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PubMed1979

Lysosomal dysfunctions associated with mutations at mouse pigment genes.

Melanosomes and lysosomes share several structural and biosynthetic properties. Therefore, a large number of mouse pigment mutants were tested to determine whether genes affecting melanosome structure of function might also affect the lysosome. Among 31 mouse pigment mutants, six had 1.5- to 2.5-fold increased concentrations of kidney beta-glucuronidase. Three mutants, pale ear, pearl and pallid, had a generalized effect on lysosomal enzymes since there were coordinate increases in kidney beta-galactosidase and alpha-mannosidase. The effects of these three mutations are lysosome specific since rates of kidney protein synthesis and activities of three nonlysosomal kidney enzymes were normal. Also, the mutants are relatively tissue specific in that all had normal liver lysosomal enzyme concentrations.--A common dysfunction in all three mutants was a lowered rate of lysosomal enzyme secretion from kidney into urine. While normal C57BL/6J mice daily secreted 27 to 30% of total kidney beta-glucuronidase and beta-galactosidase, secretion of these two enzymes was coordinately depressed to 1 to 2%, 8 to 9% and 4 to 5% of total kidney enzyme in the pale-ear, pearl and pallid mutants, respectively. Although depressed lysosomal enzyme secretion is the major pigment mutant alteration, the higher lysomal enzyme concentrations in pearl and pallid may be partly due to an increase in lysosomal enzyme synthesis. In these mutants kidney glucuronidase synthetic rate was increased 1.4- to 1.5-fold.--These results suggest that there are several critical genes in mammals that control the biogenesis, processing and/or function of related classes of subcellular organelles. The mechanism of action of these genes is amenable to further analysis since they have been incorporated into congenic inbred strains of mice.

Animals↗
PubMed2026

Intra-locus coeruleus LPS administration induces anxiety-like behavior, thermal hyperalgesia, and striatal lysosomal alterations: Relevance to Parkinson's disease.

According to Braak's staging hypothesis, Parkinson's disease (PD) pathology may originate in extranigral regions, including the locus coeruleus (LC). In parallel, PD has been associated with lysosomal dysfunction. Here, we investigated whether intra-LC lipopolysaccharide (LPS) injection may produce behavioral alterations and lysosomal protein changes in the striatum and prefrontal cortex (PFC), regions critically implicated in PD pathology. Adult male Wistar rats received unilateral injections of saline or LPS (10 µg/2 µL) into the LC or striatum and were assessed for anxiety-like behavior, thermal hyperalgesia, and motor coordination. A separate cohort was sacrificed 15 days post-injection to assess lysosomal proteins (cathepsin D, β-glucocerebrosidase, Lysosomal Associated Membrane Protein 2 (LAMP2)) and α-synuclein (α-Syn). Intra-LC LPS induced anxiety-like behavior, reflected by reduced time spent in the center of the open field, and thermal hyperalgesia, as shown by shortened tail-flick latency, whereas intra-striatal LPS impaired locomotion and motor coordination, evidenced by reduced line crossings and decreased rotarod performance. Intra-LC but not intra-striatal LPS reduced LAMP2 levels in the striatum, while all other markers remained unchanged in both regions. These findings provide experimental support for Braak's hypothesis.

Animals↗
PubMed2026

Genetic and molecular evidence linking CTSH to Alzheimer's disease pathophysiology.

INTRODUCTION: Lysosomal dysfunction contributes to Alzheimer's disease (AD) by impairing protein clearance and promoting neuroinflammation. Cathepsin H (CTSH), a lysosomal protease, recently emerged as a protective AD locus. We investigated how CTSH is regulated and how it influences early AD pathophysiology. METHODS: We analyzed genomic, transcriptomic, and proteomic data from cerebrospinal fluid (CSF) and brain tissue across three independent clinical and post mortem cohorts to assess CTSH regulation, expression, and disease associations. RESULTS: The coding variant rs2289702 acts as a cis-regulatory variant, altering CTSH mRNA and protein levels. The T allele associates with better cognition and reduced amyloid plaque burden. CSF CTSH correlates with total tau, phosphorylated tau181, neuronal markers, and multiple glial and complement-related inflammatory proteins. DISCUSSION: CTSH tracks early neurodegenerative, synaptic, and inflammatory changes, and co-expression analyses link it to broader immune-metabolic pathways. The findings position CTSH as a genetically regulated contributor to AD pathophysiology.

Humans↗
PubMed2026

Micro- and nanoplastics-induced neurotoxicity: a CNS-centered, evidence-graded adverse outcome pathway framework based on systematic weight-of-evidence assessment.

Micro- and nanoplastics (MPs/NPs) are ubiquitous anthropogenic particulate pollutants posing emerging threats to human neurological health. Severe heterogeneity in particle physicochemical properties, environmental aging status, exposure paradigms and experimental platforms has created persistent mechanistic uncertainties in MP/NP neurotoxicology, hindering reliable hazard characterization and risk translation. Here, we systematically consolidate empirical toxicological evidence and construct a dedicated central nervous system (CNS)-targeted adverse outcome pathway (AOP) network integrated with rigorous weight-of-evidence (WoE) grading to elucidate the hierarchical, particle-specific toxic cascades underlying MP/NP-induced neural injury. Our synthesis overturns the conventional linear toxicity paradigm, demonstrating that MPs/NPs trigger neurotoxicity via a complex multi-input mechanistic network. We definitively establish oxidative stress as a robust early convergent key event-rather than a universal molecular initiating event-orchestrating ROS overproduction, lipid peroxidation, mitochondrial dysfunction, and neuroinflammation to propagate neuronal damage. This core module is driven by five distinct particulate upstream triggers: particle-biomolecule interfacial perturbation, corona-facilitated cellular internalization, plastic-associated chemical leaching, aging-derived free radical reactivity, and gut-borne systemic neurotoxic signaling. Downstream pathogenic outcomes encompass glial overactivation, neurotransmitter dyshomeostasis, autophagy-lysosome dysfunction, metabolic reprogramming, regulated neuronal cell death, and behavioral impairments. Tiered WoE analysis confirms strong validation for early oxidative/inflammatory cascades, moderate support for gut-brain axis crosstalk and intracellular trafficking disruption, and nascent evidence for synaptic dysfunction and neurodegeneration-linked proteostatic defects. Extrapolation to human health risk remains constrained by the frequent use of high-dose exposure paradigms, limited validated data on internal dosimetry in the human brain, discrepancies between effective concentrations in experimental models and environmentally relevant human tissue burdens, and insufficient causal validation of distal adverse outcomes. We highlight key research priorities including aged mixed-particle exposure systems, leachate-controlled assays, quantitative internal dose evaluation, and mechanistic intervention verification. This evidence-stratified AOP framework resolves longstanding mechanistic ambiguities in particulate neurotoxicity, providing a standardized, causality-based foundation for future mechanistic exploration and health risk assessment of global plastic pollution.

Adverse outcome pathway↗
PubMed2026

Inflammatory cell death and monocyte dysfunction in VEXAS syndrome.

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a severe adult-onset autoinflammatory disease caused by somatic mutations in the UBA1 gene, disrupting cytoplasmic ubiquitin-activating enzyme E1 function in hematopoietic progenitors. Its pathogenesis remains poorly understood, particularly the mechanisms by which UBA1 mutations disrupt myeloid cell function in the context of inflammatory stimuli. Here, we combine a genetically engineered THP-1 monocytic model with ex vivo analyses of blood and tissue samples from patients with VEXAS syndrome to investigate the consequences of the canonical UBA1M41V mutation. We show that UBA1-mutated monocytes exhibit tumor necrosis factor α (TNF-α)-induced cell death, characterized by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) phosphorylation, and mixed lineage kinase domain-like- and caspase-8-mediated cell death. Importantly, we extend these findings to patient-derived CD14+ sorted cells, confirming that these cells undergo aberrant apoptotic and necroptotic cell death. Mechanistically, activation of these cell death pathways appears to be promoted by defective NF-κB-dependent transcriptional responses and reduced cFLIP(L) expression following TNF-α stimulation. UBA1-mutated monocytes also display blunted cytokine responses to Toll-like receptor (TLR) agonists despite preserved TLR expression, linked to an impaired NF-κB response. UBA1M41V-derived macrophages exhibit a proinflammatory transcriptional profile with increased chemokine secretion that promotes monocyte recruitment. In addition, these UBA1-mutated macrophages display impaired efferocytosis due to lysosomal dysfunction. Together, these findings reveal a pathogenic axis in VEXAS syndrome linking UBA1 loss of function and defective ubiquitination to RIPK1-mediated inflammatory cell death, impaired antimicrobial signaling, and defective resolution mechanisms. Our study provides novel mechanistic insights into the myeloid dysfunction underlying inflammation and cytopenia in VEXAS syndrome and supports the therapeutic targeting of inflammatory cell death pathways.

Humans↗
PubMed2026

Biologic Therapies for Alleviating Neurodegeneration in Lysosomal Storage Diseases.

Lysosomal storage diseases (LSDs) are a group of rare inherited metabolic disorders characterized by lysosomal dysfunction and progressive accumulation of undegraded substrates, leading to multisystem involvement and, in many cases, severe neurodegeneration. Because the blood-brain barrier (BBB) restricts central nervous system (CNS) access for most therapeutic modalities, neurological manifestations remain the major unmet need across LSDs. In this review, we summarize current and emerging strategies aimed at correcting CNS pathology, including enzyme replacement therapy (ERT), adeno-associated virus (AAV)-mediated gene therapy, allogeneic hematopoietic stem cell transplantation (HSCT), and autologous HSCT with gene-modified hematopoietic stem cells. While ERT provides limited CNS benefits and allogeneic HSCT mitigates neurodegeneration only partially, their overall impact on CNS outcomes remains restricted. Newer approaches, such as BBB-shuttling ERTs, CNS-tropic AAV capsids, and genetically modified autologous hematopoietic stem and progenitor cells capable of sustained supraphysiological enzyme production, offer promising avenues for enhanced CNS delivery and cross-correction. Together, these advances underscore a shift toward integrated therapeutic strategies that combine systemic and CNS-directed interventions, with the potential to transform outcomes for patients with LSDs and other neurodegenerative disorders amenable to cross-correction.

Journal Article↗
PubMed2025

Inhibition of autophagy-lysosomal function exacerbates microglial and monocyte lipid metabolism reprograming and dysfunction after brain injury.

CNS has an overall higher level of lipids than all tissues except adipose and contains up to 25% of total body cholesterol. Recent data demonstrate a complex crosstalk between lipid metabolism and inflammation, suggesting potential contribution of the lipid-rich brain environment to neuroinflammation. While recent data support the importance of brain lipid environment to inflammatory changes observed in age related chronic neurodegenerative diseases, in vivo interactions between lipid environment, lipid metabolism and neuroinflammation in acute brain disease and injury remain poorly understood. Here we utilize a mouse model of traumatic brain injury (TBI) to demonstrate that acute neurotrauma leads to widespread lipid metabolism reprograming in all microglial and brain associated and infiltrating monocyte populations. Additionally, we identify unique microglial and monocyte populations with higher degree of lipid metabolism reprograming and pronounced accumulation of neutral storage lipids, including cholesteryl esters and triglycerides. These lipids accumulate not only in lipid droplets but also in the microglial and monocyte lysosomes and are associated with lysosomal dysfunction and inhibition of autophagy after TBI. Our data indicate that lipid accumulation in these cells is the result of altered lipid handling rather than lipid synthesis and is triggered by phagocytosis of lipid-rich myelin debris generated after TBI. Finally, we use mice with autophagy defects in microglia and monocytes to demonstrate that further inhibition of autophagy leads to more pronounced lipid metabolism reprograming and exacerbated cellular lipid accumulation. Our data suggest a pathological feedback loop, where lipid phagocytosis causes inhibition of autophagy-lysosomal function, which in turn exacerbates cellular lipid retention, reprograming and inflammation.

Journal Article↗
PubMed2026

Cholesterol dysregulation in APOE4 astrocytes promotes α-synuclein pathology in miBrains.

The pathological hallmarks of neurodegeneration are the aberrant post-translational modification and aggregation of proteins. Genetic factors, like APOE4, increase the prevalence and severity of tau, amyloid, and α-synuclein pathologies. However, the human brain is largely inaccessible during this process, limiting mechanistic understanding. Here, we developed an iPSC-based 3D model that integrates neurons, glia, myelin, and cerebrovascular cells into a human brain-like tissue ("miBrain"). Single-nucleus RNA sequencing of miBrains confirmed the presence of diverse cell populations and revealed transcriptional responses to α-synuclein pathology. Like the human brain, pathogenic α-synuclein is increased in APOE4/4 miBrains. Combinatorial experiments revealed that endolysosomal dysfunction caused by cholesterol accumulation in APOE4/4 astrocytes impairs the degradation of soluble α-synuclein leading to a pathogenic transformation that seeds α-synuclein inclusions in neurons. Collectively, this study establishes a robust model for investigating protein inclusions in human iPSC-derived brain tissue and highlights the role of astrocytes and cholesterol in APOE4-mediated pathologies.

alpha-Synuclein↗
PubMed2025

Rescue of common and rare exon 2 skipping variants of the GAA gene using modified U1 snRNA.

BACKGROUND: Pompe disease (PD) is an autosomal recessive lysosomal storage disorder caused by the deficient activity of acid alpha glucosidase (GAA) enzyme due to mutations in the GAA gene. As a result, undigested glycogen accumulates within lysosomes causing their dysfunction. From a clinical point of view, the disease can be classified in infantile-onset (IO) and late-onset (LO) forms. The common GAA c.-32-13T>G variant, found in 40-70% of LO-PD alleles, is a leaky splicing mutation interfering with the correct GAA exon 2 recognition by the spliceosome leading to the production of non-functional GAA transcripts. In this study, we used modified, GAA-tailored U1 snRNAs to correct the aberrant splicing determined by the c.-32-13T>G and other GAA exon 2-skipping mutations. METHODS: A set of constructs expressing 5 different engineered U1 snRNAs was generated. A functional splicing assay using a GAA hybrid minigene carrying different variants known to affect GAA exon 2 splicing was used to test the effect of engineered U1 snRNAs on exon 2 inclusion. The effect on endogenously expressed GAA transcript and GAA enzymatic activity was assessed by transfecting patient-derived fibroblasts bearing the common c.-32-13T>G with the best performing modified U1 snRNA. RESULTS: Modified U1-3, U1+1 and U1+6 snRNAs were all able to increase, in a dose-dependent manner, the inclusion of exon 2 within the transcript derived from the GAA minigene harbouring the c.-32-13T>G variant. The U1+1 was the most effective one (2,5 fold increase). Moreover, U1+1 snRNA partially rescued the correct splicing of GAA minigenes harbouring mutations that affect the 3'ss (c.-32-3C>G, c.-32-2A>G) and the 5'ss (c.546G>A, c.546G>C, c.546G>T). Notably, the treatment of patient-derived fibroblasts carrying the c.-32-13T>G mutation with the U1+1 snRNA increased the amount of normal GAA mRNA by 1,8 fold and the GAA enzymatic activity by 70%. CONCLUSIONS: we provide the proof-of-concept for the use of modified GAA-tailored U1 snRNAs, designed to potentiate the recognition of the GAA exon 2 5'ss, as therapeutic tools to correct the aberrant transcripts carrying variants that affect exon 2 splicing, including the common c.-32-13T>G variant.

Humans↗
PubMed1979

Diffuse glomerulosclerosis--a dysfunction of the mesangium? A morphometric study of the rat's kidney.

The phagocytic capacity of the glomerular mesangial cells in 42 rats was inhibited by the prolonged injection of PVA. Morphometric evaluation of electron microscopic sections showed that this treatment leads to an accelerated thickening of the lamina densa of the glomerular basement membrane and of the mesangial matrix (early glomerulosclerosis). This suggests that a disturbance of lysosomal degradation can lead to glomerulosclerosis, and that the possibility of mesangial dysfunction should always be considered as a possible factor in the pathogenesis of all types of diffuse glomerulosclerosis.

Animals↗
PubMed2026

Neonatal gene therapy with AAV2/8-LSPhGAA improves hypertrophic cardiomyopathy in the Gaac.1826dupA knock-in murine model.

Pompe disease (PD) results from lysosomal acid α-glucosidase (GAA) deficiency, causing lysosomal glycogen accumulation in cardiac and skeletal muscles. We previously characterized a murine model carrying the orthologous human infantile-onset PD (IOPD) pathogenic variant, c.1826dupA (p.Y609*), introduced into the mouse Gaa gene. Compared to wild-type (WT; C57BL/6NJ) controls, Gaac.1826dupA mice exhibit reduced GAA activity and develop early-onset hypertrophic cardiomyopathy-evidenced by increased left ventricular wall thickness and left ventricular mass index (LVMI)- as well as impaired grip strength and gait abnormalities. To benchmark the model's disease fidelity and assess its responsiveness to established therapeutic intervention, Gaac.1826dupA mice received a single retro-orbital dose of AAV2/8-LSPhGAA (2 × 109 vg/g body weight) at postnatal day 12-14. Twelve weeks post-treatment, mice exhibited supraphysiological GAA enzymatic activity in the heart (550% of WT) and liver (400% of WT) with a 93% reduction in cardiac glycogen. No sex-dependent differences in therapeutic efficacy were observed. Echocardiography revealed robust reversal of cardiac pathology, with wall thicknesses and LVMI values approaching WT levels. In contrast to this profound cardiac rescue, skeletal muscle improvements were modest; while forelimb grip strength remained unchanged, automated gait analysis showed benefit limited to hind paw base of support. These findings demonstrate that the Gaac.1826dupA model mirrors the critical cardiomyopathy characteristic of IOPD. While systemic AAV treatment yields definitive cardiac correction, the partial skeletal muscle response highlights a clear need for optimization. Consequently, the Gaac.1826dupA mouse serves as a high-fidelity platform for evaluating next-generation genomic correction strategies targeting both cardiac and refractory neuromuscular manifestations of PD.

Acid α-glucosidase↗
PubMed2026

A human lysosomal storage disorder toolkit for decoding proteome landscapes in cortical-like and dopaminergic-like induced neurons.

Lysosomes maintain cellular homeostasis by degrading proteins delivered via endocytosis and autophagy and by recycling building blocks for organelle biogenesis. Lysosomal storage disorders (LSDs) comprise a group of diseases affecting diverse lysosomal functions. To facilitate molecular phenotyping across diverse LSD gene classes, we are developing a library of human embryonic stem cells engineered to lack individual LSD genes as a resource for the field. Here, we report our initial stem cell toolkit lacking one of 23 LSD genes, including the majority of genes associated with sphingolipidoses and neuronal ceroid lipofuscinoses, and its use in the generation of a proteomic resource for induced cortical-like and midbrain dopaminergic-like neurons. In-depth abundance and correlation profiling across organelles and suborganelle components revealed potential vulnerabilities that reflect distinct patterns of proteome alterations across both genotypes and neuronal cell types. We characterize alterations in the mitochondrial proteome associated with GBA1 and ASAH1 deficiency and identify synaptic and mitochondrial defects in ASAH1-/- induced neurons that correlate with defects in neuronal firing rates. Moreover, we developed an informatic pipeline for proteome-wide identification of individual protein-protein interactions and protein complexes that may be disrupted as a result of LSD gene deficiency. Finally, we visualized structural alterations of ASAH1-deficient endolysosomes in situ using cryoelectron tomography, revealing swollen organelles that were largely devoid of dense internal membranes characteristic of wild-type cells, but containing numerous intralumenal vesicle compartments. This toolkit and associated proteomic landscapes provide a resource for defining molecular signatures associated with LSD gene dysfunction and organelle vulnerability.

Humans↗
PubMed2025

Diffusion tensor imaging in Chediak Higashi Disease.

AIM: To define the natural history of diffusion tensor imaging (DTI) in Chediak-Higashi Disease (CHD) participants in relation to normative development. METHODS: Twenty-five DTI scans from 15 CHD participants were compared with 100 DTI scans from 100 neurotypical controls (NC). Comparisons were evaluated for DTI metrics including fractional anisotropy (FA), and mean, axial, and radial diffusivity (MD, AD, RD, respectively). Correlational tractography was also performed to identify group differences between CHD and NC. RESULTS: Pediatric CHD participants had DTI metrics similar to NC, however, progressive pathogenic increases in MD, AD, and RD were observed in CHD participants compared to NC in white matter pathways of the whole brain, corpus callosum, and cerebellum. Correlational fiber tractography identified fiber tracts with decreased FA, and increased MD, AD, and RD in CHD compared to NC. INTERPRETATION: Progressive deviations in DTI metrics highlight the progressive neurodegeneration of CHD. Aberrations in cerebellar white matter is reflective of the clinical neurologic phenotype of CHD including cerebellar dysfunction and cognitive decline. The corollary of progressive aberrations in DTI metrics and progressive clinical neurodegeneration suggest DTI may be a suitable neuroimaging marker for CHD. Future studies should evaluate functional magnetic resonance imaging and volumetric studies in this cohort.

Chediak-Higashi Disease↗
PubMed2025

Similarities and Differences in the Late-Onset GM2 Gangliosidoses: Tay-Sachs and Sandhoff Diseases.

The two predominating subtypes of late-onset GM2 gangliosidosis are late-onset Tay-Sachs (LOTS) and late-onset Sandhoff disease (LOSD). Due to shared deficiencies of ß-hexosamindase A and significant clinical overlap, the two diseases have been considered indistinguishable. However, a growing body of evidence supports the notion of several distinctions between the two diseases. In this study, we highlight these distinctions through the cross-sectional evaluation of 27 late-onset GM2 gangliosidosis participants. Twenty-one participants with LOTS and 6 with LOSD were included in this study. We performed physical examinations alongside assessments for gait, balance, muscle strength, ataxia, nerve conduction velocities, and analyzed brain magnetic resonance imaging. Lower limb weakness (95% in LOTS, 100% in LOSD) and later development of upper limb weakness (90% in LOTS, 83% in LOSD) was highly prevalent in both cohorts. Accompanying gait disturbances, balance issues, and dysmetria (as assessed by the brief ataxia rating scale [BARS]) were also prevalent in both cohorts. Strength testing for the quadriceps and hamstrings demonstrated weakness in both cohorts, primarily impacting extensor muscles. Supratentorial gray and white matter volumes in both cohorts were similar to normative data. In contrast, BARS scores for dysarthria and oculomotor dysfunction were present and heterogenous in LOTS participants and absent in LOSD participants. 24% of LOTS participants and none of the LOSD participants had a history of neuropsychiatric symptoms. Cerebellar volume including lobules V and VI were lower in LOTS compared to LOSD and normative data. However, length dependent sensory neuropathy was present in all LOSD participants but absent in LOTS participants. Dysfunction of the posterior cerebellum (lobules VI, VII, and IX) has been shown to cause cerebellar cognitive affective syndrome (CCAS), that includes cognitive and behavioral disturbances. Furthermore, cerebellar dysfunction of lobules V and VI has been linked to dysarthric speech, and dysfunction of the posterior cerebellum has been linked to oculomotor symptoms. The finding of low cerebellar lobule volumes in LOTS, suggests the distinctive features of the LOTS phenotype are related to cerebellar dysfunction. However, the sensory symptoms unique to LOSD remains a mystery. The molecular and biochemical basis for the dichotomy between the LOTS and LOSD phenotypes requires further investigation.

GM2 Gangliosidosis↗
PubMed2026

Transcriptome Analysis and Experimental Validation of Palmitoylation- Related Biomarkers in Atherosclerosis.

INTRODUCTION: Protein palmitoylation contributes to membrane localisation, signal transduction, and cell-fate regulation. It is closely associated with lipid metabolic dysfunction, immune inflammation, and vascular remodelling in atherosclerosis (AS). However, key palmitoylation-related transcriptomic markers and their potential causal associations with AS remain incompletely defined. METHODS: The Gene Expression Omnibus (GEO) dataset GSE100927 was used as the training cohort, and GSE43292 was used as an external validation cohort. Differentially expressed genes were identified using limma and intersected with palmitoylation-related genes to obtain palmitoylation-related differentially expressed genes (PRDEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were then performed using clusterProfiler. Two-sample Mendelian randomisation was used to evaluate potential causal relationships between characteristic genes and AS. Feature selection was conducted using random forest and support vector machine recursive feature elimination (SVM-RFE), and the overlapping genes selected by both methods were retained. Receiver operating characteristic (ROC) curves were used to assess diagnostic performance. A five-gene nomogram was constructed, and its clinical utility was evaluated using calibration curves and decision curve analysis (DCA). Gene set variation analysis (GSVA) was applied to compare pathway activity between high- and low-expression groups for each core gene. Single-cell analysis using Seurat and expression-based cell-cell communication analysis using CellChat were conducted with GSE159677, and upstream transcription factors were predicted using NetworkAnalyst. For in vivo validation, an AS model was established in ApoE⁸/⁸ mice fed a high-fat diet, and aortic gene and protein expression were assessed by RT-qPCR and western blotting. RESULTS: In GSE100927, 51 PRDEGs were identified. GO and KEGG enrichment analyses highlighted pathways associated with regulation of monoatomic ion transport, sarcomere and myofibril organisation, and immune inflammation. Mendelian randomisation suggested a potential protective causal association between SLC7A7 and AS. By integrating MR with random forest and SVM-RFE feature selection, we prioritised five core genes: PLCB2, GMIP, NEXN, PLN, and SLC7A7. These genes showed good diagnostic performance in GSE43292. The resulting nomogram was well calibrated and demonstrated stable net benefit in decision curve and clinical impact curve analyses. Single-gene GSVA identified consistently activated pathways across multiple genes, including innate and adaptive immune recognition, calcium signalling and myocardial contraction/cardiomyopathy, extracellular matrix-receptor interaction, cell junction pathways, autophagy-lysosome pathways, and several metabolic programmes. At the single-cell level, PLCB2 and GMIP were predominantly expressed in T cells and macrophages, NEXN and PLN were enriched in vascular smooth muscle cells, and SLC7A7 was mainly expressed in macrophages. CellChat analysis indicated increased signals for immune-related ligand-receptor interactions. In ApoE⁸/⁸ mice fed a high-fat diet, PLCB2, GMIP, and SLC7A7 were upregulated, whereas NEXN and PLN were downregulated; protein-level changes were concordant with the transcriptomic trends. DISCUSSION: These findings indicate that palmitoylation-related dysregulation in AS converges on immune inflammation, calcium signalling/contractile programmes, ECM remodelling, and autophagy-linked metabolism. The five-gene panel is supported by external validation, single-cell localisation to immune and vascular compartments, and concordant results in ApoE⁸/⁸ mice. CONCLUSION: This study identified and validated five palmitoylation-related genes associated with AS. SLC7A7 showed a potential protective causal signal in MR analysis. The enriched pathway patterns linked these genes to immune inflammation, calcium signalling-contraction coupling, ECM remodelling, cell adhesion, and autophagy- associated metabolic reprogramming. The five-gene nomogram showed potential utility for diagnostic classification and decision support, nominating candidate biomarkers and pathway targets for AS molecular subtyping, diagnosis, and mechanistic investigation.

Atherosclerosis (AS)↗