Filipin Staining Research Articles

Molecular determinants of phospholipid treatment to reduce intracellular cholesterol accumulation in NPC1 deficiency

Niemann-Pick type C (NPC) disease, caused by mutations in the NPC1 or NPC2 genes, leads to abnormal intracellular cholesterol accumulation in late endosomes/lysosomes (LE/LY). Exogenous enrichment with lysobisphosphatidic acid (LBPA), also known as bis-monoacylglycerol phosphate or BMP, either directly or via the LBPA precursor phosphatidylglycerol (PG), has been investigated as a therapeutic intervention to reduce cholesterol accumulation in NPC disease. Here we report the effects of stereoisomer configuration and acyl chain composition of LBPA on cholesterol clearance in NPC1-deficient cells. We find that S,R, S,S, and S,R LBPA stereoisomers behaved similarly, with all 3 compounds leading to comparable reductions in filipin staining in two NPC1-deficient human fibroblast cell lines. Examination of several LBPA molecular species containing one or two mono- or polyunsaturated acyl chains showed that all LBPA species containing one 18:1 chain significantly reduced cholesterol accumulation, whereas the shorter chain species di-14:0 LBPA had little effect on cholesterol clearance in NPC1 deficient cells. Since cholesterol accumulation in NPC1 deficient cells can also be cleared by PG incubation, we used non-hydrolyzable PG analogues to determine whether conversion to LBPA is required for sterol clearance, or whether PG itself is effective. The results showed that non-hydrolyzable PG species were not appreciably converted to LBPA and showed virtually no cholesterol clearance efficacy in NPC1 deficient cells, supporting the notion that LBPA is the active agent promoting LE/LY cholesterol clearance. Overall these studies are helping to define the molecular requirements for potential therapeutic use of LBPA as an option for addressing NPC disease.

β-Sitosterol Reduces the Content of Triglyceride and Cholesterol in a High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease Zebrafish (Danio rerio) Model.

Non-alcoholic fatty liver disease (NAFLD) is strongly associated with hyperlipidemia, which is closely related to high levels of sugar and fat. β-sitosterol is a natural product with significant hypolipidemic and cholesterol-lowering effects. However, the underlying mechanism of its action on aquatic products is not completely understood. A high-fat diet (HFD)-induced NAFLD zebrafish model was successfully established, and the anti-hyperlipidemic effect and potential mechanism of β-sitosterol were studied using oil red O staining, filipin staining, and lipid metabolomics. β-sitosterol significantly reduced the accumulation of triglyceride, glucose, and cholesterol in the zebrafish model. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that differential lipid molecules in β-sitosterol mainly regulated the lipid metabolism and signal transduction function of the zebrafish model. β-sitosterol mainly affected steroid biosynthesis and steroid hormone biosynthesis in the zebrafish model. Compared with the HFD group, the addition of 500 mg/100 g of β-sitosterol significantly inhibited the expression of Ppar-γ and Rxr-α in the zebrafish model by at least 50% and 25%, respectively. β-sitosterol can reduce lipid accumulation in the zebrafish model of NAFLD by regulating lipid metabolism and signal transduction and inhibiting adipogenesis and lipid storage.

Tumour-associated macrophage-derived DOCK7-enriched extracellular vesicles drive tumour metastasis in colorectal cancer via the RAC1/ABCA1 axis.

Metastasis accounts for the majority of deaths among patients with colorectal cancer (CRC). Here, the regulatory role of tumour-associated macrophages (TAMs) in CRC metastasis was explored. Immunohistochemical (IHC) analysis of the TAM biomarker CD163 was conducted to evaluate TAM infiltration in CRC. Transwell assays and an ectopic liver metastasis model were established to evaluate the metastatic ability of tumour cells. RNA sequencing (RNA-seq) and liquid chromatography-mass spectrometry (LC-MS) were applied to identify the differentially expressed genes and proteins in CRC cells and in TAM-derived extracellular vesicles (EVs). Cholesterol content measurement, a membrane fluidity assay and filipin staining were performed to evaluate cholesterol efflux in CRC cells. Our results showed that TAM infiltration is positively correlated with CRC metastasis. TAMs can facilitate the migration and invasion of MC-38 and CT-26 cells via EVs. According to the RNA-seq data, TAM-EVs increase cholesterol efflux and enhance membrane fluidity in CRC cells by regulating ABCA1 expression, thus affecting the motility of CRC cells. Mechanistically, DOCK7 packaged in TAM-EVs can activate RAC1 in CRC cells and subsequently upregulate ABCA1 expression by phosphorylating AKT and FOXO1. Moreover, IHC analysis of ABCA1 in patients with liver-metastatic CRC indicated that ABCA1 expression is significantly greater in metastatic liver nodules than in primary CRC tumours. Overall, our findings suggest that DOCK7 delivered via TAM-EVs could regulate cholesterol metabolism in CRC cells and CRC cell metastasis through the RAC1/AKT/FOXO1/ABCA1 axis. DOCK7 could thus be a new therapeutic target for controlling CRC metastasis.

Abnormal Cholesterol Metabolism and Lysosomal Dysfunction Induce Age-Related Hearing Loss by Inhibiting mTORC1-TFEB-Dependent Autophagy.

Cholesterol is a risk factor for age-related hearing loss (ARHL). However, the effect of cholesterol on the organ of Corti during the onset of ARHL is unclear. We established a mouse model for the ARHL group (24 months, n = 12) and a young group (6 months, n = 12). Auditory thresholds were measured in both groups using auditory brainstem response (ABR) at frequencies of 8, 16, and 32 kHz. Subsequently, mice were sacrificed and subjected to histological analyses, including transmission electron microscopy (TEM), H&E, Sudan Black B (SBB), and Filipin staining, as well as biochemical assays such as IHC, enzymatic analysis, and immunoblotting. Additionally, mRNA extracted from both young and aged cochlea underwent RNA sequencing. To identify the mechanism, in vitro studies utilizing HEI-OC1 cells were also performed. RNA sequencing showed a positive correlation with increased expression of genes related to metabolic diseases, cholesterol homeostasis, and target of rapamycin complex 1 (mTORC1) signaling in the ARHL group as compared to the younger group. In addition, ARHL tissues exhibited increased cholesterol and lipofuscin aggregates in the organ of Corti, lateral walls, and spiral ganglion neurons. Autophagic flux was inhibited by the accumulation of damaged lysosomes and autolysosomes. Subsequently, we observed a decrease in the level of transcription factor EB (TFEB) protein, which regulates lysosomal biosynthesis and autophagy, together with increased mTORC1 activity in ARHL tissues. These changes in TFEB and mTORC1 expression were observed in a cholesterol-dependent manner. Treatment of ARHL mice with atorvastatin, a cholesterol synthesis inhibitor, delayed hearing loss by reducing the cholesterol level and maintaining lysosomal function and autophagy by inhibiting mTORC1 and activating TFEB. The above findings were confirmed using stress-induced premature senescent House Ear Institute organ of Corti 1 (HEI-OC1) cells. The findings implicate cholesterol in the pathogenesis of ARHL. We propose that atorvastatin could prevent ARHL by maintaining lysosomal function and autophagy by inhibiting mTORC1 and activating TFEB during the aging process.

Cholesterol and matrisome pathways dysregulated by APOE4 in human glia

AbstractBackgroundThe association of Apolipoprotein E (APOE) ɛ4 risk with Alzheimer’s disease (AD) is well‐established. However, the impact of APOE ɛ4 on human brain cell function remains unclear. We hypothesized that human APOE ε4/ε4 (APOE 44) genotype contributes to disease risk through cell autonomous and non‐cell autonomous mechanisms in a human cell specific manner.MethodGlobal transcriptomic analyses were performed on astrocytes, microglia, mixed cortical cultures (neurons and astrocytes) and brain microvascular endothelial cells derived from human induced pluripotent stem cells (iPSCs) and post‐mortem AD brains. Lipid pathway dysregulation identified through GSEA, WGCNA and cell type deconvolution was validated in vitro using Gas Chromatography‐Mass Spectrometry (GC‐MS), filipin staining, autophagy fluorescent reporters with immunocytochemistry and western blotting to determine the subcellular compartment that accumulates cholesterol in iPSC‐derived astrocytes, and further confirmed by immunohistochemical analysis in human APOE4 post‐mortem brain. Matrisome pathway dysregulation was validated by Luminex multiplex immunoassays and intracellular protein levels in pure astrocytes and mixed cortical cultures.ResultGlobal transcriptomic analyses reveal that APOE 44 drives cholesterol metabolic dysregulation in astrocytes and microglia from human iPSCs and cell type‐deconvolved post‐mortem AD brain. APOE ε4 leads to elevated de novo cholesterol synthesis despite the intracellular accumulation of free cholesterol in lysosome and cholesterol ester in lipid droplets coupled with reduced efflux due to impaired lipophagy in iPSC‐derived astrocytes. Lysosomal cholesterol accumulation is found in astrocytes from APOE ε4 human brain. Activation of LXRs and induction of lipophagy ameliorates the lipid phenotypes in APOE ε4 astrocytes. Further, matrisome dysregulation is associated with upregulated chemotaxis, glial activation, and lipid biosynthesis in astrocytes co‐cultured with neurons, which recapitulates altered astrocyte matrisome signaling in human brain.ConclusionThus, human APOE ε4 causes human glia‐specific, cell autonomous and non‐cell autonomous cholesterol and matrisome pathway dysregulation that may increase AD risk.

HIF-1α activation induces cholesterol homeostasis dysfunction to accelerate progression of diabetic nephropathy in rats

To investigate the effect of hypoxia inducible factor-1α (HIF-1α) activation on cholesterol homeostasis dysfunction in diabetic nephropathy (DN). Rat models of type 1 diabetes established by intraperitoneal STZ injection were treated with intraperitoneal injection of Lificiguat (YC-1, a HIF-1α inhibitor). Human proximal tubular cell line HK-2 was incubated with cobalt chloride (CoCl2, 100 μmol/L) in the presence or absence of 30 mmol/L glucose for 24 h. Renal injury of the rats was assessed by measuring 24-h urinary total protein level and PAS staining of the renal tubules. Cholesterol deposition in rat kidneys and HK-2 cells were observed using a quantitative assay of total cholesterol and Filipin staining, and HIF-1α protein expression was detected using Western blotting, immunohistochemistry or immunofluorescence assay; the expressions of cholesterol metabolism-related proteins HMGCR, LDLr, CXCL16 and profibrogenic factors including TGF-β1 and CTGF were also analyzed. The diabetic rats showed significantly increased 24-h urinary protein level (P<0.001), obvious renal tubular injury, and increased renal cholesterol content (P<0.05) with significantly increased HIF-1α expression in the renal tubular (P<0.01). YC-1 treatment significantly ameliorated tubulointerstitial injury in the diabetic rats as shown by decreased 24-h urinary total protein (P<0.05) and reduced damage area of the tubules, and effectively decreased renal cholesterol levels and renal expression of HIF-1α (P<0.05). In HK-2 cells, CoCl2 stimulation in the presence of high glucose effectively activated HIF-1α expression (P<0.0001), aggravated cholesterol accumulation (P<0.05), and increased the expressions of HMGCR, LDLr, CXCL16, TGF-β1, and CTGF (P<0.05 or 0.01). Consistent with the in vitro study, YC-1 treatment also significantly decreased the expressions of cholesterol metabolism-related proteins and the profibrogenic factors in the renal tubules of the diabetic rats. HIF-1α activation induces cholesterol homeostasis dysregulation possibly by upregulating the de novo synthesis and uptake of cholesterol, thereby aggravating tubulointerstitial injury in DN.

Functional analysis of two SLC9A6 frameshift variants in lymphoblastoid cells from patients with Christianson syndrome.

Christianson syndrome (CS) is caused by mutations in SLC9A6 and is characterized by global developmental delay, epilepsy, hyperkinesis, ataxia, microcephaly, and behavioral disorder. However, the molecular mechanism by which these SLC9A6 mutations cause CS in humans is not entirely understood, and there is no objective method to determine the pathogenicity of single SLC9A6 variants. Trio-based whole exome sequencing (WES) was carried out on two individuals with suspicion of CS. qRT-PCR, western blot analysis, filipin staining, lysosomal enzymatic assays, and electron microscopy examination, using EBV-LCLs established from the two patients, were performed. Trio-based WES identified a hemizygous SLC9A6 c.1560dupT, p.T521Yfs*23 variant in proband 1 and a hemizygous SLC9A6 c.608delA, p.H203Lfs*10 variant in proband 2. Both children exhibited typical phenotypes associated with CS. Expression analysis in EBV-LCLs derived from the two patients showed a significant decrease in mRNA levels and no detectable normal NHE6 protein. EBV-LCLs showed a statistically significant increase in unesterified cholesterol in patient 1, but only non-significant increase in patient 2 when stained with filipin. Activities of lysosomal enzymes (β-hexosaminidase A, β-hexosaminidase A + B, β-galactosidase, galactocerebrosidase, arylsulfatase A) of EBV-LCLs did not significantly differ between the two patients and six controls. Importantly, by electron microscopy we detected an accumulation of lamellated membrane structures, deformed mitochondria, and lipid droplets in the patients' EBV-LCLs. The SLC9A6 p.T521Yfs*23 and p.H203Lfs*10 variants in our patients result in loss of NHE6. Alterations of mitochondria and lipid metabolism may play a role in the pathogenesis of CS. Moreover, the combination of filipin staining with electron microscopy examination of patient lymphoblastoid cells can serve as a useful complementary diagnostic method for CS.

Cholesterol homeostasis in hair follicle keratinocytes is disrupted by impaired ABCA5 activity

The importance of cholesterol in hair follicle biology is underscored by its links to the pathogenesis of alopecias and hair growth disorders. Reports have associated defects in ABCA5, a membrane transporter, with altered keratinocyte cholesterol distribution in individuals with a form of congenital hypertrichosis, yet the biological basis for this defect in hair growth remains unknown. This study aimed to determine the impact of altered ABCA5 activity on hair follicle keratinocyte behaviour. Primary keratinocytes isolated from the outer root sheath of plucked human hair follicles were utilised as a relevant cell model. Following exogenous cholesterol loading, an increase in ABCA5 co-localisation to intracellular organelles was seen. Knockdown of ABCA5 revealed a dysregulation in cholesterol homeostasis, with LXR agonism leading to partial restoration of the homeostatic response. Filipin staining and live BODIPY cholesterol immunofluorescence microscopy revealed a reduction in endo-lysosomal cholesterol following ABCA5 knockdown. Analysis of oxysterols showed a significant increase in the fold change of 25-hydroxycholesterol and 7-β-hydroxycholesterol following cholesterol loading in ORS keratinocytes, after ABCA5 knockdown. These data suggest a role for ABCA5 in the intracellular compartmentalisation of free cholesterol in primary hair follicle keratinocytes. The loss of normal homeostatic response, following the delivery of excess cholesterol after ABCA5 knockdown, suggests an impact on LXR-mediated transcriptional activity. The loss of ABCA5 in the hair follicle could lead to impaired endo-lysosomal cholesterol transport, impacting pathways known to influence hair growth. This avenue warrants further investigation.

Conditioned medium of human menstrual blood-derived endometrial stem cells protects against cell inflammation and apoptosis of Npc1KO N2a cells.

Niemann-Pick disease type C1 (NPC1) is a hereditary neurodegenerative disorder caused by a mutation in the NPC1 gene. This gene encodes a transmembrane protein found in lysosomes. This disease characterized by hepatosplenomegaly, neurological impairments and premature death. Recent preclinical studies have shown promising results in using mesenchymal stem cells (MSCs) to alleviate the symptoms of NPC1. One type of MSCs, known as human menstrual blood-derived endometrial stem cells (MenSCs), has attracted attention due to its accessibility, abundant supply, and strong proliferation and regeneration capabilities. However, it remains uncertain whether the conditioned medium of MenSCs (MenSCs-CM) can effectively relieve the symptoms of NPC1. To investigate this further, we employed the CRISPR-Cas9 technique to successfully create a Npc1 gene knockout N2a cell line (Npc1KO N2a). Sanger sequencing confirmed the occurrence of Npc1 gene mutation in these cells, while western blotting revealed a lack of NPC1 protein expression. Filipin staining provided visual evidence of unesterified cholesterol accumulation in Npc1KO N2a cells. Moreover, Npc1KO N2a cells exhibited significantly decreased viability, increased inflammation, and heightened cell apoptosis. Notably, our study demonstrated that the viability of Npc1KO N2a cells was most significantly improved after being cultured by 36h-collected MenSCs-CM for 0.5 days. Additionally, MenSCs-CM exhibited the ability to effectively reduce inflammation, counteract cell apoptosis, and ameliorate unesterified cholesterol accumulation in Npc1KO N2a cells. This groundbreaking finding establishes, for the first time, the protective effect of MenSCs-CM on N2a cells with Npc1 gene deletion. These findings suggest that the potential of MenSCs-CM as a beneficial therapeutic approach for NPC1 and other neurodegenerative diseases.

Extra-virgin olive oil ameliorates high-fat diet-induced seminal and testicular disorders by modulating the cholesterol pathway.

Rabbits are sensitive to dietary cholesterol and rapidly develop hypercholesterolemia, leading to prominent subfertility. Sterol regulatory element-binding protein isoform 2 drives the intracellular cholesterol pathway in many tissues, including the testicles. Its abnormal regulation could be the mainly responsible for the failure of suppressing cholesterol synthesis in a cholesterol-enriched environment, ultimately leading to testicular and seminal alterations. However, extra-virgin olive oil consumption has beneficial properties that promote lowering of cholesterol levels, including the recovery of seminal parameters altered under a high-fat diet. Our goal was to investigate the effects of high-fat diet supplementation with extra-virgin olive oil at the molecular level on rabbit testes, by analyzing sterol regulatory element-binding protein isoform 2 protein and its corresponding downstream effectors. During 12months, male rabbits were fed a control diet, high-fat diet, or 6-month high-fat diet followed by 6-month high-fat diet plus extra-virgin olive oil. Serum lipids, testosterone levels, bodyweight, and seminal parameters were tested. The mRNA and protein levels of sterol regulatory element-binding protein isoform 2, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, and low-density lipoprotein receptor were determined by semi-quantitative polymerase chain reaction and Western blotting techniques. The expression pattern of sterol regulatory element-binding protein isoform 2 protein in the rabbit testicles was studied by indirect immunofluorescence. In addition, testicular cholesterol was detected and quantified by filipin staining and gas chromatography. The data showed that the addition of extra-virgin olive oil to high-fat diet reduced testicular cholesterol levels and recovered the expression of sterol regulatory element-binding protein isoform 2, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, and low-density lipoprotein receptor initially altered by the high-fat diet. The combination of high-fat diet with extra-virgin olive oil encourages testicular recovery by modifying the expression of the enzymes related to intracellular cholesterol management.

Erythrocyte Plasma Membrane Lipid Composition Mirrors That of Neurons and Glial Cells in Murine Experimental In Vitro and In Vivo Inflammation.

Lipid membrane turnover and myelin repair play a central role in diseases and lesions of the central nervous system (CNS). The aim of the present study was to analyze lipid composition changes due to inflammatory conditions. We measured the fatty acid (FA) composition in erythrocytes (RBCs) and spinal cord tissue (gas chromatography) derived from mice affected by experimental allergic encephalomyelitis (EAE) in acute and remission phases; cholesterol membrane content (Filipin) and GM1 membrane assembly (CT-B) in EAE mouse RBCs, and in cultured neurons, oligodendroglial cells and macrophages exposed to inflammatory challenges. During the EAE acute phase, the RBC membrane showed a reduction in polyunsaturated FAs (PUFAs) and an increase in saturated FAs (SFAs) and the omega-6/omega-3 ratios, followed by a restoration to control levels in the remission phase in parallel with an increase in monounsaturated fatty acid residues. A decrease in PUFAs was also shown in the spinal cord. CT-B staining decreased and Filipin staining increased in RBCs during acute EAE, as well as in cultured macrophages, neurons and oligodendrocyte precursor cells exposed to inflammatory challenges. This regulation in lipid content suggests an increased cell membrane rigidity during the inflammatory phase of EAE and supports the investigation of peripheral cell membrane lipids as possible biomarkers for CNS lipid membrane concentration and assembly.

Cholesterol as a tool to probe the role of membrane in cochlear hair cell mechanotransduction.

Most mechanically gated channels are sensitive to force translated through the membrane. The lipid bilayer can modulate channel function directly through lipid/protein interactions or indirectly based on membrane mechanical properties. Cholesterol is an important component of the membrane and a major modulator of membrane mechanical properties. While other ion channels and mechanically gated channels can be activated or modulated by changes to the membrane cholesterol, the functional role of membrane cholesterol in regulation of mammalian cochlear mechanotransduction (MET) channels has not been closely investigated. Using whole-cell patch clamping and live-cell fluorescence lifetime imaging (FLIM) of a viscosity-sensitive molecular rotor BODIPY 1c for the first time in the inner ear, we examined the role of membrane cholesterol in modulating the MET response properties of rat cochlear hair cells. Molecular rotors are fluorophores for which the fluorescence lifetime (the average time a fluorophore remains in the excited state) increase with increasing viscosity of their immediate environment. We confirmed extraction of cholesterol, using methyl β cyclodextrin (MβCD), with reduced filipin staining in both inner and outer hair bundles. MβCD results in reversible reduction in fluorescence lifetime in hair bundles, suggesting initial reduction followed by gradual recovery in the stereocilia membrane viscosity. MβCD reversibly increases the channel resting open probability, suggesting that cholesterol depletion increases force transfer to the MET channel. Together this data suggests that the cell membrane is part of the force relay machinery to the MET channel and could possibly interact directly with components of the MET machinery. Further studies are needed to generate causal link between MET channel gating and membrane mechanics.

The Antifungal Antibiotic Filipin as a Diagnostic Tool of Cholesterol Alterations in Lysosomal Storage Diseases and Neurodegenerative Disorders.

Cholesterol is the most considerable member of a family of polycyclic compounds understood as sterols, and represents an amphipathic molecule, such as phospholipids, with the polar hydroxyl group located in position 3 and the rest of the molecule is completely hydrophobic. In cells, it is usually present as free, unesterified cholesterol, or as esterified cholesterol, in which the hydroxyl group binds to a carboxylic acid and thus generates an apolar molecule. Filipin is a naturally fluorescent antibiotic that exerts a primary antifungal effect with low antibacterial activity, interfering with the sterol stabilization of the phospholipid layers and favoring membrane leakage. This polyene macrolide antibiotic does not bind to esterified sterols, but only to non-esterified cholesterol, and it is commonly used as a marker to label and quantify free cholesterol in cells and tissues. Several lines of evidence have indicated that filipin staining could be a good diagnostic tool for the cholesterol alterations present in neurodegenerative (e.g., Alzheimer's Disease and Huntington Disease) and lysosomal storage diseases (e.g., Niemann Pick type C Disease and GM1 gangliosidosis). Here, we have discussed the uses and applications of this fluorescent molecule in lipid storage diseases and neurodegenerative disorders, exploring not only the diagnostic strength of filipin staining, but also its limitations, which over the years have led to the development of new diagnostic tools to combine with filipin approach.

Intravenous 2-hydroxypropyl-β-cyclodextrin (Trappsol® Cyclo™) demonstrates biological activity and impacts cholesterol metabolism in the central nervous system and peripheral tissues in adult subjects with Niemann-Pick Disease Type C1: Results of a phase 1 trial.

Niemann-Pick Disease Type C1 (NPC1) is a disorder of intracellular cholesterol and lipid trafficking that leads to the accumulation of cholesterol and lipids in the late endosomal/lysosomal compartment, resulting in systemic manifestations (including hepatosplenomegaly and lung infiltration) and neurodegeneration. Preclinical studies have demonstrated that systemically administered 2-hydroxypropyl-β-cyclodextrin (HPβCD; Trappsol® Cyclo™) restores cholesterol metabolism and homeostasis in peripheral organs and tissues and in the central nervous system (CNS). Here, we assessed the safety, pharmacokinetics, and pharmacodynamics of HPβCD in peripheral tissues and the CNS in adult subjects with NPC1. A Phase 1, randomized, double-blind, parallel group study enrolled 13 subjects with NPC1 who received either 1500 mg/kg or 2500 mg/kg HPβCD intravenously every 2 weeks for a total of 7 doses (14 weeks). Subjects were 18 years or older, with a confirmed diagnosis of NPC1 and evidence of systemic involvement on clinical assessment. Pharmacokinetic evaluations in plasma and cerebrospinal fluid (CSF) were performed at the first and seventh infusions. Pharmacodynamic assessments included biomarkers of systemic cholesterol synthesis (serum lathosterol) and degradation (serum 4β-hydroxycholesterol), secondary sphingomyelin storage (plasma lysosphingomyelin-509, now more accurately referred to as N-palmitoyl-O-phosphocholineserine [PPCS]), and CNS-specific biomarkers of neurodegeneration (CSF total Tau) and cholesterol metabolism (serum 24(S)-hydroxycholesterol [24(S)-HC]). Safety monitoring included assessments of liver and kidney function, infusion related adverse events, and hearing evaluations. Ten subjects completed the study, with 6 at the 1500 mg/kg dose and 4 at the 2500 mg/kg dose. One subject withdrew following the first infusion after experiencing hypersensitivity pneumonitis, and 2 subjects withdrew after meeting a stopping rule related to hearing loss. Overall, HPβCD had an acceptable safety profile. The observed pharmacokinetic profile of HPβCD was similar following the first and seventh infusions, with a plasma half-life of 2 h, a maximum concentration reached at 6 to 8 h, and no evidence of accumulation. Serum biomarkers of cholesterol metabolism showed reduced synthesis and increased degradation. Compared to Baseline, filipin staining of liver tissue showed significant reductions of trapped unesterified cholesterol at both dose levels at Week 14. Plasma PPCS levels were also reduced. HPβCD was detected at low concentrations in the CSF (maximum, 33 μM) at both dose levels and persisted longer in CSF than in plasma. Total Tau levels in CSF decreased in most subjects. Serum levels of 24(S)-HC, a cholesterol metabolite from the CNS that is exported across the blood-brain barrier and into the circulation, decreased after both the first and seventh doses. Hence, pharmacodynamic assessments in both peripheral and CNS-related tissue show target engagement. While not the aim of the study, subjects reported favorable impacts on their quality of life. The plasma pharmacokinetics and pharmacodynamics of HPβCD administered at two intravenous dose levels to subjects with NPC1 were comparable to those observed in preclinical models. HPβCD cleared cholesterol from the liver and improved peripheral biomarkers of cholesterol homeostasis. At low CSF concentrations, HPβCD appeared to be pharmacologically active in the CNS based on the increased efflux of 24(S)-HC and reduction in CSF total Tau, a biomarker of CNS neurodegeneration. These data support the initiation of longer-term clinical trials to evaluate the safety and efficacy of intravenous HPβCD in subjects with NPC1. (ClinicalTrials.gov numbers: present trial, NCT02939547; open-label extension of the present trial, NCT03893071; global pivotal trial, NCT04860960).

Lancemaside A from Codonopsis lanceolata: Studies on Antiviral Activity and Mechanism of Action against SARS-CoV-2 and Its Variants of Concern.

Several plant-derived natural products with anti-SARS-CoV-2 activity have been evaluated for the potential to serve as chemotherapeutic agents for the treatment of COVID-19. Codonopsis lanceolata (CL) has long been used as a medicinal herb in East Asian countries to treat inflammatory diseases of the respiratory system but its antiviral activity has not been investigated so far. Here, we showed that CL extract and its active compound lancemaside A (LA) displayed potent inhibitory activity against SARS-CoV-2 infection using a pseudotyped SARS-CoV-2 entry assay system. We demonstrated that this inhibitory effect of LA was due to the alteration of membrane cholesterol and blockade of the membrane fusion between SARS-CoV-2 and host cells by filipin staining and cell-based membrane fusion assays. Our findings also showed that LA, as a membrane fusion blocker, could impede the endosomal entry pathway of SARS-CoV-2 and its variants of concern (VOCs), including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529), in Vero cells with similar of IC50 values ranging from 2.23 to 3.37 μM as well as the TMPRSS2-mediated viral entry pathway in A549 cells overexpressing ACE2 and TMPRSS2 with IC50 value of 3.92 μM. We further demonstrated that LA could prevent the formation of multinucleated syncytia arising from SARS-CoV-2 spike protein-mediated membrane fusion. Altogether, the findings reported here suggested that LA could be a broad-spectrum anti-SARS-CoV-2 therapeutic agent by targeting the fusion of viral envelope with the host cell membrane.

First Discovery of Cholesterol-Lowering Activity of Parthenolide as NPC1L1 Inhibitor.

Elevated cholesterol significantly increases the risk of developing atherosclerosis and coronary heart disease. The key to treating hypercholesterolemia is lowering plasma cholesterol levels. There have been no studies on the cholesterol-lowering potential of parthenolide (PTL), a naturally occurring small molecule from Tanacetum parthenium. Here, we first put forth PTL’s cholesterol-lowering ability to inhibit cellular uptake of cholesterol in a dose-dependent manner. Its performance was on par with the positive control drug, ezetimibe. Niemann–Pick C1 Like-1 (NPC1L1) has been identified as a potential therapeutic target for hypercholesterolemia. The interaction of PTL with NPC1L1 could be explained by the results of molecular docking and filipin staining further reinforces this hypothesis. Furthermore, PTL reduced the expression of NPC1L1 in HepG2 cells in a concentration-dependent manner, which suggests that PTL functions as a potential NPC1L1 inhibitor with therapeutic potential for hypercholesterolemia.

A Simple Fluorescent Cholesterol Labeling Method to Cryoprotect and Detect Plasma Lipoprotein-X.

Lipoprotein-X (LpX) are abnormal nephrotoxic lipoprotein particles enriched in free cholesterol and phospholipids. LpX with distinctive lipid compositions are formed in patients afflicted with either familial LCAT deficiency (FLD) or biliary cholestasis. LpX is difficult to detect by standard lipid stains due to the absence of a neutral lipid core and because it is unstable upon storage, particularly when frozen. We have recently reported that free cholesterol-specific filipin staining after agarose gel electrophoresis sensitively detects LpX in fresh human plasma. Herein, we describe an even more simplified qualitative method to detect LpX in both fresh and frozen-thawed human FLD or cholestatic plasma. Fluorescent cholesterol complexed to fatty-acid-free BSA was used to label LpX and was added together with trehalose in order to cryopreserve plasma LpX. The fluorescent cholesterol bound to LpX was observed with high sensitivity after separation from other lipoproteins by agarose gel electrophoresis. This methodology can be readily developed into a simple assay for the clinical diagnosis of FLD and biliary liver disease and to monitor the efficacy of treatments intended to reduce plasma LpX in these disease states.

Analysis of Brain Lipids in the Early-Onset Tay-Sachs Disease Mouse Model With the Combined Deficiency of β-Hexosaminidase A and Neuraminidase 3.

Introduction: Tay–Sachs disease is an autosomal recessively inherited lysosomal storage disease that results from loss-of-function mutations in the HEXA gene coding β-hexosaminidase A. HEXA gene deficiency affects the central nervous system owing to GM2 ganglioside accumulation in lysosomes resulting in progressive neurodegeneration in patients. We recently generated a novel mice model with a combined deficiency of β-hexosaminidase A and neuraminidase 3 (Hexa−/−Neu3−/−) that mimics both the neuropathological and clinical abnormalities of early-onset Tay–Sachs disease. Here, we aimed to explore the secondary accumulation of lipids in the brain of Hexa−/−Neu3−/− mice. Materials and Methods: In the cortex and hippocampus of five-month-old WT, Hexa−/−, Neu3−/−, and Hexa−/−Neu3−/− mice, lipid levels belonging to glycerolipids, glycerophospholipids, and sterol lipids were evaluated using a shotgun lipidomics approach. The levels of myelin were also assessed by luxol fast blue staining and immunohistochemistry using antibodies against myelin basic protein. We further examined glycoconjugate and cholesterol levels by periodic acid–Schiff and filipin staining, respectively. Toluidine blue staining was also performed to display axonal degeneration. Results: Among glycerophospholipids, we demonstrated elevated levels of phosphatidylcholine-ether and lysophosphatidylcholine while decreased levels of phosphatidylcholine and phosphatidylserine in both cortex and hippocampus of Hexa−/−Neu3−/− mice. In the glycerolipid class, we showed an alleviated level of sphingomyelin in both cortex and hippocampus, but the higher levels of diacylglycerol and triacylglycerol were detected in only the hippocampus of Hexa−/−Neu3−/− mice. The lower level of sterol was also detected in the cortex of Hexa−/−Neu3−/− mice but not in the hippocampus.Histochemical studies showed a decrease in the myelin level and axonal degeneration indicating neuronal pathology in the brain of Hexa−/−Neu3−/− mice. Although glycoconjugate accumulation was evident both in the cortex and hippocampus, we did not detect any changes in the level of cholesterol. Conclusion: Our results indicate that alterations in lipid metabolism and neuropathology, such as demyelination and axonal degeneration, might be related to the dysfunctionality of lipid-related cellular pathways like autophagy. Understanding of brain-specific lipid alterations contributes to evaluating the effectiveness of treatments in Hexa−/−Neu3−/− mice in future studies.

Inflammation Induced by Lipopolysaccharide and Palmitic Acid Increases Cholesterol Accumulation via Enhancing Myeloid Differentiation Factor 88 Expression in HepG2 Cells.

Recently, multiple studies have shown that chronic inflammation disturbs cholesterol homeostasis and promotes its accumulation in the liver. The underlying molecular mechanism remains to be revealed. The relationship between the toll-like receptor 4 (TLR4) inflammatory signaling pathway and cholesterol accumulation was investigated in HepG2 cells treated with lipopolysaccharide (LPS) or palmitic acid (PA) for different lengths of time. In addition, the effects of pretreatment with 20μmol/L ST2825 (MyD88 inhibitor) were also studied in LPS- or PA-treated HepG2 cells and myeloid differentiation factor 88 (MyD88)-overexpressing HEK293T cells. The intracellular total and free cholesterol levels were measured using a commercial kit and filipin staining, respectively. The expression levels of sterol regulatory element-binding protein-2 (SREBP-2) and components in the TLR4 signaling pathway were determined using Western blotting. The treatments with LPS for 12 h and with PA for 24 h significantly increased the contents of intracellular total and free cholesterol, as well as the expression levels of SREBP-2 and components in the TLR4 signaling pathway. The inhibition of MyD88 by ST2825 significantly decreased the cholesterol content and the expression levels of SREBP-2 and components of the TLR4/MyD88/NF-κB pathway in HepG2 cells, as well as MyD88-overexpressing HEK293T cells. These results indicated that LPS and PA treatments increase SREBP-2-mediated cholesterol accumulation via the activation of the TLR4/MyD88/NF-κB signaling pathway in HepG2 cells.

Chemical Exchange Saturation Transfer (CEST) Signal at -1.6 ppm and Its Application for Imaging a C6 Glioma Model.

The chemical exchange saturation transfer (CEST) signal at −1.6 ppm is attributed to the choline methyl on phosphatidylcholines and results from the relayed nuclear Overhauser effect (rNOE), that is, rNOE(−1.6). The formation of rNOE(−1.6) involving the cholesterol hydroxyl is shown in liposome models. We aimed to confirm the correlation between cholesterol content and rNOE(−1.6) in cell cultures, tissues, and animals. C57BL/6 mice (N = 9) bearing the C6 glioma tumor were imaged in a 7 T MRI scanner, and their rNOE(−1.6) images were cross-validated through cholesterol staining with filipin. Cholesterol quantification was obtained using an 18.8-T NMR spectrometer from the lipid extracts of the brain tissues from another group of mice (N = 3). The cholesterol content in the cultured cells was manipulated using methyl-β-cyclodextrin and a complex of cholesterol and methyl-β-cyclodextrin. The rNOE(−1.6) of the cell homogenates and their cholesterol levels were measured using a 9.4-T NMR spectrometer. The rNOE(−1.6) signal is hypointense in the C6 tumors of mice, which matches the filipin staining results, suggesting that their tumor region is cholesterol deficient. The tissue extracts also indicate less cholesterol and phosphatidylcholine contents in tumors than in normal brain tissues. The amplitude of rNOE(−1.6) is positively correlated with the cholesterol concentration in the cholesterol-manipulated cell cultures. Our results indicate that the cholesterol dependence of rNOE(−1.6) occurs in cell cultures and solid tumors of C6 glioma. Furthermore, when the concentration of phosphatidylcholine is carefully considered, rNOE(−1.6) can be developed as a cholesterol-weighted imaging technique.