MLN64 is a late endosomal cholesterol-binding membrane protein that has been implicated in cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria, in toxin-induced resistance, and in mitochondrial dysfunction. Down-regulation of MLN64 in Niemann-Pick C1 deficient cells decreased mitochondrial cholesterol content, suggesting that MLN64 functions independently of NPC1. However, the role of MLN64 in the maintenance of endosomal cholesterol flow and intracellular cholesterol homeostasis remains unclear. We have previously described that hepatic MLN64 overexpression increases liver cholesterol content and induces liver damage. Here, we studied the function of MLN64 in normal and NPC1-deficient cells and we evaluated whether MLN64 overexpressing cells exhibit alterations in mitochondrial function. We used recombinant-adenovirus-mediated MLN64 gene transfer to overexpress MLN64 in mouse liver and hepatic cells; and RNA interference to down-regulate MLN64 in NPC1-deficient cells. In MLN64-overexpressing cells, we found increased mitochondrial cholesterol content and decreased glutathione (GSH) levels and ATPase activity. Furthermore, we found decreased mitochondrial membrane potential and mitochondrial fragmentation and increased mitochondrial superoxide levels in MLN64-overexpressing cells and in NPC1-deficient cells. Consequently, MLN64 expression was increased in NPC1-deficient cells and reduction of its expression restore mitochondrial membrane potential and mitochondrial superoxide levels. Our findings suggest that MLN64 overexpression induces an increase in mitochondrial cholesterol content and consequently a decrease in mitochondrial GSH content leading to mitochondrial dysfunction. In addition, we demonstrate that MLN64 expression is increased in NPC cells and plays a key role in cholesterol transport into the mitochondria.

Niemann–Pick type C disease (NPCD) is a lysosomal storage disease (LSD) characterized by abnormal cholesterol accumulation in lysosomes, impaired autophagy flux, and lysosomal dysfunction. The activation of transcription factor EB (TFEB), a master lysosomal function regulator, reduces the accumulation of lysosomal substrates in LSDs where the degradative capacity of the cells is compromised. Genistein can pass the blood–brain barrier and activate TFEB. Hence, we investigated the effect of TFEB activation by genistein toward correcting the NPC phenotype. We show that genistein promotes TFEB translocation to the nucleus in HeLa TFEB-GFP, Huh7, and SHSY-5Y cells treated with U18666A and NPC1 patient fibroblasts. Genistein treatment improved lysosomal protein expression and autophagic flux, decreasing p62 levels and increasing those of the LC3-II in NPC1 patient fibroblasts. Genistein induced an increase in β-hexosaminidase activity in the culture media of NPC1 patient fibroblasts, suggesting an increase in lysosomal exocytosis, which correlated with a decrease in cholesterol accumulation after filipin staining, including cells treated with U18666A and NPC1 patient fibroblasts. These results support that genistein-mediated TFEB activation corrects pathological phenotypes in NPC models and substantiates the need for further studies on this isoflavonoid as a potential therapeutic agent to treat NPCD and other LSDs with neurological compromise.

Niemann-Pick type C disease (NPCD) is a lysosomal storage disorder caused by mutations in the NPC1 gene. The most affected tissues are the central nervous system and liver, and while significant efforts have been made to understand its neurological component, the pathophysiology of the liver damage remains unclear. In this study, hepatocytes derived from wild type and Npc1^−/− mice were analyzed by mass spectrometry (MS)-based proteomics in conjunction with bioinformatic analysis. We identified 3832 proteins: 416 proteins had a p-value smaller than 0.05, of which 37% (n = 155) were considered differentially expressed proteins (DEPs), 149 of them were considered upregulated, and 6 were considered downregulated. We focused the analysis on pathways related to NPC pathogenic mechanisms, finding that the most significant changes in expression levels occur in proteins that function in the pathways of liver damage, lipid metabolism, and inflammation. Moreover, in the group of DEPs, 30% (n = 47) were identified as lysosomal proteins and 7% (n = 10) were identified as mitochondrial proteins. Importantly, we found that lysosomal DEPs, including CTSB/D/Z, LIPA, DPP7 and GLMP, and mitocondrial DEPs, AKR1B10, and VAT1 had been connected with liver fibrosis, damage, and steatosis in previous studies, validiting our dataset. Our study found potential therapeutic targets for the treatment of liver damage in NPCD.

The global prevalence of Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is increasing, now affecting 25%–30% of the population worldwide. MASLD, characterized by hepatic steatosis, results from an imbalance in lipid metabolism, leading to oxidative stress, lipoperoxidation, and inflammation. The activation of autophagy, particularly lipophagy, alleviates hepatic steatosis by regulating intracellular lipid levels. Lutein, a carotenoid with antioxidant and anti-inflammatory properties, protects against liver damage, and individuals who consume high amounts of lutein have a lower risk of developing MASLD. Evidence suggests that lutein could modulate autophagy-related signaling pathways, such as the transcription factor EB (TFEB). TFEB plays a crucial role in regulating lipid homeostasis by linking autophagy to energy metabolism at the transcriptional level, making TFEB a potential target against MASLD. STARD3, a transmembrane protein that binds and transports cholesterol and sphingosine from lysosomes to the endoplasmic reticulum and mitochondria, has been shown to transport and bind lutein with high affinity. This protein may play a crucial role in the uptake and transport of lutein in the liver, contributing to the decrease in hepatic steatosis and the regulation of oxidative stress and inflammation. This review summarizes current knowledge on the role of lutein in lipophagy, the pathways it is involved in, its relationship with STARD3, and its potential as a pharmacological strategy to treat hepatic steatosis.

Introduction: Skeletal muscle differentiation in the C2C12 myoblast model requires extensive mitochondrial remodeling to meet rising bioenergetic demands through coordinated changes in biogenesis, dynamics, and respiratory adaptation. Urolithin A (UA), a gut microbiota-derived metabolite of ellagitannins, improves mitochondrial health, but its role in late-stage myogenic differentiation remains unclear. Methods: C2C12 myotubes were treated with UA (2 μM) for 72 h during late-stage differentiation (days 3–6). Mitochondrial signaling, respiratory capacity, myogenic morphology, and ultrastructure were assessed by Western blot, high-resolution respirometry, hematoxylin–eosin staining, and transmission electron microscopy. Results: UA was non-cytotoxic and increased AMPKα phosphorylation and PGC-1α expression, whereas TOM20, MFN2, and OPA1 were unchanged. Mitophagy/autophagy-related markers (p-ULK1, p62, BNIP3L/NIX, LC3-II/I) were not altered, indicating no detectable changes in steady-state autophagy under the conditions tested. UA selectively increased OXPHOS Complex I and II abundance and enhanced maximal uncoupled respiration, and was associated with increased myotube diameter and myogenic marker abundance. No overt ultrastructural differences were observed by electron microscopy. Discussion: These findings suggest that UA promotes mitochondrial functional adaptation during myogenic differentiation, with accompanying changes in myogenic phenotype, without clear evidence of altered steady-state mitophagy/autophagy markers or mitochondrial morphology.

When the original text contained incorrect information, to preserve the scientific record, please include that text when editing the below templates. For example:There was a mistake in the Funding statement, an incorrect number was used. The correct number is '2015C03Bd051'. The publisher apologizes for this mistake.The original version of this article has been updated.In the published article, there was a mistake in the Funding statement. The funding statement for the Key Development Project of the Department of Science and Technology was displayed as '2015CBd051'. The correct statement is 'Key Development Project of Department of Science and Technology (2015C03Bd051)'.Figure 2: Proposed mechanisms involved in lutein-mediated hepatoprotective effects. We propose that independent of its antioxidant and antiapoptotic activity, lutein stimulates LD autophagy in the liver, reducing their accumulation and improving steatosis. This action is mediated through the activation of TFEB. Given lutein's hydrophobic nature as a carotenoid, an intracellular transporter is required to exert its activity within the cell. We propose that STARD3 serves as this transporter. 1) To enter the cell, lutein in lipoproteins, primarily QM and HDL, binds to their transporters on the hepatocyte membrane (SRBI, LDLR, LRP1). 2) Additionally, lutein may directly enter the cell by binding to STARD3 on the plasma membrane. 3) Once inside the cell, lutein bound to STARD3 would directly or indirectly interact with TFEB, mediating its activation. 4) Another pathway for lutein entry involves endosomal uptake, where it binds to STARD3 in the lysosome, activating TFEB via the MCOLN1/Ca2+/calcineurina pathway.End of template. If you would like to request a correction for a reason not seen here, please contact the journal's editorial office.