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

The transport of about 70 enzymes to lysosomes depends on mannose 6-phosphate signals formed by GNPTAB. Editing of Gnptab in an intestinal mouse cell line revealed the loss of multiple lysosomal enzymes associated with the accumulation of sphingomyelins, ceramides, and cholesterol, and reduced levels of bis(monoacylglycero)phosphate (BMP) in lysosomal proteomes and lipidomes. By cross-correlation we identified two subsets of lysosomal lipid-modifying enzymes linked with mixed unsaturated or di-monosaturated BMP. Autophagy-related proteins functioning in early stages of autophagosome formation associated with neutral ceramides, whereas proteins involved in autophagosome-lysosome fusion correlated with negatively charged BMPs. Therefore, the high cholesterol and low BMP level of lysosomes might be causal for impaired autophagic flux in GNPTAB deficient cells. We propose a functional axis of three lysosomal proteins as potential target to improve the autophagic flux in cells with dysfunctional lysosomes and in a newly established intestinal organoid model suitable as novel experimental tool.

Glaucoma is an optic neuropathy characterized by progressive retinal degeneration. Population genetic analyses describe an association of variants in angiopoietin-like protein 7 (ANGPTL7) with lower intraocular pressure (IOP) and decreased glaucoma susceptibility. In addition to its reported role in the trabecular meshwork (TM), we demonstrate that ANGPTL7 is also expressed within disease-relevant cell types of the human posterior eye, specifically prelaminar optic nerve head astrocytes (ONHA). Contrasting with previously published findings, we observe no difference in IOP between Angptl7-/- mice and wild-type littermates. Despite absence of an IOP phenotype, loss of ANGPTL7 protects from IOPinduced retinal ganglion cell (RGC) damage in a rodent glaucoma neurodegeneration model. ANGPTL7 may signal through LAIR1-positive immune cells, potentially contributing to pro-inflammatory signaling and phenotypic modulation of resident immune cells, with evidence suggesting that oligomerization of ANGPTL7 is necessary for its activity. Comprehensively, our data highlight an unappreciated role of ANGPTL7 in modulating neuroinflammation in the posterior eye, contributing to glaucoma progression.

Age-related macular degeneration (AMD) is the leading cause of irreversible central vision loss in older adults. The highest genetic risk for AMD maps to Chromosome 10q26. However, due to strong linkage disequilibrium between two genes, ARMS2 and HTRA1 located in this region, it has been difficult to determine whether one or both genes contribute to the AMD-disease risk. Functional studies of ARMS2 have been hampered due to the lack of animal models, as orthologs are only found in the higher primates and the gene is absent in rodents. To model ARMS2 biology in vivo, we used CRISPR-Cas9 to insert the complete human ARMS2 gene carrying the A69S risk variant (rs10490924), into a region of homology between humans and mice, on mouse Chromosome 7, 3.5 kb upstream of Htra1, preserving its native promoter, spacing, and orientation. We also generated a mouse model bearing its wild-type counterpart ARMS2A69 (ARMS2WT). RNA analysis revealed tissue- and cell-type-specific pattern of ARMS2 expression enriched in neural tissues, testis, and eye, and within the eye, in retinal horizontal cells and the retinal pigment epithelium (RPE). ARMS2 expression was consistent with human expression data and unaltered by the A69S mutation. By 12 months, ARMS2A69S mice develop AMD-relevant phenotypes including increased fundus spots, A69S-specific thinning of the outer nuclear layer, and reduced scotopic ERG amplitudes. Transcriptomic profiling of postnatal day 14 retina and RPE-enriched fractions revealed reduced mitochondrial oxidative-phosphorylation genes, corroborated by reduced retinal mtDNA copy number and lower maximal respiration, alongside preliminary results that suggest sphingolipid remodeling in the RPE. This humanized model links the ARMS2 A69S variant to mitochondrial dysfunction and provides an in vivo platform for defining AMD mechanisms and testing therapies.

Non-coding variants altering mRNA splicing are increasingly recognized as important cause of Mendelian disorders. Deep intronic variants (DIV) that activate cryptic exons (CEs) cause ~20% inherited retinal diseases (IRD) cases, yet it remains challenging to predict intronic variant pathogenicity accurately, thus we used a high throughput splicing assay (HTSA) to measure the effects of rare deep intronic variants in recessive IRDs with one confirmed pathogenic variant. 640 very rare deep intronic variants were chosen in trans-position with known mutation from 76 patients. We tested them with HTSA, which consists of a split-GFP minigene, separated by an SMN1 gene intron into which the 270bp sequences flanking the DIVs were cloned. The plasmid library was transfected into 293HEK cells, and 48 hours later total RNA was extracted from the transfected cells. RNA transcripts produced by HTSA minigene were amplified by RT-PCR and sequenced. The intron sequences spliced between the two GFP exons were identified and quantified. 98 variants activated CE >100 times more in altered oligo compared to reference oligo and were classified as pathogenic in this experimental setting, validated in longer context, 26 variants were classified as VUS. Interestingly, only 6 from 90 variants (6,6%) were predicted to be pathogenic by in silico algorithms (Splice AI) which leaves room for significant improvement of prediction algorithms. The results of this experiment confirmed diagnosis for 50 of 78 patients (64%). So, 19,4% of deep intronic variants were shown to cause CE activation. This can lead to conclusion that significant portion of undiagnosed patients with recessive IRDs carry pathogenic intronic mutation which causes the disease.

A cardinal feature of nonexudative age-related macular degeneration (AMD) is the accumulation of drusen between the retinal pigment epithelium (RPE) and Bruch's membrane, yet the mechanisms that initiate and promote drusen formation remain to be fully understood. Hydroxyapatite (HAP), a calcium-phosphate mineral identified within human drusen, has been proposed to contribute to drusen nucleation and is associated with increased likelihood of RPE atrophy. However, mechanistic studies providing insight into RPE-associated mineralization have been limited by a lack of easy-to-use and manipulatable culture models. Here, we established a rapid, inducible ARPE-19-based model of HAP mineralization. We found that ARPE-19 cells cultured in minimum essential media (MEM) supplemented with nicotinamide (NIC), sodium phosphate, and ascorbic acid (NaPAA) reproducibly formed HAP positive deposits, whereas mineralization was not observed while using standard DMEM/F12 medium, DMEM/F12 with NaPAA, MEM-NIC medium, or MEM-NIC medium with alternative osteogenic supplements (such as beta-glycerophosphate, ascorbic acid, and dexamethasone). Scanning electron microscopy with energy dispersive X ray spectroscopy demonstrated that the formed deposits were enriched in calcium (Ca) and phosphorus (P) with Ca/P ratios comparable to drusen-associated HAP deposits from AMD donor tissue. ARPE-19-based HAP deposits were also closely associated with neutral lipid-rich material, staining positive for BODIPY around their core structure. Bulk RNA sequencing of mineralized ARPE-19 cells revealed extracellular matrix, angiogenic, inflammatory, and metabolic transcriptional changes in the absence of strong enrichment of canonical osteogenic pathways. Co-treatment of mineralizing cells with either pyrophosphate, EDTA, or sodium citrate significantly reduced mineral deposition, but these approaches were not able to reverse it. Together, these findings establish a tractable RPE mineralization model that recapitulates key features of AMD-associated HAP and provides a platform for investigating mechanisms of drusen-associated mineral formation.

Methylmalonic aciduria (MMA) is caused by deficiency of methylmalonyl-CoA mutase (MMUT), which catalyses the final step of propionate catabolism and fuels the tricarboxylic acid cycle for energy production. Previous studies reported disrupted mitochondrial homeostasis in MMA, including reduced mitochondrial membrane potential and increased oxidative stress, especially in energetically demanding and chronically affected tissues like brain and kidneys. However, how these changes impact mitochondrial energy production remains unclear. Here, we systematically investigated mitochondrial energy production using extracellular flux analysis in cellular models of MMA, including 293T cells, patient-derived fibroblasts, urine-derived epithelial kidney cells and induced pluripotent stem cells (iPSCs) as well as iPSC-derived neurons harbouring either complete loss (knockout, KO) or pathogenic missense variants in MMUT. We found no impact on mitochondrial energy production in MMUT-KO 293T cells and fibroblasts compared to controls. In contrast, fibroblasts and 293T cells expressing the pathogenic MMUT-p.N219Y variant showed decreased energy production. This corresponds to the altered mitochondrial membrane potential in 293T cells, but contrasts with their unchanged mitochondrial abundance. Depletion of glucose, glutamine and pyruvate from the media or provision of each individually as sole fuel source exacerbated the phenotype of MMUT-p.N219Y clones, but did not induce a phenotype in MMUT-KO 293T cells. Finally, we confirmed reduced mitochondrial energy production in patient-derived kidney cells but found no evidence in MMUT-p.N219Y iPSCs and their derived neurons. Overall, our work suggests that the impact of MMUT-deficiency on mitochondrial energy production is cell type- and variant-dependent. Further investigations should clarify molecular mechanisms and their clinical impact.

Background. Activation of de novo lipogenesis (DNL) is a major hallmark of steatotic liver disease (SLD), a highly prevalent group of diseases ranging from a harmless steatosis to more severe steatohepatitis, cirrhosis, and hepatocellular carcinoma. We interrogated herein the early alterations in liver proteome, transcriptome, lipidome, inflammation, fibrosis, and oxidative status in a mouse model of progressive SLD induced by constitutive activation of PI3K-AKT signaling, the major inducer of DNL. Methods. Male, 8 weeks-old mice bearing phosphatase and tensin homolog (Pten) deletion and therefore constitutive activation of PI3K-AKT in hepatocytes (albumin-Cre) and littermate controls were evaluated for liver steatosis, injury, lipid peroxidation, metabolism, proteome, lipidome and transcriptome either upon feeding with a chow or high-fat diets supplemented or not with linoleic acid or after treatment with vehicle or acetyl-CoA carboxylase (ACC) inhibitor (ND-630, 20 mg/kg/day, i.p.) Results. Mice bearing Pten deletion and therefore constitutive activation of PI3K-AKT in hepatocytes display steatohepatitis characterized by enhanced DNL, severe steatosis, hepatocyte ballooning, inflammation, fibrosis and hepatomegaly, which, unexpectedly, was associated with reduced lipid peroxidation, increased GSH content and enhanced rates of triacylglycerol turnover, fatty acid {beta}-oxidation, tricarboxylic acid cycle flux, and mitochondrial respiration, altogether promoting a robust lipidome remodeling mainly defined by enrichment of DNL-derived fatty acids in detriment of a broad depletion of essential fatty acids (linoleic and -linolenic acids) and cardiolipins. Dietary restoration of liver linoleic acid content mildly attenuated liver inflammation, without however affecting lipid peroxidation and improving steatosis and fibrosis, whereas pharmacological acetyl-CoA carboxylase (ACC) and DNL inhibition markedly reduced steatosis, inflammation and fibrosis, despite further lowering hepatic linoleic acid content. Conclusions. Collectively, these findings support a harmful role of essential fatty acids depletion fomenting liver inflammation and identify DNL as a major culprit of steatohepatitis induced by constitutive PI3K-AKT activation.

In our past study on human cilia from primary ciliary dyskinesia (PCD) patients using cryo-electron tomography and mass spectrometry, defects in the DNAH5 gene were proven to cause the loss of the entire outer dynein arm along with other proteins. This phenomenon was not observed in the unicellular green alga Chlamydomonas. In this study, we examined the loss of ciliary components caused by defects in various genes, based on proteomic and structural analyses of motile cilia from multiple PCD patients. The absence of single outer arm dynein genes causes a loss or significant decrease in other inner and outer arm dyneins. Interestingly, some patients with outer dynein defects show decreased intraflagellar transport (IFT) proteins, suggesting an influence of cargo components or assembly on transport. These phenomena were not observed in Chlamydomonas, suggesting a more complex mechanism of ciliogenesis in humans despite the high similarity in the final 3D architecture of the 9+2 axoneme. Defects in certain central pair proteins unexpectedly reduced components on the doublets as well. Our results demonstrate protein-protein interactions during stages of human motile ciliogenesis that are more complex than those in unicellular organisms.

Achromatopsia (ACHM) is a rare autosomal recessive retinal disorder characterized by absent cone photoreceptor function from early life, leading to severe visual impairment. Mutations in genes involved in the cone phototransduction cascade frequently result in elevated cyclic guanosine monophosphate (cGMP) levels and activation of stress pathways, including endoplasmic reticulum (ER) stress and the unfolded protein response. Targeting common downstream mechanisms rather than individual mutations may provide a broadly applicable therapeutic strategy. Here, we investigated whether pharmacological inhibition of valosin-containing protein (VCP), a key regulator of ER and protein homeostasis, can prevent cone degeneration in the spontaneous cone photoreceptor function loss 1 (cpfl1) mouse model of ACHM. Organotypic culture of retinal explants from cpfl1 mice were treated with the selective VCP inhibitor ML240. Cone survival, cell death, opsin expression and localization were assessed by TUNEL assay, immunohistochemistry, and quantitative image analysis. ML240 treatment significantly increased cone density and improved cone opsin expression and trafficking to the outer segments (OSs) in cpfl1 explants compared to controls. Importantly, rhodopsin trafficking in rod photoreceptors was unaffected, indicating that VCP inhibition did not impair normal rod phototransduction. These findings demonstrate that VCP inhibition by ML240 effectively preserves cone photoreceptors and improves cone-specific functional markers in the cpfl1 model. Targeting VCP may represent a mutation-independent therapeutic strategy for preventing cone death in ACHM.

Purpose. To determine which proposed keratoconus (KC) mechanisms carry its genetic susceptibility and how it relates to corneal structure, and which describe its corneal tissue. Methods. Six mechanism modules and two attribution sets (transforming growth factor-{beta} [TGF-{beta}] and canonical type-2 atopy) were tested for enrichment in three independent European KC genome-wide association studies (GWAS) and in GWAS of central corneal thickness (CCT) and corneal resistance factor (CRF), with colocalization at matrix loci. Tissue expression was pooled across nine arms from eight studies with control-source meta-regression; activator protein-1 (AP-1)/immediate-early regulatory activity was scored in 107 donors, spatial expression in 21, and serum responses in human and rabbit stromal cells. Results. Extracellular matrix (ECM) was the only module enriched in all three KC sources (P [≤] 5.46 x 10-) and for CCT, CRF, and thickness-independent CRF (P [≤] 2.07 x 10-). COL5A1 shared causal variants with CCT in all three sources (posterior probability 0.985-0.991), and FNDC3B and COL1A1 with CRF or its thickness-independent component (0.961-0.986). Pooled, KC corneas had lower general and corneal-local inflammatory and stress/immediate-early expression (log2 fold-change -0.97, -0.98, -0.82; P [≤] 0.049). In whole-cornea and stromal tissue, ECM expression reversed between postmortem-donor and pathological controls (log2 fold-change +0.36, -2.34), whereas inflammatory and TGF-{beta} programs were lower against both. AP-1/immediate-early regulatory activity was lower in four donor cohorts (Hedges' g -1.50) and stress/immediate-early expression in two spatial cohorts; serum raised immediate-early readouts in human and rabbit cells. Conclusions. KC combines matrix-mechanical genetic susceptibility with a low-inflammatory, low-immediate-early corneal state whose matrix expression direction depends on the control source. Because this susceptibility is genetically shared with corneal thickness and resistance, interventions on the inherited disease are judged by corneal structure and mechanics.

Objective: Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) frequently present as overlap syndromes, characterized by shared sexual dimorphism and elevated intestinal Veillonella dispar (V. dispar) abundance, yet exhibiting distinct hallmark pathological manifestations. This study aimed to elucidate the shared pathogenesis, phenotypic differentiation mechanisms and sex-specific predisposition of AIH and PBC induced by V. dispar. Methods: Clean-grade and germ-free (GF) mice were employed to establish AIH and PBC models. Model induction involved oral gavage of V. dispar and intraperitoneal injection of either liver-specific antigen S100 (for AIH) or a pyruvate dehydrogenase complex E2 subunit (PDC-E2) mimetic peptide (for PBC). Single-cell RNA sequencing, metabolomics, transmission electron microscopy, western blotting, and flow cytometry were utilized to elucidate the underlying mechanisms. Results: V. dispar successfully induced autoimmune liver disease-like features in mice, characterized by significant intrahepatic B cell infiltration. Mechanistically, V. dispar caused systemic arginine reduction, leading to intrinsic apoptosis of gonadal stromal cells and a subsequent reduction in testosterone. This decreased testosterone weakened its inhibitory effect on B cell activating factor (BAFF) secretion, thereby amplifying B cell-mediated immune responses. Furthermore, in the presence of S100 or PDC-E2 mimetic peptide, V. dispar also facilitated Th17 cell activation, which guided the phenotypic differentiation into AIH or PBC, respectively. Consistent with these findings, our clinical observations revealed that patients with AIH and PBC tended to harbor enriched V. dispar, along with lower serum arginine and testosterone. In addition, elevated serum levels of BAFF and IL-17 were also observed in these patients. Conclusion: V. dispar induces reduced testosterone levels and upregulation of BAFF, leading to intrahepatic B cell expansion, which serves as the common pathogenic basis for AIH and PBC. The presence of S100 and PDC-E2 subsequently drives further maturation of these expanded B cells and promotes Th17 cell activation, thereby determining the phenotypic divergence between AIH and PBC.

Background and aims: Cyclin E1 is a canonical regulator of cell cycle progression known to promote liver fibrosis and hepatocarcinogenesis. However, its precise contribution to hepatic immune responses remains poorly understood. We investigated whether Cyclin E1 regulates macrophage-associated inflammation and acute immune-mediated liver injury. Methods: Hepatic immune populations were analyzed in untreated and concanavalin A-treated wildtype and constitutive Ccne1-/- mice by flow cytometry. Liver injury was assessed by survival, histopathology, and caspase activation. Inflammatory mediators were quantified by qPCR and ELISA. Bone marrow-derived macrophages were examined during differentiation and pro-inflammatory polarization using flow cytometry, cytokine measurements, and RNA-sequencing. Lipid nanoparticle-mediated delivery of Ccne1-specific siRNA was evaluated in J774A.1 macrophage-like cells. Results: Under homeostatic conditions, Ccne1-/- mice exhibited reduced hepatic immune cell numbers, including Kupffer cells and CD4+ and CD8+ lymphoid cells. Following concanavalin A administration, Ccne1-/- mice showed improved survival, reduced hepatic necrosis, and diminished caspase-3 and caspase-8 activation. Protection was associated with reduced Kupffer and CD8+ lymphoid cell abundance and attenuated inflammatory mediator expression. Cyclin E1 deficiency did not substantially alter bone marrow-derived macrophage differentiation or cell cycle distribution. However, pro-inflammatory Ccne1-/- macrophages showed reduced interleukin-6 expression, attenuated inflammatory gene programs, and lower inferred IRF1 and NF-{kappa}B1 activity. Ccne1 knockdown similarly reduced interleukin-6 expression in pro-inflammatory J774A.1 cells. Conclusions: Cyclin E1 contributes to hepatic immune homeostasis and promotes macrophage-associated inflammatory signaling during immune-mediated hepatitis. Its inflammatory function is uncoupled from overt changes in macrophage cell cycle progression and may involve regulation of IRF1- and NF-{kappa}B-associated transcriptional programs. Targeting Cyclin E1 may therefore provide a strategy to attenuate selected inflammatory responses in liver disease.

Background: Barth syndrome (BTHS) is an X-linked mitochondrial disorder caused by loss-of-function mutations in TAFAZZIN (TAZ), resulting in defective cardiolipin (CL) remodeling, cardiomyopathy, and premature death. The mechanisms linking TAZ deficiency to impaired mitochondrial quality control remain incompletely understood, and there are no disease-modifying therapies available for BTHS. Here, we investigated the role of Acyl-CoA Lysocardiolipin Acyltransferase-1 (ALCAT1), a stress-inducible phospholipid-remodeling enzyme, in BTHS cardiomyopathy. Methods: Inducible TAZ knockdown (TAZKD) and cardiomyocyte-specific TAZ knockout (TAZcKO) mice were used to determine the effects of genetic ALCAT1 deletion or pharmacological inhibition with Juvenatin, a highly selective small molecule ALCAT1 inhibitor. Cardiac function, exercise capacity, mitochondrial function, lipid remodeling, lysosomal function, and mitophagic flux were assessed. Results: ALCAT1 was markedly upregulated in TAZ-deficient hearts. Genetic ALCAT1 deletion attenuated cardiac dysfunction in TAZKD mice. Juvenatin similarly improved cardiac function in TAZKD mice and reversed established cardiomyopathy in TAZcKO mice. Genetic or pharmacological ALCAT1 inhibition improved mitochondrial ultrastructure and respiration and reduced mitochondrial oxidative stress. These benefits occurred without correcting the characteristic CL abnormalities caused by TAZ deficiency. Instead, ALCAT1 deletion or inhibition corrected pathological phosphatidylinositol remodeling, normalized phosphoinositide signaling, and restored lysosomal function and mitophagic flux, leading to improved mitochondrial quality control in TAZ-deficient cells. Conclusions: ALCAT1 contributes to BTHS cardiomyopathy through a CL-independent pathway involving dysregulated phosphatidylinositol/phosphoinositide signaling, lysosomal dysfunction, and impaired mitophagy. Genetic and pharmacological targeting of ALCAT1 restores mitochondrial and cardiac function despite persistent CL abnormalities, establishing ALCAT1 as a promising disease-modifying therapeutic target for BTHS.

Purpose: Age is the leading factor for age-related macular degeneration (AMD), in which retinal pigmented epithelium (RPE) atrophy is a hallmark. The nuclear factor-erythroid 2-related factor 2 (Nrf2), a major anti-stress transcription factor, decreases in AMD, suggesting bioenergetic stress interacts with age during AMD pathogenesis. We profiled visual function and gene expression of the retina and RPE-choroid with Nrf2 deletion and aging. Methods: We tested 3mo and 14mo Nrf2 null (Nrf2 KO) and Nrf2 WT mice for optokinetic tracking (OKT) and dark-adapted electroretinography (ERG) responses. Eyes were harvested for retina outer nuclear layer (ONL) thickness analysis and retina and RPE-choroid cup RNA isolation for RNAseq profiling. Results: Visual acuity was reduced with aging with lower values in the Nrf2 KO vs Nrf2 WT comparison at 14mo. We observed aged-related reduction of a-, b-, and c-wave ERG amplitudes in both WT and KO mice. Age-related c-wave decline was exacerbated by Nrf2 loss. By RNAseq, two-way ANOVA of RPE-choroid samples revealed differentially expressed genes (DEGs) with age and genotype main effects, as well as DEGs with age and genotype interaction effects. Age DEGs were over-represented in pathways related to inflammation and phagocytosis. Genotype DEGs were associated with alterations in antioxidant and inflammatory processes. Retina RNAseq showed age and genotype DEGs. Conclusions: Ablation of Nrf2 exacerbates age-related declines suggesting that decrease in Nrf2 with aging has functional consequences for inflammation/resiliency. We provide retina and RPE-choroid bulk-RNAseq datasets from the same cohort of mice that can be interrogated to better understand degeneration of retina/RPE-choroid.

Phenylketonuria (PKU) is an autosomal recessive disorder caused by PAH gene defects and can cause irreversible neurological damage as early as the fetal period; however, non-invasive prenatal monitoring methods are lacking. In this study, Pah+/- female mice were mated with Pah+/-; male mice and wild-type male mice, respectively. Urine samples were collected longitudinally from pregnant mice at 10 time points during gestation from Day 1 to Day 19. Data-independent acquisition (DIA) quantitative proteomics combined with GO/KEGG analysis, WGCNA, and a dual-strategy LASSO-random forest approach was used to screen candidate biomarkers. The results showed that stable differentially expressed proteins between groups were detectable as early as Day 1, and the differences persisted throughout gestation and exhibited temporal dynamics. Permutation tests indicated that the probability of random generation of the differential protein combination was low (0.04-0.06), suggesting that it was not randomly generated. The intersection of the two strategies yielded five proteins, GINM1, PRG4, LY6A, NTM, and YIPF3, and after including GLO1, a total of six candidate biomarkers were obtained. The candidate proteins had interaction or co-expression associations with 14 proteins involved in PKU mechanisms, with GLO1 serving as a network hub. This study found that the maternal urine proteome can non-invasively reflect the systemic response induced by fetal-origin PKU, providing proof of concept and candidate biomarker resources for non-invasive early-pregnancy screening of PKU.

This study evaluated the Delayed Cmax Drug Delivery System (DCMax), an erosion-based controlled- release formulation designed to delay peak plasma concentration (Cmax) and sustain drug release. The tablet matrix contained hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate succinate, and polyethylene oxide. In-vitro dissolution and erosion studies confirmed the delayed release profile. In-vivo performance was assessed in six healthy volunteers by measuring salivary caffeine concentrations after administration of immediate-release caffeine tablets and DCMax tablets. DCMax induced a delayed Cmax of 5 hours compared with 1 hour for the immediate-release formulation, while peak concentrations were lower compared to immediate release. These findings demonstrate that erosion-based drug delivery can achieve delayed release, with potential application in chronotherapy for endocrine disorders.

Background: Calcific aortic valve disease (CAVD) is the most common valvular heart disease in older adults and currently has no approved pharmacological therapy to prevent or slow disease progression. Aortic valve replacement, either surgically or by transcatheter aortic valve implantation (TAVI), therefore remains the only effective treatment for severe CAVD. Monocytes play an important role in driving inflammation and disease progression in CAVD. As the disease progresses and the aortic valve becomes increasingly stenotic, circulating monocytes passing through the narrowed valve are repeatedly exposed to pathological haemodynamic forces, including markedly elevated shear stress, before their recruitment into the valve tissue. However, how exposure to these mechanical forces reprograms circulating monocytes influences their subsequent responses within the valve remain poorly understood. Methods and Results: In this study, we used an engineered model of aortic valve stenosis combined with phosphoproteomic profiling to investigate how prolonged exposure to pathological shear stress reprograms circulating monocytes and influences their subsequent phenotype and function following recruitment into the valve matrix. We further used TAVI as a unique clinical model to determine whether restoration of physiological haemodynamics in patients with severe aortic stenosis reverses the shear-induced monocyte phenotype observed in our experimental model. We found that prolonged exposure of monocytes to pathological shear stress induced extensive remodelling of the proteomic and phosphoproteomic landscape, with enrichment of pathways associated with cellular metabolism, cytoskeletal organisation, and inflammatory signalling. Kinase activity analysis further identified increased protein kinase C (PKC) activity accompanied by suppression of AKT signalling. Consistent with these molecular changes, pathological shear stress increased actin polymerisation, mitochondrial membrane potential, intracellular calcium signalling, and PKC activity. Using a bioengineered extracellular matrix model that recapitulates the glycosaminoglycan-rich microenvironment of the stenotic aortic valve we found that prior exposure to pathological shear stress altered monocyte-to-macrophage differentiation, reducing oxidised LDL uptake and foam-cell formation while promoting co-expression of CD80 and CD163, indicative of a mixed macrophage phenotype. Finally, using a myeloid-specific Piezo1 knockout mouse model, we identified Piezo1 as a key mechanosensor linking pathological shear stress to PKC activation, mitochondrial responses, and persistent changes in monocyte phenotype following migration into the extracellular matrix. Conclusions: These findings establish a mechanistic link between abnormal valve haemodynamics, metabolic and inflammatory reprogramming of circulating monocytes, extracellular matrix remodelling, and immune dysregulation during CAVD progression, highlighting monocyte mechanotransduction and downstream PKC/Piezo1 signalling as potential therapeutic targets.

Objectives: The bile acid nuclear receptor Farnesoid X Receptor (FXR, NR1H4) is a major regulator of metabolism and energy homeostasis in peripheral organs. It modulates bile acid, glucose, and lipid metabolism, as well as fat mass and body weight. However, FXR is also expressed in the brain, particularly in the hypothalamus, a key center for the regulation of energy homeostasis. Although one study has demonstrated a role for brain FXR activation in energy balance, its specific hypothalamic role is still unknown. Here, we examined the role of FXR in the mediobasal hypothalamus in the regulation of energy balance. Methods: We used a genetic approach combined with metabolic phenotyping to determine the effect of FXR invalidation in the mediobasal hypothalamus on metabolic parameters involved in the central regulation of energy homeostasis. Results: Our results demonstrate that hypothalamic FXR deficiency induces a positive energy balance, resulting in a reduction in energy expenditure due to alterations in glucose metabolism accompanied by structural changes in white adipose tissues. Conclusion: This study uncovers a previously unrecognized role for hypothalamic FXR in the central homeostatic control of energy balance, providing new insights into its contribution to peripheral glucose metabolism and adipose tissue structural remodeling.

Reliance on invasive liver biopsy endpoints is a major limiting factor in new therapeutic development for metabolic dysfunction-associated steatohepatitis (MASH). While liver fat accumulation is considered a driver of early disease, liver fat reduction is not recognized as a surrogate endpoint by health authorities. Here, we conducted the largest-to-date genome-wide association study (GWAS) of liver fat in 79,642 people with magnetic resonance imaging and developed a polygenic score with liver fat-lowering alleles across 55 genomic loci. In a separate set of 714,886 individuals, a 30% relative liver fat reduction due to the polygenic score was associated with 39% lower odds of liver cirrhosis and 41% lower odds of hepatocellular carcinoma, consistent with a strong causal relationship between lifelong liver fat differences and advanced liver clinical outcomes. A systematic review and meta-analysis of 20 randomized controlled trials including a total of 3,502 MASH patients showed that treatment-induced liver fat reductions were associated with MASH resolution (meta-regression slope in % units of the outcome for each 1% relative reduction in liver fat from baseline, {beta} = -0.70%, p<0.001) and fibrosis improvement ({beta} = -0.24%, p<0.001). These findings demonstrate the profound etiologic impact of liver fat in MASH and provide compelling evidence for liver fat reductions as a reasonably likely surrogate endpoint for future clinical development.

Individuals carrying a G4C2 repeat expansion in C9orf72 can develop amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD), at various and unpredictable ages. The contribution of the expansion to this extreme clinical variability remained a matter of debate. In this work, we validated the use of optical genome mapping to efficiently size this expansion from blood and applied this method to a cohort of 54 repeat expansion carriers including patients with FTD (n = 23), ALS (n = 22) and presymptomatic individuals (n = 9). Despite the high variability in expansion profiles, distinct groups can be inferred. Although consensus repeat profiles were similar between FTD and ALS patients, relationships between repeat number, indexes of repeat instability, and the age at sampling differed between clinical conditions. An age at sampling-dependent increase of repeat instability in blood was observed in presymptomatic carriers and FTD patients (p = 0.01 and p = 0.04 respectively), in contrast to patients with ALS (p = 0.9). The proportion of shorter repeats ([500-1500 repeats]) increased with the age at sampling in ALS patients but decreased in presymptomatic and FTD cohorts, while the proportion of larger repeats (>2000 G4C2), in particular those exceeding 4000 repeats, increased along with the age at sampling in presymptomatic carriers and FTD cases (p <1.10-e4 and p = 0.006 respectively) conversely to patients with ALS (p = 0.98). In conclusion, this work strongly supports different dynamics of repeat instability of the C9orf72 pathogenic expansion in blood linked to the clinical evolution. These findings provide new insights into the relationship between expansion profiles and clinical variability.