Intracellular Cholesterol Transport Research Articles

Comprehensive Multi-Omics Analysis Reveals NPC2 and ITGAV Genes as Potential Prognostic Biomarkers in Gastrointestinal Cancers.

Gastrointestinal cancers (GICs) continue to dominate in terms of both incidence and mortality worldwide. Due to the absence of efficient and accurate prognostic biomarkers, the prognosis and treatment outcomes of many GICs are poor. Identifying biomarkers to predict individual clinical outcomes efficiently is a fundamental challenge in clinical oncology. Although several biomarkers have been continually discovered, their predictive accuracy is relatively modest, and their therapeutic use is restricted. In light of this, the discovery of reliable biomarkers for predicting prognosis and outcome in GIC is urgently required. We evaluated the Human Protein Atlas dataset and identified NPC Intracellular Cholesterol Transporter 2 (NPC2) and Integrin Subunit Alpha V (ITGAV) as probable poor predictive genes for these cancers. In addition, we used the GEPIA2, cBioPortal, UALCAN, LinkedOmics, STRING, Enrichr, TISDB, TIMER2.0, hTFTarget, miRTarBase, circBank, and drug-gene interaction database databases to conduct a comprehensive and systematic analysis of the NPC2 and ITGAV genes. Our results found high expression levels of NPC2 and ITGAV in most GICs. The aforementioned gene expressions were linked to several clinicopathological characteristics of GICs as well as poorer prognosis in LIHC and STAD. The most common alteration type of NPC2 was amplification, and for ITGAV was deep deletion. Significant promotor hypermethylation was also seen in NPC2 and ITGAV in PAAD and COAD, respectively. For the immunologic significance, NPC2 and ITGAV were positively correlated with the abundance of tumor-infiltrating lymphocytes and macrophages. Furthermore, various immunomodulators showed strong correlations with the expression of these genes. There were currently 10 small molecule drugs targeting ITGAV. Consequently, our bioinformatics analysis showed that NPC2 and ITGAV might be used as potential biomarkers to determine the prognosis of various GICs and are also related to immune infiltration.

Graphene Microelectrode Arrays, 4D Structured Illumination Microscopy, and a Machine Learning Spike Sorting Algorithm Permit the Analysis of Ultrastructural Neuronal Changes During Neuronal Signaling in a Model of Niemann-Pick Disease Type C.

Simultaneously recording network activity and ultrastructural changes of the synapse is essential for advancing understanding of the basis of neuronal functions. However, the rapid millisecond-scale fluctuations in neuronal activity and the subtle sub-diffraction resolution changes of synaptic morphology pose significant challenges to this endeavor. Here, specially designed graphene microelectrode arrays (G-MEAs) are used, which are compatible with high spatial resolution imaging across various scales as well as permit high temporal resolution electrophysiological recordings to address these challenges. Furthermore, alongside G-MEAs, an easy-to-implement machine learning algorithm is developed to efficiently process the large datasets collected from MEA recordings. It is demonstrated that the combined use of G-MEAs, machine learning (ML) spike analysis, and 4D structured illumination microscopy (SIM) enables monitoring the impact of disease progression on hippocampal neurons which are treated with an intracellular cholesterol transport inhibitor mimicking Niemann-Pick disease type C (NPC), and show that synaptic boutons, compared to untreated controls, significantly increase in size, leading to a loss in neuronal signaling capacity.

EXTL3 and NPC1 are mammalian host factors for Autographa californica multiple nucleopolyhedrovirus infection

Baculovirus is an obligate parasitic virus of the phylum Arthropoda. Baculovirus including Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been widely used in the laboratory and industrial preparation of proteins or protein complexes. Due to its large packaging capacity and non-replicative and non-integrative natures in mammals, baculovirus has been proposed as a gene therapy vector for transgene delivery. However, the mechanism of baculovirus transduction in mammalian cells has not been fully illustrated. Here, we employed a cell surface protein-focused CRISPR screen to identify host dependency factors for baculovirus transduction in mammalian cells. The screening experiment uncovered a series of baculovirus host factors in human cells, including exostosin-like glycosyltransferase 3 (EXTL3) and NPC intracellular cholesterol transporter 1 (NPC1). Further investigation illustrated that EXTL3 affected baculovirus attachment and entry by participating in heparan sulfate biosynthesis. In addition, NPC1 promoted baculovirus transduction by mediating membrane fusion and endosomal escape. Moreover, in vivo, baculovirus transduction in Npc1−/+ mice showed that disruption of Npc1 gene significantly reduced baculovirus transduction in mouse liver. In summary, our study revealed the functions of EXTL3 and NPC1 in baculovirus attachment, entry, and endosomal escape in mammalian cells, which is useful for understanding baculovirus transduction in human cells.

The role of NPC2 gene in glioma was investigated based on bioinformatics analysis

Glioblastoma (GBM) is one of the most malignant primary brain tumors in adults. The NPC2 gene (Niemann–Pick type C intracellular cholesterol transporter 2) is a protein-coding gene with a lipid recognition domain. The NPC2 gene was found to be significantly increased in gliomas (LGG and GBM), and it is now thought to be a risk factor. COX analysis demonstrated that NPC2 was a significant risk factor for glioma. Functional enrichment analysis of genes that were differentially expressed between high and low expression groups revealed that genes were primarily enriched in the regulation of trans-synaptic signaling, Retrograde endocannabinoid signaling and other pathways. According to the findings of the immunoinfiltration investigation, the NPC2 gene and macrophage, DC, etc. have a strong positive association. In addition, patients with high NPC2 expression had higher levels of immune cell expression. Medication sensitivity research revealed that NPC2’s differential expression had some bearing on patients’ medication sensitivity. There was a strong correlation between the prognosis of glioma patients and the gene sets NUDT19 and NUME. In brief, the NPC2 gene was identified to be a possible biomarker of glioma, and preliminary analysis was done on the role of the NPC2 gene in immunological microenvironment of glioma.

Transcriptional landscape of a hypoxia response identifies cell-specific pathways for adaptation.

How the HIF-1 (Hypoxia-Inducible) transcription factor drives and coordinates distinct responses to low oxygen across diverse cell types is poorly understood. We present a multi-tissue single-cell gene-expression atlas of the hypoxia response of the nematode Caenorhabditis elegans . This atlas highlights how cell-type-specific HIF-1 responses overlap and diverge among and within neuronal, intestinal, and muscle tissues. Using the atlas to guide functional analyses of candidate muscle-specific HIF-1 effectors, we discovered that HIF-1 activation drives downregulation of the tspo-1 ( TSPO, Translocator Protein) gene in vulval muscle cells to modulate a hypoxia-driven change in locomotion caused by contraction of body-wall muscle cells. We further showed that in human cardiomyocytes HIF-1 activation decreases levels of TSPO and thereby alters intracellular cholesterol transport and the mitochondrial network. We suggest that TSPO-1 is an evolutionarily conserved mediator of HIF-1-dependent modulation of muscle and conclude that our gene-expression atlas can help reveal how HIF-1 drives cell-specific adaptations to hypoxia.

Associations of lipids and lipid-modifying drug target genes with atrial fibrillation risk based on genomic data

BackgroundThe causal associations of lipids and the drug target genes with atrial fibrillation (AF) risk remain obscure. We aimed to investigate the causal associations using genetic evidence.MethodsMendelian randomization (MR) analyses were conducted using summary-level genome-wide association studies (GWASs) in European and East Asian populations. Lipid profiles (low-density lipoprotein cholesterol, triglyceride, and lipoprotein[a]) and lipid-modifying drug target genes (3-hydroxy-3-methylglutaryl-CoA reductase, proprotein convertase subtilisin/kexin type 9, NPC1-like intracellular cholesterol transporter 1, apolipoprotein C3, angiopoietin-like 3, and lipoprotein[a]) were used as exposures. AF was used as an outcome. The inverse variance weighted method was applied as the primary method. Summary-data-based Mendelian randomization analyses were performed for further validation using expression quantitative trait loci data. Mediation analyses were conducted to explore the indirect effect of coronary heart disease.ResultsIn the European population, MR analyses demonstrated that elevated levels of lipoprotein(a) increased AF risk. Moreover, analyses focusing on drug targets revealed that the genetically proxied target gene LPA, which simulates the effects of drug intervention by reducing lipoprotein(a), exhibited an association with AF risk. This association was validated in independent datasets. There were no consistent and significant associations observed for other traits when analyzed in different datasets. This finding was also corroborated by Summary-data-based Mendelian randomization analyses between LPA and AF. Mediation analyses revealed that coronary heart disease plays a mediating role in this association. However, in the East Asian population, no statistically significant evidence was observed to support these associations.ConclusionsThis study provided genetic evidence that Lp(a) may be a causal factor for AF and that LPA may represent a promising pharmacological target for preventing AF in the European population.

NPC Intracellular Cholesterol Transporter 1 Regulates Ovarian Maturation and Molting in Female Macrobrachium nipponense.

NPC intracellular cholesterol transporter 1 (NPC1) plays an important role in sterol metabolism and transport processes and has been studied in many vertebrates and some insects, but rarely in crustaceans. In this study, we characterized NPC1 from Macrobrachium nipponense (Mn-NPC1) and evaluated its functions. Its total cDNA length was 4283 bp, encoding for 1344 amino acids. It contained three conserved domains typical of the NPC family (NPC1_N, SSD, and PTC). In contrast to its role in insects, Mn-NPC1 was mainly expressed in the adult female hepatopancreas, with moderate expression in the ovary and heart. No expression was found in the embryo (stages CS-ZS) and only weak expression in the larval stages from hatching to the post-larval stage (L1-PL15). Mn-NPC1 expression was positively correlated with ovarian maturation. In situ hybridization showed that it was mainly located in the cytoplasmic membrane and nucleus of oocytes. A 25-day RNA interference experiment was employed to illustrate the Mn-NPC1 function in ovary maturation. Experimental knockdown of Mn-NPC1 using dsRNA resulted in a marked reduction in the gonadosomatic index and ecdysone content of M. nipponense females. The experimental group showed a significant delay in ovarian maturation and a reduction in the frequency of molting. These results expand our understanding of NPC1 in crustaceans and of the regulatory mechanism of ovarian maturation in M. nipponense.

Presenilin Deficiency Results in Cellular Cholesterol Accumulation by Impairment of Protein Glycosylation and NPC1 Function.

Presenilin proteins (PS1 and PS2) represent the catalytic subunit of γ-secretase and play a critical role in the generation of the amyloid β (Aβ) peptide and the pathogenesis of Alzheimer disease (AD). However, PS proteins also exert multiple functions beyond Aβ generation. In this study, we examine the individual roles of PS1 and PS2 in cellular cholesterol metabolism. Deletion of PS1 or PS2 in mouse models led to cholesterol accumulation in cerebral neurons. Cholesterol accumulation was also observed in the lysosomes of embryonic fibroblasts from Psen1-knockout (PS1-KO) and Psen2-KO (PS2-KO) mice and was associated with decreased expression of the Niemann-Pick type C1 (NPC1) protein involved in intracellular cholesterol transport in late endosomal/lysosomal compartments. Mass spectrometry and complementary biochemical analyses also revealed abnormal N-glycosylation of NPC1 and several other membrane proteins in PS1-KO and PS2-KO cells. Interestingly, pharmacological inhibition of N-glycosylation resulted in intracellular cholesterol accumulation prominently in lysosomes and decreased NPC1, thereby resembling the changes in PS1-KO and PS2-KO cells. In turn, treatment of PS1-KO and PS2-KO mouse embryonic fibroblasts (MEFs) with the chaperone inducer arimoclomol partially normalized NPC1 expression and rescued lysosomal cholesterol accumulation. Additionally, the intracellular cholesterol accumulation in PS1-KO and PS2-KO MEFs was prevented by overexpression of NPC1. Collectively, these data indicate that a loss of PS function results in impaired protein N-glycosylation, which eventually causes decreased expression of NPC1 and intracellular cholesterol accumulation. This mechanism could contribute to the neurodegeneration observed in PS KO mice and potentially to the pathogenesis of AD.

Abstract 2071: Identification Of Genetic Loci Influencing Lethal Atherothrombosis In Mice

Introduction: Atherothrombosis leading to myocardial infarction and stroke is a major cause of mortality. Ruptured atherosclerotic plaques release prothrombotic molecules such as tissue factor, which initiates thrombosis. Both genetic and environmental factors contribute to atherothrombosis. Combined complete deficiency of the Scavenger Receptor Class B type 1 (Scarb1) and Apolipoprotein E (ApoE) genes (dKO) on a mixed C57BL/6J (B6) and 129S1/SvImJ (129S1) background leads to an atherothrombotic phenotype and early death before 8 weeks of age. However, the dKO phenotype is dramatically different if the Scarb1 and ApoE deficient mice are backcrossed extensively to the B6 mouse strain, with no dKOs surviving to the weaning age. Hypothesis/Goals: We hypothesized that 129S1 genetic variants reduce atherothrombosis severity in dKO mice and used mouse genetics to identify these loci. Methods: To identify potential genetic factors, we produced and analyzed Scarb1 and ApoE dKO mice from 6 backcross/intercross generations with progressively more B6 genomic contribution. Weights were taken for 38 dKO mice and littermates from 3 weeks old until death of the dKO mice at 8 weeks of age. dKO mice from F1-F3 generations were divided into 3 distinct phenotypes based on their weights relative to their littermates. dKO mouse DNA was subjected to genome wide marker genotyping using the GigaMUGA platform. Quantitative trait loci analysis (QTL) was performed to identify loci responsible for the phenotypic weight differences. Results: dKO weights fell into 3 categories: indistinguishable from littermates, 14%-39% less than littermates, and greater than 39% less than littermates. QTL analyses identified significant interactions of Chr 12 with Chrs 1, 2, 3, and 14. Niemann Pick type C2 (Npc2), an intracellular cholesterol transporter, is within the interaction region on Chr 12 and is a prime candidate dKO modifier. Conclusion: We used weight data as a representative of overall health to categorize dKO phenotypes relative to non dKO littermates. We identified 6 significant genetic loci, with Npc2 as a candidate modifier gene for the Chromosome 12 locus. Identification of atherothrombosis modifier genes will provide novel therapeutic targets for this complex disease.

Endogenous Protein-Protein Interaction Network of the NPC Cholesterol Transporter 1 in the Cerebral Cortex.

NPC intracellular cholesterol transporter 1 (NPC1) is a multipass, transmembrane glycoprotein mostly recognized for its key role in facilitating cholesterol efflux. Mutations in the NPC1 gene result in Niemann-Pick disease, type C (NPC), a fatal, lysosomal storage disease. Due to the progressively expanding implications of NPC1-related disorders, we investigated endogenous NPC1 protein-protein interactions in the mouse cortex and human-derived iPSCs neuronal models of the disease through coimmunoprecipitation-coupled with LC-MS based proteomics. The current study investigated protein-protein interactions specific to the wild-type and the most prevalent NPC1 mutation (NPC1I1061T) while filtering out any protein interactor identified in the Npc1-/- mouse model. Additionally, the results were matched across the two species to map the parallel interactome of wild-type and mutant NPC1I1061T. Most of the identified wild-type NPC1 interactors were related to cytoskeleton organization, synaptic vesicle activity, and translation. We found many putative NPC1 interactors not previously reported, including two SCAR/WAVE complex proteins that regulate ARP 2/3 complex actin nucleation and multiple membrane proteins important for neuronal activity at synapse. Moreover, we identified proteins important in trafficking specific to wild-type and mutant NPC1I1061T. Together, the findings are essential for a comprehensive understanding of NPC1 biological functions in addition to its classical role in sterol efflux.

Sterol O-Acyltransferase 1 (SOAT1): A Genetic Modifier of Niemann-Pick Disease, Type C1.

Niemann-Pick disease type C1 (NPC1) is a lysosomal disorder due to impaired intracellular cholesterol transport out of the endolysosomal compartment.. Marked heterogeneity has been observed in individuals with the same NPC1 genotype, thus suggesting a significant effect of modifier genes. Prior work demonstrated that decreased SOAT1 activity decreased disease severity in an NPC1 mouse model. Thus, we hypothesized that a polymorphism associated with decreased SOAT1 expression might influence the NPC1 phenotype. Phenotyping and genomic sequencing of 117 individuals with NPC1 was performed as part of a Natural History trial. Phenotyping included determination of disease severity and disease burden. Significant clinical heterogeneity is present in individuals homozygous for the NPC1I1061T variant and in siblings. Analysis of the SOAT1 polymorphism, rs1044925 (A>C), showed a significant association of the C-allele with earlier age of neurological onset. The C-allele may be associated with a higher Annualized Severity Index Score as well as increased frequency of liver disease and seizures. A polymorphism associated with decreased expression of SOAT1 appears to be a genetic modifier of the NPC1 phenotype. This finding is consistent with prior data showing decreased phenotypic severity in Npc1-/-:Soat1-/- mice and supports efforts to investigate the potential of SOAT1 inhibitors as a potential therapy for NPC1.

ALOX15B controls macrophage cholesterol homeostasis via lipid peroxidation, ERK1/2 and SREBP2

Macrophage cholesterol homeostasis is crucial for health and disease and has been linked to the lipid-peroxidizing enzyme arachidonate 15-lipoxygenase type B (ALOX15B), albeit molecular mechanisms remain obscure. We performed global transcriptome and immunofluorescence analysis in ALOX15B-silenced primary human macrophages and observed a reduction of nuclear sterol regulatory element-binding protein (SREBP) 2, the master transcription factor of cellular cholesterol biosynthesis. Consequently, SREBP2-target gene expression was reduced as were the sterol biosynthetic intermediates desmosterol and lathosterol as well as 25- and 27-hydroxycholesterol. Mechanistically, suppression of ALOX15B reduced lipid peroxidation in primary human macrophages and thereby attenuated activation of mitogen-activated protein kinase ERK1/2, which lowered SREBP2 abundance and activity. Low nuclear SREBP2 rendered both, ALOX15B-silenced and ERK1/2-inhibited macrophages refractory to SREBP2 activation upon blocking the NPC intracellular cholesterol transporter 1. These studies suggest a regulatory mechanism controlling macrophage cholesterol homeostasis based on ALOX15B-mediated lipid peroxidation and concomitant ERK1/2 activation.

Long-term efficacy of intrathecal cyclodextrin in patients with Niemann-Pick disease type C

Background and objectivesNiemann–Pick type C (NPC) is a rare lysosomal storage disease characterized by hepatosplenomegaly and progressive neurological deterioration due to abnormal intracellular cholesterol transport. Cyclic oligosaccharide 2-hydroxypropyl-β-cyclodextrin (HPBCD) is an effective treatment for NPC; however, few reports have shown its long-term efficacy and safety. To demonstrate long-term efficacy and safety of intrathecal HPBCD (IT-HPBCD) treatment for NPC, we herein reports five patients with NPC treated using IT-HPBCD for 4–11 years. Cases and resultsPatients’ ages at the onset ranged from 1.5 to 20 years. Notably, all patients showed rapid disease progression despite treatment with miglustat before IT-HPBCD treatment. Similarly, some patients showed transient improvement; however, all patients’ conditions stabilized after long-term IT-HPBCD therapy. Mild-to-moderate hearing loss was observed in three patients. Furthermore, long-term treatment with IT-HPBCD may suppress neurological deterioration in patients with NPC; however, patients still experience some disease progression. ConclusionsLong-term treatment with IT-HPBCD may suppress neurological deterioration in patients with NPC; however, the treatment outcome is dependent on the neurological status at the time of diagnosis, and disease progression is not completely inhibited. Awareness of the disease and newborn screening is needed for earlier disease detection. In addition, further optimization of the treatment protocol and additional treatments are needed to improve patient outcomes.

Sperm traits and seminal plasma proteome of locally adapted hairy rams subjected to intermittent scrotal insulation

The present study evaluated the effects of heat stress on reproductive parameters of hairy rams. Six animals were subjected to scrotal insulation during four consecutive nights (6 PM – 6 AM). Day (D) 0 was the first day of insulation. Scrotal circumference increased from 30.5 ± 0.3 cm (at pre-insulation) to 31.8 ± 0.4 cm on D4, decreased 3.9 cm on D28, returning to 30.6 ± 0.6 cm on D57. Sperm concentration decreased from 3.7 ± 0.12 ×109 sperm/mL before insulation to 2.6 ± 0.1 ×109 on D23, returning to normal on D57. Sperm motility averaged 75 ± 2.9% before insulation, was undetectable on D23, and became normal on D77. Sperm with normal morphology reached 5.9 ± 2.6% on D35 but recovered (86.8 ± 2.1%) on D91. Sperm DNA integrity decreased from 86.5 ± 4.7% before insulation to 11.1 ± 3.7% on D63, returning to pre-insulation values on D120. Sperm BSP immunostaining was reduced after scrotal insulation. Variations in seminal protein abundances coincided with changes in sperm parameters. Seminal plasma superoxide dismutase, carboxypeptidase Q-precursor and NPC intracellular cholesterol transporter 2 decreased on D18, returning to normal after D28. Albumin, inhibitor of carbonic anhydrase precursor, EGF-like repeat and discoid I-like domain-containing protein 3 and polymeric immunoglobulin receptor increased after insulation. In summary, intermittent scrotal insulation drastically altered ram sperm attributes and seminal proteins, especially those associated with oxidative stress. Knowledge of animal´s response to thermal stress is vital in the scenario of climate changes.

Repositioning of ezetimibe for the treatment of idiopathic pulmonary fibrosis.

We previously identified ezetimibe, an inhibitor of Niemann-Pick C1-like intracellular cholesterol transporter 1 and European Medicines Agency-approved lipid-lowering agent, as a potent autophagy activator. However, its efficacy against pulmonary fibrosis has not yet been evaluated. This study aimed to determine whether ezetimibe has therapeutic potential against idiopathic pulmonary fibrosis. Primary lung fibroblasts isolated from both humans and mice were employed for mechanistic in vitro experiments. mRNA sequencing of human lung fibroblasts and gene set enrichment analysis were performed to explore the therapeutic mechanism of ezetimibe. A bleomycin-induced pulmonary fibrosis mouse model was used to examine in vivo efficacy of the drug. Tandem fluorescent-tagged microtubule-associated protein 1 light chain 3 transgenic mice were used to measure autophagic flux. Finally, the medical records of patients with idiopathic pulmonary fibrosis from three different hospitals were reviewed retrospectively, and analyses on survival and lung function were conducted to determine the benefits of ezetimibe. Ezetimibe inhibited myofibroblast differentiation by restoring the mechanistic target of rapamycin complex 1-autophagy axis with fine control of intracellular cholesterol distribution. Serum response factor, a potential autophagic substrate, was identified as a primary downstream effector in this process. Similarly, ezetimibe ameliorated bleomycin-induced pulmonary fibrosis in mice by inhibiting mechanistic target of rapamycin complex 1 activity and increasing autophagic flux, as observed in mouse lung samples. Patients with idiopathic pulmonary fibrosis who regularly used ezetimibe showed decreased rates of all-cause mortality and lung function decline. Our study presents ezetimibe as a potential novel therapeutic for idiopathic pulmonary fibrosis.

Fut2 Deficiency Promotes Intestinal Stem Cell Aging by Damaging Mitochondrial Functions via Down-Regulating α1,2-Fucosylation of Asah2 and Npc1.

Fut2-mediated α1,2-fucosylation is important for gut homeostasis, including the intestinal stem cell (ISC). The stemness of ISC declines with age, and aging-associated ISC dysfunction is closely related to many age-related intestinal diseases. We previously found intestinal epithelial dysfunction in some aged Fut2 knockout mice. However, how Fut2-mediated α1,2-fucosylation affects ISC aging is still unknown. On this basis, the herein study aims to investigate the role of Fut2-mediated α1,2-fucosylation in ISC aging. Aging models in ISC-specific Fut2 knockout mice were established. ISCs were isolated for proteomics and N-glycoproteomics analysis. ISC functions and mitochondrial functions were examined in mice and organoids. Ulex europaeus agglutinin I chromatography and site-directed mutagenesis were used to validate the key target fucosylated proteins of Fut2. As a result, Fut2 knockout impaired ISC stemness and promoted aging marker expression in aged mice. Proteomics analysis indicated mitochondrial dysfunction in Fut2 knockout ISC. More injured mitochondria, elevated levels of reactive oxygen species, and decreased levels of adenosine 5'-triphosphate (ATP) in Fut2 knockout ISC were found. Moreover, respiratory chain complex impairment and mitophagy dysfunction in Fut2 knockout ISC were further noted. Finally, Fut2 was demonstrated to regulate mitochondrial functions mainly by regulating the α1,2-fucosylation of N-acyl sphingosine amidohydrolase 2 (Asah2) and Niemann-Pick type C intracellular cholesterol transporter 1 (Npc1). In conclusion, this study demonstrated the substantial role of Fut2 in regulating ISC functions during aging by affecting mitochondrial function. These findings provide novel insights into the molecular mechanisms of ISC aging and therapeutic strategies for age-related intestinal diseases.

Glucose-mediated mitochondrial reprogramming by cholesterol export at TM4SF5-enriched mitochondria-lysosome contact sites.

Transmembrane 4 L six family member 5 (TM4SF5) translocates subcellularly and functions metabolically, although it is unclear how intracellular TM4SF5 translocation is linked to metabolic contexts. It is thus of interests to understand how the traffic dynamics of TM4SF5 to subcellular endosomal membranes are correlated to regulatory roles of metabolisms. Here, we explored the metabolic significance of TM4SF5 localization at mitochondria-lysosome contact sites (MLCSs), using in vitro cells and in vivo animal systems, via approaches by immunofluorescence, proximity labelling based proteomics analysis, organelle reconstitution etc. RESULTS: Upon extracellular glucose repletion following depletion, TM4SF5 became enriched at MLCSs via an interaction between mitochondrial FK506-binding protein 8 (FKBP8) and lysosomal TM4SF5. Proximity labeling showed molecular clustering of phospho-dynamic-related protein I (DRP1) and certain mitophagy receptors at TM4SF5-enriched MLCSs, leading to mitochondrial fission and autophagy. TM4SF5 bound NPC intracellular cholesterol transporter 1 (NPC1) and free cholesterol, and mediated export of lysosomal cholesterol to mitochondria, leading to impaired oxidative phosphorylation but intact tricarboxylic acid (TCA) cycle and β-oxidation. In mouse models, hepatocyte Tm4sf5 promoted mitophagy and cholesterol transport to mitochondria, both with positive relations to liver malignancy. Our findings suggested that TM4SF5-enriched MLCSs regulate glucose catabolism by facilitating cholesterol export for mitochondrial reprogramming, presumably while hepatocellular carcinogenesis, recapitulating aspects for hepatocellular carcinoma metabolism with mitochondrial reprogramming to support biomolecule synthesis in addition to glycolytic energetics.

Identification of Genetic Loci That Modify the Scarb1 and Apoe Atherothrombotic Phenotype

Introduction: Atherothrombosis, or the formation of a thrombus on a ruptured atherosclerotic plaque, is a major health problem in western societies and can lead to myocardial infarction and ischemic stroke. Atherosclerotic plaques form on the inner walls of blood vessels and consist of macrophages, lipids, and prothrombotic molecules such as tissue factor that initiate the blood clotting process. Despite the complexity of atherothrombotic disease, with major (and evolving) environmental contributors, genetic variants play a role in modifying the disease process. In mice, deficiency of the Scavenger Receptor Class B type 1 (Scarb1) and Apolipoprotein E (ApoE) genes play important roles in the proper maintenance of lipid levels in the body. The Scarb1 protein is an HDL receptor known for its role in the process of transporting cholesterol from peripheral tissues toward the liver for excretion. The ApoE protein combines with lipids to form lipoproteins that package cholesterol and carry it throughout the circulation. In order to study mice deficient in these proteins, our lab produced Scarb1 and ApoE double knockout (dKO) mice on the C57BL/6J (B6) and 129S1/SvImJ (129S1) background and discovered major phenotypic differences in size and survival. Mice with a mixed 129S1/B6 background succumb to lethal atherothrombosis at approximately 8 weeks of age (as seen in Figure 1), whereas dKOs generated on the B6 background die at approximately 5 days old. We hypothesized that specific 129S1 genetic variants modify the severity of atherothrombosis in dKO mice. Methods: We produced F1-F3 generation dKO mice on the mixed 129S1/B6 background for experiments. Weights were taken for the 38 dKO mice and their littermates from 3 weeks old until the death of the knockout mice at around 8 weeks of age. Purified DNA from the dKO mice was subjected to whole genome genotyping for 143,000 markers using the GigaMUGA genotyping platform (Neogen). Weight data were used as a phenotype to perform quantitative trait loci analysis (QTL) to identify potential loci responsible for the phenotypic weight differences. Results: dKO mice from F1-F3 generations were born at the expected Mendelian frequencies but exhibited 3 distinct phenotypes based on their weights relative to their littermates. dKO mouse weights fell into 3 distinct categories: (1) indistinguishable from littermates, (2) 14%-39% less than littermates, (3) greater than 39% less than littermates. QTL analysis identified 6 loci above the significance threshold of 4.04 on Chromosomes (Chr) 1 (LOD 6.82), 2 (LOD 6.44) , 3 (LOD 4.65), 6 (LOD 4.30), 12 (LOD 4.22) and 14 (LOD 5.36; Figure 2). In addition, interaction analyses identified significant interactions of Chr 12 with Chrs 1, 2, 3, and 14. The Chr 12 candidate region is between 83 and 93 Mb consisting of 89 genes. Niemann Pick type C2 ( Npc2), an intracellular cholesterol transporter, is within the candidate region. Thus, 129S1 variants in this region affecting Npc2 transcription are strong candidates for modifying the dKO phenotypes. Discussion: We have analyzed 38 dKO mice for genetic variants affecting their health, as measured by weights relative to their non dKO littermates. We identified 6 significant genetic loci, with Npc2 as a candidate gene for the Chromosome 12 locus. Studies to reinforce these findings include the analysis of an additional cohort of dKO mice to narrow the candidate QTL intervals, analysis of Npc2 expression in the B6 and 129 strain, histology of the liver to visualize lipid accumulation, blood smears to examine the blood cell size difference in dKOs, and sequencing with GigaMUGA. The identification of atherothrombosis modifier genes could result in novel treatment strategies for atherothrombosis.

Identifying genes involved in the secretory physiological response to feeding in Pacific White Shrimp (Litopenaeus vannamei) using transcriptomics

The physiological response to feeding is important for production aspects that include feed utilization and growth, and the responses require the action of numerous secretory factors. However, as an important aquaculture animal, the secretory response of Pacific White Shrimp (Litopenaeus vannamei) after feeding has not been comprehensively characterized. In this study, transcriptome analysis showed that 3172 differentially expressed genes were involved in the post-feeding response, including 289 new genes not annotated in the L. vannamei reference genome. Subsequently, 715 differentially expressed secretory reference genes and 18 new differentially expressed secretory genes were obtained through the identification of signal peptides in secreted proteins. Functional classification revealed that differentially expressed secretory genes were enriched in pathways pertaining to lipid metabolism (20 genes), carbohydrate metabolism (21 genes), glycan biosynthesis and metabolism (27 genes), digestive system (40 genes), and transport and metabolism (43 genes). The 14 pathways most enriched by differentially expressed secretory genes involved 83 genes, 71 of which encoded enzymes involved in food digestion and metabolism. Specific enzymes such as lipase 3-like and NPC intracellular cholesterol transporter 1-like in lipid metabolism, alpha-amylase-like and glucosylceramidase-like in carbohydrate metabolism, and cysteine proteinase 4-like and trypsin-1-like in the digestive system were found to be differentially expressed. Furthermore, we discovered a new gene, MSTRG.2504, that participates in the digestive system and carbohydrate metabolism. The study provides valuable insights into the secretory response (especially metabolism-related enzymes) to feeding in L. vannamei, uncovering the significant roles of both known and new genes. Furthermore, this study will improve our understanding of the feeding physiology of L. vannamei and provide a reference basis for further feeding endocrine research in the future.

Ocular manifestation of an adult Niemann-Pick disease type B

Niemann-Pick disease is a rare, autosomal recessive inherited lysosomal storage disorder. The pathophysiological background for this condition is the deficiency or reduced function of the enzyme sphingomyelinase, as well as a deficiency in the intracellular cholesterol transporter protein. Due to the breakdown defect, sphingomyelin and cholesterol accumulate in the lysosomes of cells. The disease is divided into 5 subtypes (A, A/B, B, C, D). The authors present the case of a 24-year-old young man diagnosed with Niemann-Pick disease type B as a child, focusing on the ophthalmic manifestation of the disease. During the examination of the patient, fundus photographs and fundus autofluorescence imaging were taken, and optical coherence tomography (OCT), optical coherence tomography angiography (OCTA), and visual field (perimetry) examinations were performed. The characteristic macular halo and the cherry-red spot in the fovea were clearly visible during ophthalmoscopy and on the fundus photographs. The OCT images showed focal thickening with high reflectivity in the ganglion cell layer corresponding to the macular halo, and the area of the foveola was spared. With visual field examination, an intact field of vision was found on both eyes. Similar to the presented patient, symptoms in patients with the B subtype are milder, and besides the visceral symptoms, there are no neurological symptoms, and the specific ophthalmic abnormalities do not cause visual impairment. Currently, Niemann-Pick disease is considered a rare disease, and the diagnosis of the patients is often delayed or even missed due to non-specific or mild symptoms. Through consultation between medical specialties, ophthalmological examination can also contribute to the correct diagnosis in cases with mild general symptoms. Timely diagnosis can potentially lead to mitigation of symptoms thanks to the ever-expanding therapeutic options, stabilization of the disease progression, and increase of the patients' life expectancy. Orv Hetil. 2023; 164(46): 1838-1844.