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

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.

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.

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

Mucopolysaccharidosis IIIC (Sanfilippo syndrome type C) is a rare lysosomal storage disorder caused by loss-of-function mutations in HGSNAT, which encodes an enzyme involved in heparan sulfate (HS) degradation, leading to impaired HS catabolism, lysosomal accumulation, and progressive neurodegeneration. Because enzyme replacement therapies have limited penetration across the blood-brain barrier, substrate-reduction therapy represents an alternative therapeutic strategy. Here, N-deacetylase/N-sulfotransferase 1 (NDST1), a key enzyme responsible for HS biosynthesis, was investigated as a potential substrate-reduction target. A structure-based computational pipeline was used to identify and evaluate inhibitors targeting the NDST1 sulfotransferase domain. Approximately 4.1 million drug-like compounds and FDA-approved drugs were screened by molecular docking, followed by pharmacokinetic filtering, molecular dynamics simulations, and MM/PBSA binding free energy calculations. In parallel, peptide binders targeting the same site were generated using diffusion-based protein design and evaluated using molecular dynamics and MM/GBSA analysis. Four chemically distinct small-molecule scaffolds and three peptide candidates were identified as stable binders to the NDST1 active site. The lead small-molecule candidate exhibited a predicted binding free energy of -13.36 {+/-} 5.87 kcal mol-1. These provide a focused set of candidates for further investigation and support the feasibility of targeting NDST1 as a substrate-reduction strategy for MPS IIIC.

BackgroundAcrodysostosis type 2 (ACRDYS2) is a rare autosomal dominant disease characterized by skeletal defects and cognitive deficit, with clinical symptoms observed in multiple other tissues including the skin. It is caused by mutations in a phosphodiesterase, PDE4D, a key regulator of cAMP/PKA (cyclic adenosine monophosphate / protein kinase A) signalling. Despite its well-defined genetic causes, the molecular mechanisms underlying the disease remain poorly understood, with studies based largely on engineered cellular models reaching conflicting interpretations. MethodsTo investigate how endogenous dynamics are affected by PDE4D mutations in unmanipulated cells, we studied PDE4D transcript and protein expression, activity and downstream signalling in native dermal fibroblast from ACRDYS2 patients and healthy controls. ResultsSignificant reduction in total PDE4D expression in patient cells was observed both at the transcript and protein level, with marked decreases in the long isoforms PDE4D4 and PDE4D7; a reduction in PDE4D9 mRNA was also observed. PDE4D enzymatic activity was reduced in ACRDYS2 fibroblasts, though total PDE activity was largely preserved. Reduced PDE4D expression was associated with an increase in the phosphorylated form of the cAMP-responsive transcription factor CREB and elevated PRKAR1A (PKA type 1 regulatory subunit alpha) transcript levels, suggesting altered downstream signalling. Interestingly, expression of the related phosphodiesterase family member PDE4B was increased, consistent with a compensatory response to reduced PDE4D function. ConclusionsThis is the first study demonstrating reduced PDE4D expression and isoform-specific dysregulation in native ACRDYS2 cells. Together, our results support a model in which reduction in PDE4D activity and compensatory changes in other PDE4 family members contribute to the molecular pathology of ACRDYS2, providing new insights into the molecular mechanisms underlying this disorder.

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

Circadian disruption, notably sleep disturbances, serves as an early indicator of Alzheimers disease (AD), preceding cognitive symptoms like memory loss. The suprachiasmatic nucleus (SCN) governs biological rhythms and receives direct retinal input via melanopsin-expressing retinal ganglion cells (mRGCs) to synchronize with environmental light cycles. The anatomical and functional basis for circadian disruption in AD remains unclear. Here, we explored the multi-level relationships between gene expression, the SCN connectome, and regulations of sleep and circadian rhythms in the APP/PS1 mouse model. The sleep architecture of APP/PS1 mice displayed significantly reduced rapid eye movement sleep (REM), associated with a reduced daily core body temperature amplitude and locomotor hyperactivity. Lastly, APP/PS1 mice showed an impaired response to acute light pulse stimulation and present hyperactivity of mRGCs at a young age and hypoactivity of these cells at older ages. These physiological functions are known to be, at least in part, regulated by the SCN, the main target of mRGCs. We noted several modifications in SCN connectomics using serial blockface electron microscopy (SBEM), including a reduction of the dendro-dendritic chemical synapse (DDCS) network that receives a large part of the retinal input and is thought to be crucial for synchronicity between SCN neurons. In addition, we observed multiple signs of dystrophy, including modifications of the shape of dendrites and cell soma, accumulation of aggregated lysosomes, and swelling of axons. At the same time, we investigated the changes in gene expression using spatial transcriptomics. The SCN presents changes in the expression of genes associated with synapse formation, cell adhesion, and neurite growth. These results suggest that, despite the absence of amyloid plaques in the ventral hypothalamus, the SCN of APP/PS1 mice still undergo profound gene expression changes, impacting connectomics and physiological functions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/744599v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@ceedb0org.highwire.dtl.DTLVardef@156cfaaorg.highwire.dtl.DTLVardef@5bc262org.highwire.dtl.DTLVardef@36df4d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Long noncoding RNAs encode for microproteins that regulate cellular functions. Small regulatory peptide of amino acid response (SPAR) is a microprotein in the lysosome that responds to amino acid availability of the cell. In this study, we investigated the interactions between SPAR and SLC38A9, a lysosomal amino acid transporter and receptor involved in the mechanistic target of rapamycin 1 (mTORC1) pathway. We found that SPAR binds SLC38A9 and inhibits arginine transport in SLC38A9. Moreover, the downstream recruitment of Rag GTPases is also inhibited when SPAR is present in SLC38A9 liposomes. Docking model shows potential interactions between SPAR and SLC38A9. Together, these findings reveal the mechanism of mTORC1 inhibition through microprotein SPAR and illustrates the power of non long coding RNAs in altering cellular functions. Statement of SignificanceMicroproteins encoded from long noncoding RNAs are emerging as critical regulators of many pathways. This study investigates a novel mechanism of SPAR microprotein that directly regulates the mechanistic target of rapamycin complex1 (mTORC1) signaling pathway through the lysosomal amino acid transporter SLC38A9. SPAR blocks both arginine transport and the downstream recruitment of Rag GTPases. These findings provide critical results in how SPAR controls cellular amino acid availability, while broadly highlighting the powerful regulatory mechanism of microproteins in cellular processes.

Background and AimsPregnancy requires dynamic, stage-specific adaptations in maternal liver metabolism and growth to sustain fetal development while preserving systemic homeostasis. Estrogen signaling, which significantly increases during pregnancy, is primarily mediated in hepatocytes by estrogen receptor (ER). Although hepatic ER regulates female liver metabolism under non-pregnant conditions, its role in pregnancy-induced hepatic remodeling remains unclear. MethodsWe studied non-pregnant and pregnant control and liver-specific ER knockout (LERKO) mice across gestational stages using longitudinal physiological measurements, liver transcriptomics, targeted metabolomics, histological assessment of cell proliferation, and metabolic phenotyping. ResultsIn control mice, pregnancy elicited sequential hepatic remodeling characterized by early induction of cell-cycle programs, a mid-gestational peak in hepatocyte proliferation with transient suppression of selected metabolic pathways, and late reactivation of specific metabolic programs. Chronic hepatic ER deficiency alters this temporal pattern. LERKO livers showed premature activation of proliferative and anabolic transcriptional programs, changes in amino acid- and fatty acid-related metabolic pathways, and altered temporal regulation of AKT-mTORC1-related signaling. At mid-gestation, LERKO mice displayed reduced hepatocyte proliferation, altered expression of metabolic and insulin-related genes, blunted gestational glucose adaptation without overt evidence of systemic insulin resistance, and changes in the light/dark-phase metabolic patterns. ConclusionsThese findings suggest that hepatic ER is required for the appropriate stage-specific coupling of liver growth, metabolic remodeling, and insulin-responsive signaling during pregnancy. Its loss is associated with gestational hepatic maladaptation and systemic metabolic phenotypes, providing a framework for investigating estrogen-dependent mechanisms underlying pregnancy-associated metabolic and liver disorders. HighlightsHepatic ER is required for stage-specific liver remodeling during pregnancy. Loss of hepatic ER alters temporal coupling of liver growth and metabolism. LERKO mice show early changes in amino acid- and fatty acid-related pathways. Hepatic ER loss reduces proliferation and alters gestational glucose adaptation. Hepatic ER loss is associated with altered light/dark-phase metabolic organization. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/743939v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@d52bborg.highwire.dtl.DTLVardef@b27511org.highwire.dtl.DTLVardef@23b286org.highwire.dtl.DTLVardef@19d9314_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cross-beaks are deemed a threat to poultry health, productivity, and animal welfare. Nevertheless, due to sporadic cases, heterogeneity of gene loci and incomplete dominance, the molecular mechanism of cross-beak formation, especially the degree of cross, is not yet clear. Thus, we screen key genes and reveal the possible phenotypic formation mechanism of cross-beak by comparison with different degrees of deformity in Huiyang Bearded chickens by compare whole-genome resequencing-based variant analysis. Comparative analysis between cross-beak and normal-beaked chickens identified differential variants in several candidate genes, including CDH11, CTNNAL1, NRXN3, NRXN1, CDH5, SDC3, and DHFR. Genes harboring these variants were enriched in pathways related to cell adhesion molecules and metabolic processes, with functional annotations involving cell-cell adhesion and neural crest cell migration. Comparative analysis between chickens with severe and slight cross-beak deformities identified additional candidate genes, including MRPL21, NSUN2, DDX55, GNB3, and NFKB2. These genes were associated with enriched terms and pathways related to focal adhesion, amyotrophic lateral sclerosis, steroid 7 -hydroxylase activity, and skin-barrier establishment. These findings provide a preliminary catalogue of genetic variants and candidate genes for future functional studies of cross-beak development and severity in chickens.

Different conformations, or strains, of -synuclein (-syn) aggregates are believed to be responsible for the distinct seeding propensities, propagation profiles, and clinical presentations in Lewy body diseases (LBD) and multiple system atrophy (MSA). While biochemical properties and strain differences of insoluble deposits have been extensively characterized, the understanding of what influence soluble -syn species may have on these processes is limited to a small number of studies focusing on complex mixtures of soluble species or on a single - synucleinopathy. Given that soluble oligomers are considered highly pathologically relevant, we isolated and characterized the biochemical, seeding, and toxicity properties of size-fractionated soluble -syn species from MSA and LBD brains, comparing them to species from control brains without known neurological disease (Ctrl). We observed that levels of differently sized oligomers phosphorylated at Ser129, as well as soluble large oligomers (>450 kDa), were increased in LBD compared to both MSA and Ctrl brains. Nevertheless, species derived from MSA brain exhibited seeding activity across the spectrum of -syn species (oligomers, monomers, and truncated forms) in the seed amplification assay, whereas only oligomeric species (>150 kDa) from LBD cases were seeding-prone. In the HEK293 -syn (A53T)-YFP biosensor line, as well as in murine primary neurons, only large oligomers (>450 kDa) from MSA cases induced seeding and aggregation of -syn. Taken together, our study suggests that soluble -syn species derived from MSA and LBD brains show different biochemical, aggregation and seeding patterns, presumably due to strain variations of the respective oligomers. Our findings provide novel insight into the pathogenesis of different -synucleinopathies, which may guide us in the development of targeted therapeutics.

Background. GBA2-associated hereditary spastic paraplegia (SPG46) is a rare autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in GBA2, encoding the non-lysosomal glucocerebrosidase 2. GBA2 deficiency leads to glucosylceramide (GlcCer) accumulation and glucosylated cholesterol (GlcChol) depletion, causing cytoskeletal defects in immature neurons. However, the mechanisms linking lipid dysregulation to neuronal dysfunction remain poorly understood. Methods. We modelled GBA2 loss of function by chronic pharmacological inhibition in mouse cerebellar granule neurons (CGNs) and assessed neuronal morphology, synaptic organization, Ca2+ dynamics, mitochondrial function and actin cytoskeleton during maturation. Proteomic profiling was performed in GBA2-inhibited and GlcChol-supplemented neurons. Findings were validated in a zebrafish gba2 crispant model by evaluating motor behavior, cerebellar development, neuronal organization and mitochondrial function, and in patient-derived fibroblasts carrying a homozygous pathogenic GBA2 variant (NM_020944). The role of RAC1 was studied in both neurons and patients' cultured skin fibroblasts, and upon rac1 pharmacological inhibition in zebrafish crispants. Results. Chronic GBA2 inhibition impaired axonal outgrowth in immature CGNs but not neurite complexity in mature neurons, suggesting morphological compensation. Nevertheless, mature neurons displayed enlarged presynaptic terminals, impaired synaptic vesicle clustering and altered Ca2+ responses to potassium and glutamate, the latter associated with NMDA receptor redistribution without changes in total receptor levels. Mitochondrial alterations were observed in CGNs, patient fibroblasts and zebrafish, consistent with defective architecture of the mitochondrial network. Proteomics revealed convergent alterations in actin cytoskeleton, synaptic pathways and cellular metabolism following both GBA2 inhibition and GlcChol supplementation. GlcChol bidirectionally regulated RAC1 function, likely altering its spatial distribution rather than its global activation. Confocal imaging confirmed abnormal RAC1 and F-actin localization in patient fibroblasts. Zebrafish gba2 crispants recapitulated motor deficits, Purkinje cell loss, motor neuron disorganization and mitochondrial abnormalities. Pharmacological Rac1 inhibition rescued motor behavior and neuronal organization, linking cytoskeletal disorganization to the observed phenotype in the zebrafish model. Conclusions. Our findings identify a pathogenic GlcChol-RAC1-actin signalling axis linking lipid imbalance to synaptic disorganization, NMDA receptor redistribution and mitochondrial dysfunction in SPG46. The selective vulnerability of corticospinal neurons, cerebellar granule neurons and Purkinje cells may reflect their dependence on this pathway. Rac1 inhibition rescues disease phenotypes in vivo, highlighting this pathway as a promising therapeutic target.

Cyclic adenosine monophosphate (cAMP) is a second messenger that regulates various cellular processes, including the activity of hyperpolarization-activated channels (HCN), which are implicated in cardiac physiology and neurodegenerative diseases such as Parkinsons disease (PD). In this study, we used a photoactivated adenylyl cyclase (PAC) S27A mutant to optogenetically control intracellular cAMP levels. We demonstrated that light-induced elevation of cAMP activated HCN4 channels, leading to increased beating rates in cardiomyocytes. Unilateral expression of PAC(S27A) in the substantia nigra pars compacta of mice induced rotation behavior upon light stimulation, which could be attenuated by HCN inhibitors. Furthermore, PAC(S27A) activation partially recovered motor deficits in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model, accompanied by increased HCN2 channel expression in ipsilateral basal ganglia. Our findings highlight the potential of using optogenetics to modulate cAMP and HCN channel activity for the treatment of cardiac and neurological disorders.

PurposeFuchs endothelial corneal dystrophy (FECD) is a progressive disease, causing premature death of corneal endothelial cells (CECs). Iron-dependent lipid peroxidation and ferroptosis mediate cell death in FECD. We aimed to determine whether FECD progression is mediated by derangements in ferritinophagy - a form of autophagy that degrades ferritin to release labile ferrous iron - and whether ultraviolet A (UVA) exposure drives FECD progression by activating ferritinophagy. MethodsEndothelium-Descemet membrane (EDM) tissues were collected from patients with end-stage FECD undergoing endothelial keratoplasty and from healthy age-matched donor corneas. Separately, immortalized FECD and healthy control CEC lines were cultured. Cellular levels of NCOA4 production and LC3 activation, both markers of ferritinophagy, were quantified using western blotting and PCR. UVA-exposed immortalized cells were plated on coverslips, stained for immunohistochemistry (IHC), and analyzed using confocal microscopy. Corneal endothelial peels were stained and analyzed using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). ResultsSurgically explanted FECD CECs showed significantly increased levels of NCOA4 compared to healthy controls. LC3 activation was increased in FECD immortalized CECs; UV exposure further increased LC3 activation. Additionally, UVA exposure showed trends of increased expression of NCOA4 in immortalized FECD and healthy CECs. On IHC of FECD surgical explant tissue, ferritin was decreased markedly, NCOA4 localized in a dramatic punctate pattern, and both ferritin and LC3 localized within cell nuclei. Spectrometry images showed higher iron levels correlating with areas of higher FECD disease burden. ConclusionsOur results demonstrate ferritinophagy in FECD indicated by the increase of NCOA4 and LC3 ferritinophagy markers in FECD patient and cell culture models. Our finding that UVA activates ferritinophagy implicates this mechanism in UVA-mediated FECD progression. Altogether, aberrant iron dysregulation associated with FECD and ferroptosis may be mediated by ferritinophagy, providing a biomarker to assess disease severity as well as a potential target for future medical therapeutics.

NOTCH3 is a transmembrane receptor highly expressed in vascular mural cells where it contributes to blood vessel formation and homeostasis. NOTCH3 expression declines in the vasculature with aging, and dysregulated NOTCH3 signalling is implicated in pulmonary arterial hypertension, cancer progression and CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy). RNA-based approaches targeting NOTCH3 are emerging as potential therapeutic strategies, however, the consequences of NOTCH3 suppression in mature vascular smooth muscle cells (VSMCs) remain incompletely understood. Here, we investigated the molecular and functional effects of siRNA-mediated NOTCH3 knockdown in human aortic smooth muscle cells. Transfection with NOTCH3-targeting siRNA efficiently suppressed NOTCH3 transcript and protein levels. Quantitative proteomics revealed remodelling of extracellular matrix (ECM), cytoskeletal and metabolic pathways, with enrichment of collagen biosynthesis and inhibition of glycolytic signalling. Specifically, NOTCH3 knockdown increased ECM components, including COL3A1, elevated F-actin, and upregulated the actin regulator, CTTN. In parallel, glycolytic capacity was reduced, accompanied by decreased expression of the glycolytic enzyme ENO2. Despite reduced VEGFA and alteration in angiogenic signalling proteins, endothelial network formation in co-cultures, as well as VSMC proliferation and migration remained unaffected. Finally, NOTCH3 interactome analysis revealed key collagen and actin-regulating proteins. These findings identify NOTCH3 as an important regulator of ECM homeostasis, cytoskeletal organisation, and glycolytic metabolism. The preservation of primary cellular functions despite molecular remodelling highlights the adaptive capacity of VSMCs. These findings demonstrate that therapeutic modulation of NOTCH3 may alter vascular cell biology which warrants consideration during development of RNA-based therapeutics for CADASIL and other NOTCH3-associated diseases.

BackgroundThe African green monkey (AGM) is increasingly used as a model for early-stage Alzheimers disease (AD), with cerebrospinal fluid (CSF) targeted for biomarker discovery and longitudinal disease monitoring of shifts in the central nervous system. MicroRNAs (miRNAs) are particularly informative indicators of early neuropathological change. Despite the complementary value of an early-stage disease model and a molecular marker capable of capturing early change, the miRNA composition (miRNome) of AGM remains undefined. We established the AGM CSF miRNome from antemortem samples using miRNA sequencing and a qRT-PCR-based array. We also developed a hierarchical annotation pipeline to classify miRNAs as either family-conserved or unclassified and to assess sequence alignment across humans and other species. ResultsWe used untargeted miRNA sequencing to characterize the AGM CSF miRNome and identified 205 miRNAs that could be classified into three family-conserved categories: canonical, noncanonical, and 3'-terminal variants. Of these, 150 were also detected using a human-targeted qRT-PCR array, providing independent support for the sequence-derived miRNome. Sequencing abundance and qRT-PCR array Ct values showed significant cross-platform concordance overall, although concordance was lower for 3'-terminal isomiRs than for canonical miRNAs. Comparison with human GTEx tissue-expression data indicated that several human homologs of AGM CSF miRNAs exhibited brain-preferential expression. Notably, predicted targets of many of these miRNAs were enriched for pathways implicated in neurodegenerative disease. Finally, we identified 20 unclassified candidates that could not be assigned to established miRNA families, two of which we propose as putatively novel miRNAs. ConclusionThe AGM CSF miRNome is substantially conserved with the human miRNome but also contains 3'-terminal isomiRs and unclassified miRNA candidates. AGM CSF contains miRNAs homologous to human miRNAs associated with AD and other neuropathologies, highlighting the translational potential of this model. However, our study also reveals challenges related to species-specific sequence variation and reduced cross-platform concordance for isomiRs. Thus, comparative studies will be needed to validate the functional and biomarker relevance of these miRNAs across species. More generally, this initial miRNome provides a reference resource for future studies of miRNAs in AGM across disease-related, physiological, experimental, and evolutionary contexts.

Excessive alcohol intake is frequently associated with hypertriglyceridemia, a condition that increases the risk of severe complications including acute pancreatitis and cardiovascular disease. The very low-density lipoprotein (VLDL) receptor (VLDLR) promotes uptake of apoE-containing VLDL particles by peripheral tissues and plays an important role in maintaining plasma triglyceride (TG) homeostasis. Brown adipose tissue (BAT) is a major metabolic organ that contributes to circulating lipid clearance during thermogenic activation. It was reported that cold-induced thermogenesis upregulates VLDLR expression in BAT and reduces plasma TG via VLDL uptake. However, whether BAT VLDLR-mediated VLDL uptake regulates alcohol-induced hypertriglyceridemia remains unknown. Here, we generated BAT-specific fatty acid synthase (FASN) knockout mice (FASNBKO) and subjected them to binge and acute-on-chronic alcohol feeding paradigms. We found that BAT FASN deficiency enhanced thermogenic function and promoted VLDL uptake, resulting in attenuation of alcohol-induced elevations in plasma TG. Consistent with these findings, pharmacological inhibition of FASN by TVB3664 treatment in differentiated brown adipocytes (bADs) increased thermogenic gene expression and VLDL uptake under both control and alcohol-exposed conditions. In addition, FASNBKO mice were protected from alcohol-induced hepatic steatosis, which was accompanied by increased hepatic AMP-activated-protein kinase (AMPK) activation and enhanced {beta}-oxidation. Furthermore, FASNBKO mice exhibited upregulated FGF21 mRNA expression in the BAT and elevated circulating FGF21 levels. Similarly, TVB3664-treated differentiated bADs showed higher FGF21 expression and increased FGF21 content in culture medium. Taken together, these findings identify the important role of brown adipocyte FASN in regulating thermogenic function and TG homeostasis during alcohol exposure and suggest that enhancing thermogenic lipid utilization in BAT may represent a potential therapeutic strategy for mitigating alcohol-associated increases in plasma TG and hepatic fat accumulation.

Childhood-onset DYT1 dystonia is a neurodevelopmental movement disorder caused by a three-base-pair deletion ({Delta}GAG; {Delta}E) in the TOR1A gene, which encodes TorsinA, a membrane-associated AAA+ (ATPase associated with diverse cellular activities) ATPase. However, the mechanisms by which the {Delta}E mutation causes neuronal dysfunction remain poorly understood. Using patient-derived neurons, we previously demonstrated that TorsinA-{Delta}E disrupts the nucleocytoplasmic transport (NCT) of both RNA and protein cargos. In the present study, proteomic analysis of induced human motor neurons revealed a markedly enhanced association between {Delta}E and exportin 1 (XPO1), a major nuclear export receptor. This aberrant association was enriched at the nuclear envelope and accompanied by impaired XPO1-mediated nuclear export. By integrating AlphaFold-based structural modeling with molecular, biochemical, and cellular analyses, we identified the N-terminal hydrophobic segment (HS) of TorsinA as a critical contributor to its interaction with XPO1. Deletion of the HS from {Delta}E reduced its association with XPO1, altered its nuclear envelope enrichment, and restored nuclear export. Moreover, expression of HS-derived peptides in patient-derived DYT1 neurons improved nuclear export, neurite outgrowth and branching, maturation-associated gene expression, and neuronal survival. Together, these findings identify an aberrant gain-of-function association between TorsinA-{Delta}E and XPO1 as a mechanism contributing to NCT dysfunction in DYT1 dystonia and establish the HS-dependent {Delta}E-XPO1 interaction as a potential therapeutic target.

Mitochondrial plasticity, characterized by the dynamic balance between glycolysis and oxidative phos-phorylation in response to genetic and microenvironmental changes, is a hallmark of melanoma progression. Sphingolipids play a significant role in various aspects of cancer cell biology, including metabolic reprogramming. Previous observations had shown that the lysosomal sphingolipid-metabolizing enzyme {beta}-galactosylceramidase (GALC) rewires the lipid profile of mouse melanoma cells, exerting pro-oncogenic functions, gene silencing leading to a decreased oncogenic activity in murine and human melanoma cells. Here, we have focused on the mitochondrial sphingolipid composition and energetic metabolism in GALC knockout (KO) A2058 human melanoma cells. Targeted analysis of the mitochondrial sphingolipid profile, transcriptomic data, and mitochondrial structural and functional studies indicate that GALC loss drives a sphingolipid-mediated reprogramming of mitochondrial metabolism in absence of major structural alterations, characterized by bioenergetic insufficiency possibly due to ceramide- and sphingomyelin-driven impairment of respiratory chain function. Overall, these data indicate that GALC KO leads to a sphin-golipid-driven mitochondrial metabolic suppression and may provide novel information for the development of efficacious approaches in mitochondrial targeting melanoma therapies.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent liver disorder worldwide, yet therapeutic options remain limited. TMPRSS6, a liver serine protease best known for its role in iron homeostasis, has recently emerged as a potential therapeutic target for MASLD. However, the molecular mechanisms linking TMPRSS6 to hepatic lipid metabolism remain incompletely understood. To identify novel TMPRSS6 substrates, we performed extracellular proteomic analyses of TMPRSS6-overexpressing cells. Among the proteins identified, {beta}-klotho (KLB), a co-receptor required for FGF19 and FGF21 signaling, emerged as a compelling candidate substrate. We demonstrate that TMPRSS6 interacts with KLB and promotes its proteolytic shedding in a catalytic activity-dependent manner. Functionally, TMPRSS6 reduced full-length KLB abundance at the cell surface and attenuated FGF19-dependent FGFR4 signaling in a heterologous expression system. Together, these findings identify KLB as a novel functional substrate of TMPRSS6, providing a mechanistic framework through which this protease may influence hepatic lipid metabolism. These results provide a rationale for investigating the regulation of KLB and other candidate substrates by TMPRSS6 in physiological models and further support its evaluation as a therapeutic target for MASLD.