Cellular and Molecular Life Sciences最新文献

Background: Premature ovarian insufficiency (POI) profoundly compromises female reproductive health through accelerated follicle depletion and endocrine disruption. Emerging evidence highlights the therapeutic potential of mesenchymal stem cell-derived exosomes (MSC-Exs), particularly when their function is enhanced by hypoxic preconditioning. In this study, the ability of hypoxia-preconditioned MSC-Exs (H-Exs) to ameliorate oxidative damage to granulosa cells (GCs) and restore ovarian function, was systematically evaluated, and a POI rat model was used to investigate the underlying mechanism.

Methods: CircRNAs specifically expressed in H-Exs were identified and validated. The ability of H-Exs and their corresponding circPTP4A2 to repair oxidative damage and restore mitochondrial function were evaluated by antioxidant enzyme assays, reactive oxygen species (ROS) assays, JC-1 staining, ATP level assays, oxygen consumption rate (OCR) measurements and TEM. The interaction between circPTP4A2 and YBX1 was analysed by molecular dynamics simulations, RIP, CHX assays, and MG132 assays, and the restorative effect of the circPTP4A2/YBX1 axis on ovarian function was verified.

Results: Our findings revealed that compared with normoxic MSC-Exs (N-Exs), H-Exs exerted superior protective effects, significantly attenuating oxidative stress and restoring mitochondrial bioenergetics in KGN cells. Mechanistically, circPTP4A2 was identified as a hypoxia-responsive cargo selectively enriched in H-Exs. This circular RNA stabilized Y-box binding protein 1 (YBX1) through direct interaction, increasing its antioxidative capacity and mitochondrial regulatory functions. Hypoxia-inducible factor 1-alpha (HIF-1α) was further shown to transcriptionally upregulate circPTP4A2 via direct binding to the promoter region of its host gene PTP4A2.

Conclusion: These results establish the circPTP4A2/YBX1 axis as a critical mediator of the therapeutic efficacy of H-Exs for POI, providing both mechanistic insights and a translational framework for exosome-based regenerative strategies.

Retinal ganglion cell (RGC) degeneration caused by optic nerve injury and diseases such as glaucoma leads to irreversible vision loss, yet effective neuroprotective treatments remain elusive. Secondary degeneration driven by astrocytic gliosis and neuroinflammation contributes substantially to neuronal death. Connexin 43 (Cx43), a gap junction protein abundantly expressed in astrocytes, is a key mediator of these secondary responses. Using an optic nerve crush (ONC) mouse model that recapitulates traumatic optic neuropathy, we found that Cx43 haploinsufficiency significantly preserved visual function, limited inner retina thinning, and protected RGCs from apoptosis and macrophage infiltration. Mechanistically, cytokine stimulation of astrocytes triggered Cx43 hemichannel opening and the release of inflammatory ATP and neurotoxic glutamate, which in turn promote RGC apoptosis. A novel Cx43(M1) antibody selectively inhibited astrocytic hemichannels, prevented the release of these factors, and reduced RGC death. Remarkably, a single administration of Cx43(M1) 30 min after ONC improved visual function and RGC survival for at least four weeks, accompanied by attenuated gliosis and reduced Cx43 expression. Together, these findings identify astrocytic Cx43 hemichannels as key mediators of secondary RGC neurodegeneration and demonstrate that their targeted inhibition confers sustained neuroprotection following optic nerve injury.

TLR-targeted immunotherapy represents a promising strategy for combating infectious diseases by initiating or enhancing protective antimicrobial immunity. Here, we identified the first frog-derived TLR2 and TLR4 agonist, Cathelicidin-Ka (Cath-Ka), from the skin of Kaloula pulchra. The presence of Cath-Ka significantly enhanced proliferation, cytokine production, polarization, chemotaxis, phagocytosis, and intracellular bacterial killing of macrophages and peritoneal cells by targeting TLR2 and TLR4, rather than other pattern recognition receptors, and subsequently activated the downstream MyD88-MAPKs pathway. Cath-Ka also promoted macrophage polarization towards the M1 rather than M2 phenotype, and its intraperitoneal injection significantly promoted the chemotaxis of pro-inflammatory monocytes/macrophages into the peritoneal cavity. Finally, the mutant of Cath-Ka with amination at C-terminus had stronger effects on macrophage function modulation than the original peptide. These findings suggest that Cath-Ka and its amidated mutant are promising candidates for the treatment of TLR2 and TLR4-related diseases, including infections.

Autophagy, the process for recycling cytoplasm in the lysosome, relies on tightly regulated membrane trafficking. During autophagy, autophagosomes either fuse with endosomes generating amphisomes and then lysosomes, or directly fuse with lysosomes, in both cases generating autolysosomes that degrade their contents. It remains unclear whether specific mechanisms or conditions determine these alternate routes. Here, we demonstrate that the endosomal regulator SNX3 specifically regulates basal autophagy under nutrient-adequate conditions in both Caenorhabditis elegans (C. elegans) and cultured mammalian cells. In C. elegans, SNX-3 depletion elevates autophagy independently of the UNC-51/ULK1 complex and leads to the accumulation of both autophagosomes and amphisomes, which consequently impairs the clearance of autophagic cargo, including SQST-1/p62 and protein aggregates. Mechanistically, SNX-3 depletion differentially regulates the machineries required for autophagosome-lysosome fusion. In snx-3 mutants, the Q-SNARE components SYX-17 and SNAP-29 translocate to autophagosomes, where they assemble with the endosomal R-SNAREs VAMP-7 and VAMP-8 to promote amphisome formation. Conversely, loss of SNX-3 impairs the lysosomal delivery of VAMP-8 and RAB-7, both essential for autophagosome/amphisome-lysosome fusion, thereby generating fusion-incompetent lysosomes. However, starvation restores the lysosomal fusion capability compromised by snx-3 depletion. Our findings reveal that autophagosome-lysosome fusion is preferentially regulated by nutrient status, and identify an endosomal regulator that tunes membrane trafficking with changing autophagy demands.

Aberrant protein regulatory pathways disrupt bone development and contribute to skeletal diseases. The cysteine protease family of deubiquitinating enzymes (DUBs) are critical for regulation of bone-resorbing osteoclasts and bone-forming osteoblasts. Here, we demonstrate that the DUB ubiquitin-specific protease 8 (USP8) is highly expressed in osteoclasts and its deletion impairs osteoclast development and bone resorption activity. Deletion of Usp8 in osteoclasts (Usp8^Ctsk) results in low trabecular bone mass due to defective endochondral bone formation and short stature resulting from abnormal growth plate structure. Usp8 deficiency in osteoclasts reduces the number of mitochondrial, mitochondrial activity, oxidative phosphorylation, and mitophagy, while ROS production and inflammatory responses increased. USP8 mediates the regulation of mitophagy in osteoclasts through the stabilization of Parkin. Moreover, Usp8-deficient osteoclasts in metaphysis secrete factors that impair both growth plate development and trabecular bone formation. Collectively, these findings identify USP8 as a key regulator of osteoclast development and secretory factor production, shaping the microenvironment essential for skeletal development.

Cancer stem cells (CSCs) are closely related to tumor drug resistance, recurrence, and metastasis, presenting significant challenges in cancer treatment. Although numerous long noncoding RNAs (lncRNAs) have been implicated in colorectal cancer (CRC), only a few have been reported to regulate cancer stemness. The lncRNA ELFN1-AS1 is known to play a crucial role in CRC development; however, its function in maintaining cancer stemness remains uncertain. In this study, we established colorectal cancer stem cell-like (CSC-like) cells enrichment models using human SW620 and HCT116 cell lines and found that ELFN1-AS1 was significantly overexpressed in CD44⁺CD133⁺ CSC-like cells. Functional assays, including cell spheroid formation, colony formation, and subcutaneous tumor transplantation, demonstrated that ELFN1-AS1 knockdown effectively suppressed tumorigenic capacity. Limiting dilution assays, both in vitro and in vivo, further validated the critical role of ELFN1-AS1 in tumor initiation. Mechanistic investigations, including RNA pulldown, mass spectrometry, RNA immunoprecipitation (RIP), and colocalization staining, revealed that ELFN1-AS1 directly interacts with hnRNPA1 in the nucleus of CSC-like cells. ELFN1-AS1 promoted the expression of downstream proteins CD44v6 and PKM2 by competitively binding to hnRNPA1, thereby inhibiting the interaction between the E3 ubiquitin ligase β-TrCP and hnRNPA1. Our findings demonstrated that ELFN1-AS1 facilitated CRC malignancy by maintaining and enhancing tumor cell stemness, suggesting that targeting ELFN1-AS1 may represent a potential therapeutic strategy for CRC.

The neuropeptide Y₄ receptor (Y₄R) and its endogenous ligand pancreatic polypeptide (PP) are primarily involved in the regulation of satiety and energy balance and present relevant pharmacological targets. We characterized the novel Y₄R positive allosteric modulator C1 that enhances Y₄R G-protein signaling, ligand binding, and arrestin-3 recruitment to Y₄R. Comparison with a close analog revealed the structural importance of an ethyl acetate moiety for Y₄R affinity and PAM activity at the G-protein pathway. C1 shows a high selectivity for the Y₄R, while signaling of the related subtypes Y₁R, Y₂R, and Y₅R is not affected. Y₄R G-protein signaling is even potentiated by the low-affinity agonists neuropeptide Y and peptide YY. Binding affinity of the endogenous ligands to Y₄R is enhanced by C1, indicating a stabilization of the ligand-bound Y₄R conformation. Using Y₄R/Y₁R chimera, important Y₄R domains for C1 activity were identified. Single point mutagenesis and computational docking pinpointed hot-spot residues at Y₄R important for stabilizing the active ligand-bound conformation.

Usher syndrome type 1B (USH1B) is a rare inherited disorder characterized by congenital deafness and progressive retinitis pigmentosa, caused by biallelic pathogenic variants in the MYO7A gene. We explored extracellular vesicles (EVs) from two sources: human tears and iPSC-derived RPE cells from USH1B patients and controls. Tear EVs were assessed as a non-invasive biomarker source, while RPE-derived EVs provided insights into disease mechanisms in a controlled, cell-type-specific context. Although RPE differentiation was successful and MYO7A expression levels were similar between patients and controls, Myosin VIIA was not detected by western blot in the patient-derived cells. We examined the EV cargo by small non-coding RNAs (sncRNAs) sequencing from iPSC-RPE apical site and tears to identify molecular signatures of retinal degeneration. Tear EVs showed higher load and diversity of miRNAs than RPE-derived EVs, reflecting a broader ocular origin. Comparative analysis revealed shared retinal sncRNAs (hsa-miR-204, hsa-miR-211, hsa-miR-181a-5p) and group-specific differences. Notably, when comparing to controls, hsa-miR-200a-3p and hsa-miR-194-5p were upregulated in patient tear EVs, while let-7i/c-5p and hsa-miR-320a/b, were downregulated in-patient RPE-derived EVs. Pathway analysis linked these sncRNAs to retinal structure and function, including cytoskeletal remodeling and junctional integrity. Our findings highlight the potential of tear EVs as a non-invasive source of biomarkers that capture retinal molecular alterations in USH1B, with applications for diagnosis, monitoring, and therapeutic development. Although this is a pilot study focused on uncovering promising biomarkers rather than establishing definitive cause-effect mechanisms, it provides a foundation for future research with larger cohorts to validate and expand these findings.