Cellular and Molecular Life Sciences最新文献

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.

The progression of spermatogenesis is under dynamic transcriptional regulation. As a subunit of the transcription-export complex 2 (TREX-2), PCI domain-containing protein 2 (PCID2), participates in RNA processing. However, the physiological functions of PCID2 in spermatogenesis remain poorly understood. Here, we generate germline conditional knockout (Pcid2-SKO) mice using Stra8-Cre, and it is found that Pcid2-SKO mice are infertile, exhibit extensive germ cell apoptosis, impaired spermatogonial differentiation, and failure of meiosis initiation. Single-cell transcriptome analysis reveals developmental arrest at the transition from type A to type B spermatogonia in Pcid2-SKO mice. Gene Set Enrichment Analysis (GSEA) demonstrates a significant decrease in the enrichment of mRNA splicing pathway in Pcid2-SKO germ cells. IP-MS results indicate candidate proteins interacting with PCID2 are significantly enriched in RNA splicing pathway. Co-IP results indicate that PCID2 interacts with SNRPG, hnRNPH1 and SF3B1 to modulate alternative splicing in germ cells. Combining RNA sequencing and PCR identifies four key genes (Prpf3, Nek3, Dvl2, and Slc30a9) as splicing targets of PCID2. Collectively, PCID2 is essential for normal spermatogenesis and male fertility by regulating the alternative splicing (AS) of genes critical for cell cycle progression, spliceosome assembly, and mitochondrial homeostasis. This study provides novel insights into the molecular mechanisms underlying spermatogenesis and highlights the importance of AS in germ cell development.

Neurons have adapted the transport and positioning of mitochondria to fit their extended shape and high energy needs. To sustain mitochondrial function, neurons developed systems that allow local biogenesis and adaption to locally regulate mitochondrial form and function. Likewise, fine-tuned degradative systems are required to protect the neurons from mitochondrial dysfunction. Throughout both domains of mitostasis, the local synthesis of the mitochondrial damage-induced kinase PINK1 emerges as a central player. Along with other nuclear encoded mitochondrial proteins, its mRNA associates with mitochondria to sustain mitochondrial function locally. It also regulates mitochondrial degradation, via regulation of proteases, the generation of mitochondria-derived vesicles and mitophagy. In this review, we provide a general overview of the mechanisms governing mitochondrial health in neurons, with a special focus on the role of PINK1 in this endeavor.

Human cytomegalovirus (HCMV) is the leading viral cause of congenital defects. The triggers of viral neuropathogenesis during congenital infection (cCMV) are still unclear, and treatment options are limited. We used both a two-dimensional model of dynamic neurogenesis and cerebral organoids (COs), recapitulating the developing brain in the first trimester of gestation, to investigate the neuropathogenesis induced by HCMV. We also evaluated antiviral and neuroprotective effects of different compounds, both approved, direct-acting drugs and investigational, host-directed antivirals. In differentiating neurons, treatment with direct-acting antivirals blocked HCMV active replication and provided some protection from virus-induced defects. COs exposed to two different strains of HCMV showed viral spread throughout the organoids, dysregulation of key players of neurogenesis, alteration of the tissue cytoarchitecture, and triggering of innate antiviral and pro-inflammatory responses. Inter-strain differences in virus release and growth attenuation were detected in infected COs. Regardless of the strain, treatment with direct-acting antivirals, particularly letermovir, completely abolished HCMV replication, protected COs from virus-induced disorganization of tissue architecture, and dampened innate immune and pro-inflammatory response activation. Importantly, we also demonstrated the efficacy of the antiviral treatment in HCMV-infected COs in blocking an already established infection. This study contributes to shed light on HCMV-induced neuropathogenesis that occurs during congenital infection. Importantly, we demonstrated the neuroprotective effects of letermovir in models of human developing brain, holding promise for its evaluation as a candidate therapeutic agent to ameliorate cCMV-associated neurodevelopmental defects.