMD cells. Notably, ANXAl (p < 0.05) and ANXA2 (p < 0.0l) were not only significantly overexpressed but also aberrantly localized to the extracellular space, a putative consequence of defective membrane repair. Since extracellular ANXA2 has been associated with adipocyte accumulation in the muscle tissue of patients with dysferlinopathy, a similar pathological mechanism may be at play in DMD. ConclusionsOur findings propose that ANXA2 contributes to muscle degeneration in DMD and highlight it as a potential therapeutic target to prevent adipogenesis and muscle loss.

Gastroschisis is a congenital abdominal wall defect in which fetal intestines herniate into the amniotic cavity. Despite 97% surgical repair success rate, 40% of affected infants require hospital readmission due to gastrointestinal complications, where underlying mechanisms remain poorly characterized. We hypopthesized that the cord blood metabolome of neonates with gastroschisis differs systematically from controls and may reveal pathway-level alterations relevant to neonatal physiology. Cord blood plasma collected at delivery (23 samples each group) was analyzed using ultra-performance liquid chromatography coupled with tandem mass spectrometry. Unsupervised principal component analysis and hierarchical clustering demonstrated significant separation between groups (PERMANOVA pseudo-F = 4.632, R{superscript 2} = 0.095, p = 0.001). 53 metabolites met criteria for differential abundance, 75% were lipids. Key alterations included reduced free fatty acids, increased fatty acid amides and ceramides, disrupted steroid and bile acid metabolism, and decreased biliverdin and bilirubin isomers. Our findings provide insight into gastroschisis pathophysiology and identify potential biomarkers for future investigation.

BackgroundCockayne syndrome (CS), primarily caused by autosomal recessive pathogenic variants in ERCC6 (CSB) or ERCC8 (CSA), is a transcription-coupled nucleotide excision repair disorder. CS frequently presents with features similar to primary mitochondrial disease (PMD), including leukodystrophy, lactic acidemia, and skeletal muscle mitochondrial DNA (mtDNA) depletion. How this mitochondrial phenotype arises at the cellular level, and whether it can be pharmacologically targeted, is not yet clear. MethodsWe characterized mtDNA content, respiratory chain (RC) protein abundance, mitochondrial biogenesis signaling pathways, and oxidative phosphorylation capacity in primary fibroblasts from two siblings with identical compound heterozygous ERCC6 pathogenic variants (c.1526+1G>T; c.2800C>A, p.Pro934Thr) despite marked intrafamilial phenotypic divergence. A combined metabolic stress exposure (galactose, reduced glutamine, and buthionine sulfoximine, (BSO)) which reduced CS cell survival was used to screen for therapeutic leads among twenty-three candidate mitochondrial disease therapeutic compounds. Lead compounds were mechanistically validated at the level of mitochondrial superoxide, total cellular oxidative stress, glutathione, and autophagic flux. ResultsPatient fibroblasts exhibited several hallmarks of PMD, including reduced mtDNA content, decreased expression of complex I subunit NDUFB8, elevated expression of TOM20 with paradoxically decreased PGC1 suggestive of impaired mitophagic clearance, and decreased mitochondrial respiratory capacity. Under combined metabolic stress, ATP-levels indicative of survival in CS patient fibroblasts selectively collapsed to [~]20% of controls. Five dual-rescue compounds, defined as agents that reproducibly restored ATP-based cell survival in both patient fibroblast lines under stress, were identified, including N-acetylcysteine (NAC), coenzyme Q10 (CoQ10), rapamycin, taurine, and (-)-epicatechin. Mechanistic profiling resolved three functional classes of therapeutic effects in CS cells: (1) upstream mitochondrial reactive oxygen species reduction (NAC, CoQ10); (2) mTORC1 inhibition bypassing defective stress-induced autophagic induction (rapamycin); and (3) extra-mitochondrial improvement in cellular stress resilience ((-)- epicatechin, taurine). ConclusionsERCC6-based CSB deficiency produced a stress-sensitive and physiologically complex mitochondrial phenotype in patient fibroblasts that was pharmacologically treatable by targeting three mechanistically distinct pathways. Oxidative and broader stress buffering, autophagy modulation via mTORC1 inhibition, and enhanced cellular resilience highlight novel therapeutic opportunities to be advanced to clinical trials in CSB patients.

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.