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JCI Insight Jun 2024Autosomal dominant optic atrophy plus (ADOA+) is characterized by primary optic nerve atrophy accompanied by a spectrum of degenerative neurological symptoms. Despite...
Autosomal dominant optic atrophy plus (ADOA+) is characterized by primary optic nerve atrophy accompanied by a spectrum of degenerative neurological symptoms. Despite ongoing research, no effective treatments are currently available for this condition. Our study provided evidence for the pathogenicity of an unreported c.1780T>C variant in the OPA1 gene through patient-derived skin fibroblasts and an engineered HEK293T cell line with OPA1 downregulation. We demonstrated that OPA1 insufficiency promoted mitochondrial fragmentation and increased DRP1 expression, disrupting mitochondrial dynamics. Consequently, this disruption enhanced mitophagy and caused mitochondrial dysfunction, contributing to the ADOA+ phenotype. Notably, the Drp1 inhibitor, mitochondrial division inhibitor-1 (Mdivi-1), effectively mitigated the adverse effects of OPA1 impairment. These effects included reduced Drp1 phosphorylation, decreased mitochondrial fragmentation, and balanced mitophagy. Thus, we propose that intervening in DRP1 with Mdivi-1 could correct mitochondrial abnormalities, offering a promising therapeutic approach for managing ADOA+.
PubMed: 38916953
DOI: 10.1172/jci.insight.180582 -
Microbiology Spectrum Jun 2024() as well as nontuberculous mycobacteria are intracellular pathogens whose treatment is extensive and increasingly impaired due to the rise of mycobacterial drug...
UNLABELLED
() as well as nontuberculous mycobacteria are intracellular pathogens whose treatment is extensive and increasingly impaired due to the rise of mycobacterial drug resistance. The loss of antibiotic efficacy has raised interest in the identification of host-directed therapeutics (HDT) to develop novel treatment strategies for mycobacterial infections. In this study, we identified amiodarone as a potential HDT candidate that inhibited both intracellular and in primary human macrophages without directly impairing bacterial growth, thereby confirming that amiodarone acts in a host-mediated manner. Moreover, amiodarone induced the formation of (auto)phagosomes and enhanced autophagic targeting of mycobacteria in macrophages. The induction of autophagy by amiodarone is likely due to enhanced transcriptional regulation, as the nuclear intensity of the transcription factor EB, the master regulator of autophagy and lysosomal biogenesis, was strongly increased. Furthermore, blocking lysosomal degradation with bafilomycin impaired the host-beneficial effect of amiodarone. Finally, amiodarone induced autophagy and reduced bacterial burden in a zebrafish embryo model of tuberculosis, thereby confirming the HDT activity of amiodarone . In conclusion, we have identified amiodarone as an autophagy-inducing antimycobacterial HDT that improves host control of mycobacterial infections.
IMPORTANCE
Due to the global rise in antibiotic resistance, there is a strong need for alternative treatment strategies against intracellular bacterial infections, including () and non-tuberculous mycobacteria. Stimulating host defense mechanisms by host-directed therapy (HDT) is a promising approach for treating mycobacterial infections. This study identified amiodarone, an antiarrhythmic agent, as a potential HDT candidate that inhibits the survival of and in primary human macrophages. The antimycobacterial effect of amiodarone was confirmed in an tuberculosis model based on infection of zebrafish embryos. Furthermore, amiodarone induced autophagy and inhibition of the autophagic flux effectively impaired the host-protective effect of amiodarone, supporting that activation of the host (auto)phagolysosomal pathway is essential for the mechanism of action of amiodarone. In conclusion, we have identified amiodarone as an autophagy-inducing HDT that improves host control of a wide range of mycobacteria.
PubMed: 38916320
DOI: 10.1128/spectrum.00167-24 -
Microbiology Spectrum Jun 2024is a microaerophilic Gram-negative bacterium that resides in the human stomach and is classified as a class I carcinogen for gastric cancer. Numerous studies have...
is a microaerophilic Gram-negative bacterium that resides in the human stomach and is classified as a class I carcinogen for gastric cancer. Numerous studies have demonstrated that infection plays a role in regulating the function of host cells, thereby contributing to the malignant transformation of these cells. However, infection is a chronic process, and short-term cellular experiments may not provide a comprehensive understanding of the situation, especially when considering the lower oxygen levels in the human stomach. In this study, we aimed to investigate the mechanisms underlying gastric cell dysfunction after prolonged exposure to under hypoxic conditions. We conducted a co-culture experiment using the gastric cell line GES-1 and for 30 generations under intermittent hypoxic conditions. By closely monitoring cell proliferation, migration, invasion, autophagy, and apoptosis, we revealed that sustained stimulation under hypoxic conditions significantly influences the function of GES-1 cells. This stimulation induces epithelial-mesenchymal transition and contributes to the propensity for malignant transformation of gastric cells. To confirm the results, we conducted an experiment involving Mongolian gerbils infected with for 85 weeks. All the results strongly suggest that the Nod1 receptor signaling pathway plays a crucial role in -related apoptosis and autophagy. In summary, continuous stimulation by affects the functioning of gastric cells through the Nod1 receptor signaling pathway, increasing the likelihood of cell carcinogenesis. The presence of hypoxic conditions further exacerbates this process.IMPORTANCEDeciphering the collaborative effects of infection on gastric epithelial cell function is key to unraveling the development mechanisms of gastric cancer. Prior research has solely examined the outcomes of short-term stimulation on gastric epithelial cells under aerobic conditions, neglecting the bacterium's nature as a microaerophilic organism that leads to cancer following prolonged stomach colonization. This study mimics a more genuine infection scenario by repeatedly exposing gastric epithelial cells to under hypoxic conditions for up to 30 generations. The results show that chronic exposure to in hypoxia substantially increases cell migration, invasion, and epithelial-mesenchymal transition, while suppressing autophagy and apoptosis. This highlights the significance of hypoxic conditions in intensifying the carcinogenic impact of infection. By accurately replicating the gastric environment, this study enhances our comprehension of 's pathogenic mechanisms in gastric cancer.
PubMed: 38916312
DOI: 10.1128/spectrum.00311-24 -
Frontiers in Molecular Neuroscience 2024Advancements in understanding the pathogenesis of C9orf72-associated frontotemporal dementia (C9orf72-FTD) have highlighted the role of repeat-associated non-ATG (RAN)...
Advancements in understanding the pathogenesis of C9orf72-associated frontotemporal dementia (C9orf72-FTD) have highlighted the role of repeat-associated non-ATG (RAN) translation and dipeptide repeat proteins (DPRs), with models providing valuable insights. While studies have primarily focused on RAN translation and DPR toxicity, emerging areas of investigation in fly models have expanded to neuronal dysfunction, autophagy impairment, and synaptic dysfunction, providing potential directions for new therapeutic targets and mechanisms of neurodegeneration. Despite this progress, there are still significant gaps in models of C9orf72-FTD, namely in the areas of metabolism and circadian rhythm. Metabolic dysregulation, particularly lipid metabolism, autophagy, and insulin signaling, has been implicated in disease progression with findings from animal models and human patients with C9orf72 repeat expansions. Moreover, circadian disruptions have been observed in C9of72-FTD, with alterations in rest-activity patterns and cellular circadian machinery, suggesting a potential role in disease pathophysiology. models offer unique opportunities to explore these aspects of C9orf72-FTD and identify novel therapeutic targets aimed at mitigating neurodegeneration.
PubMed: 38915937
DOI: 10.3389/fnmol.2024.1434443 -
Frontiers in Cellular and Infection... 2024is a major causative pathogen of osteomyelitis. Intracellular infections of resident bone cells including osteocytes can persist despite gold-standard clinical...
is a major causative pathogen of osteomyelitis. Intracellular infections of resident bone cells including osteocytes can persist despite gold-standard clinical intervention. The mechanisms by which intracellular evades antibiotic therapy are unknown. In this study, we utilised an infection model of human osteocytes to investigate whether antibiotic-mediated dysregulation of autophagy contributes to this phenomenon. Infected or non-infected osteocyte-like cells were exposed to combinations of rifampicin, vancomycin, and modulators of autophagy. Intracellular bacterial growth characteristics were assessed using colonyforming unit (CFU) analysis, viable bacterial DNA abundance, and the rate of escape into antibiotic-free medium, together with measures of autophagic flux. Rifampicin, alone or in combination with vancomycin, caused a rapid decrease in the culturability of intracellular bacteria, concomitant with stable or increased absolute bacterial DNA levels. Both antibiotics significantly inhibited autophagic flux. However, modulation of autophagic flux did not affect viable bacterial DNA levels. In summary, autophagy was shown to be a factor in the host-pathogen relationship in this model, as its modulation affected the growth state of intracellular with respect to both their culturability and propensity to escape the intracellular niche. While rifampicin and vancomycin treatments moderately suppressed autophagic flux acutely, this did not explain the paradoxical response of antibiotic treatment in decreasing culturability whilst failing to clear bacterial DNA and hence intracellular bacterial load. Thus, off-target effects of rifampicin and vancomycin on autophagic flux in osteocyte-like cells could not explain the persistent infection in these cells.
Topics: Autophagy; Staphylococcus aureus; Osteocytes; Anti-Bacterial Agents; Humans; Vancomycin; Rifampin; Staphylococcal Infections; Host-Pathogen Interactions; DNA, Bacterial
PubMed: 38915921
DOI: 10.3389/fcimb.2024.1403289 -
BioRxiv : the Preprint Server For... Jun 2024Cdk5 is a highly-conserved, noncanonical cell division kinase important to the terminal differentiation of mammalian cells in multiple organ systems. We previously...
Cdk5 is a highly-conserved, noncanonical cell division kinase important to the terminal differentiation of mammalian cells in multiple organ systems. We previously identified Pef1, the ortholog of cdk5, as regulator of chronological lifespan. To reveal the processes impacted by Pef1, we developed APEX2-biotin phenol-mediated proximity labeling in Efficient labeling required a short period of cell wall digestion and eliminating glucose and nitrogen sources from the medium. We identified 255 high-confidence Pef1 neighbors in growing cells and a novel Pef1-interacting partner, the DNA damage response protein Rad24. The Pef1-Rad24 interaction was validated by reciprocal proximity labeling and co-immunoprecipitation. Eliminating Pef1 partially rescued the DNA damage sensitivity of cells lacking Rad24. To monitor how Pef1 neighbors change under different conditions, cells induced for autophagy were labeled and 177 high-confidence Pef1 neighbors were identified. Gene ontology (GO) analysis of the Pef1 neighbors identified proteins participating in processes required for autophagosome expansion including regulation of actin dynamics and vesicle-mediated transport. Some of these proteins were identified in both exponentially growing and autophagic cells. Pef1-APEX2 proximity labeling therefore identified a new Pef1 function in modulating the DNA damage response and candidate processes that Pef1 and other cdk5 orthologs may regulate.
PubMed: 38915521
DOI: 10.1101/2024.06.12.598664 -
BioRxiv : the Preprint Server For... Jun 2024Epigenetic modifications to DNA and chromatin control oncogenic and tumor suppressive mechanisms in melanoma. EZH2, the catalytic component of the Polycomb repressive...
Epigenetic modifications to DNA and chromatin control oncogenic and tumor suppressive mechanisms in melanoma. EZH2, the catalytic component of the Polycomb repressive complex 2 (PRC2), which mediates methylation of lysine 27 on histone 3 (H3K27me3), can regulate both melanoma initiation and progression. We previously found that mutant interacts with the immune regulator Stat3 and together they affect anti-tumor immunity. However, given the numerous downstream targets and pathways affected by EZH2, many mechanisms that determine its oncogenic activity remain largely unexplored. Using genetically engineered mouse models we further investigated the role of pathways downstream of EZH2 in melanoma carcinogenesis and identified significant enrichment in several autophagy signatures, along with increased expression of autophagy regulators, such as Atg7. In this study, we investigated the effect of Atg7 on melanoma growth and tumor immunity within the context of an epigenetic state. We found that expression of Atg7 is largely dependent on Stat3 expression and that deletion of Atg7 slows down melanoma cell growth , but not . Atg7 deletion also results in increased CD8+ T cells and reduced myelosuppressive cell infiltration in the tumor microenvironment, suggesting a strong immune system contribution in the role of Atg7 in melanoma progression. These findings highlight the complex interplay between genetic mutations, epigenetic regulators, and autophagy in shaping tumor immunity in melanoma.
PubMed: 38915518
DOI: 10.1101/2024.06.10.598284 -
Frontiers in Pharmacology 2024Cervical cancer (CC) ranks as the fourth most prevalent malignant tumor among women worldwide, and is the fourth leading cause of cancer-related mortality. GuiErBai...
Cervical cancer (CC) ranks as the fourth most prevalent malignant tumor among women worldwide, and is the fourth leading cause of cancer-related mortality. GuiErBai (GEB), a compound preparation developed by our research team, is derived from the ancient Chinese medicine of the Miao nationality and is comprised of podophyllotoxin (PTOX), imperatorin, isoimperatorin, and alkaloids. These individual components have demonstrated notable efficacy in tumor treatment. However, the specific anti-tumor effect of the compound Chinese medicine GEB in the context of CC has yet to be validated. HeLa and SiHa cell lines were utilized for experiments and treated with 5 mg/mL and 10 mg/mL GEB concentrations, respectively. The cell cycle changes after GEB treatment were assessed using flow cytometry. Transmission electron microscopy was employed to observe autophagic bodies and apoptotic bodies, while MDC staining evaluated the occurrence of autophagy. CCK-8 was used to observe the effect of GEB on cell proliferation, and Transwell assays assessed cell migration and invasion. Western blotting detected cell cycle and apoptosis-related protein expression, along with the expression level of autophagy-related protein LC3I/II. Changes in ROS and mitochondrial membrane potential in cervical cancer cells following GEB treatment were determined using ROS detection and mitochondrial membrane potential detection kits. For the experiment, a nude mouse model of cervical cancer transplantation based on HeLa cells was established. Experimental animals were divided into negative control, positive control, high-dose GEB (10 mg/mL), and low-dose GEB (5 mg/mL) groups. In HeLa and SiHa cell lines, the G0/G1 phase of tumor cells significantly decreased ( < 0.001), while the G2/M phase increased notably ( < 0.001) following various GEB treatments. Electron microscopy showed GEB promoted apoptotic body and autophagosome formation in both cell lines. Compared to untreated HeLa and SiHa cells, GEB-treated cells exhibited significantly reduced caspase3 protein expression, and substantially increased autophagy-related protein LC3I/II expression. GEB treatment significantly reduced migration and invasion capabilities in both cell lines ( < 0.001), while ROS content and mitochondrial membrane potential were significantly elevated ( < 0.001). GEB effectively inhibited cervical cancer cell proliferation, with the optimal concentration being 10 mg/mL. A successful nude mouse model of cervical cancer transplantation was established using HeLa cells. Post-GEB treatment, the tumor volume and weight in nude mice significantly decreased ( < 0.001), with diminished expression of CD34, VEGF, and caspase3 proteins in tumor tissues. GEB exhibits a robust antitumor effect against cervical cancer, both and , in a concentration-dependent manner, by regulating autophagy and apoptosis of tumor cells.
PubMed: 38915466
DOI: 10.3389/fphar.2024.1296588 -
Frontiers in Microbiology 2024Herpes Simplex Virus type 1 (HSV-1) 1 is a neurotropic virus that has been associated with neurodegenerative disorders. The dysregulation of autophagy by HSV-1 has been...
Herpes Simplex Virus type 1 (HSV-1) 1 is a neurotropic virus that has been associated with neurodegenerative disorders. The dysregulation of autophagy by HSV-1 has been proposed as a potential cause of neurodegeneration. While studies have extensively tackled the interaction between autophagy and HSV-1 in neurons, research in glial cells is currently limited. Our studies demonstrate that HSV-1 inhibits, but not completely blocks, the formation of autophagosomes in human oligodendroglioma- and astrocytoma- derived cell lines. These findings have been confirmed in murine oligodendrocyte precursor cells (OPCs). Finally, this study investigates the impact of autophagy on HSV-1 infection in glial cells. While the lack of basal autophagy in LC3B knockout glial cells does not have a significant effect on viral infection, cells without the autophagy-related protein ATG5 exhibit reduced viral production. The absence of ATG5 leads to a decrease in the transcription and replication of viral genes, as well as a delay in the initial stages of the formation of HSV-1 replication compartments. These findings indicate that while autophagy may not play a significant role in antiviral defense in glial cells, HSV-1 may be inhibiting autophagy to exploit non-canonical functions of certain components of the autophagic machinery, such as ATG5, to benefit its lifecycle.
PubMed: 38915300
DOI: 10.3389/fmicb.2024.1411655 -
Cell Division Jun 2024Nitrogen permease regulator-like 2 (NPRL2/TUSC4) is known to exert both tumor-suppressing and oncogenic effects in different types of cancers, suggesting that its...
Nitrogen permease regulator-like 2 (NPRL2/TUSC4) is known to exert both tumor-suppressing and oncogenic effects in different types of cancers, suggesting that its actions are context dependent. Here, we delineated the molecular and functional effects of NPRL2 in malignantly transformed bronchial epithelial cells. To do so, we depleted NPRL2 in oncogenic HRas-transduced and malignantly transformed human bronchial epithelial (BEAS2B), Ras-AI-T2 cells. Intriguingly, depletion of NPRL2 in these cells induced activation of mTORC1 downstream signaling, inhibited autophagy, and impaired Ras-AI-T2 cell proliferation both in vitro and in vivo. These results suggest that NPRL2 is required for oncogenic HRas-induced cell transformation. Depletion of NPRL2 increased levels of the DNA damage marker γH2AX, the cell cycle inhibitors p21 and p27, and the apoptosis marker cleaved-PARP. These NPRL2-depleted cells first accumulated at G1 and G2, and later exhibited signs of mitotic catastrophe, which implied that NPRL2 depletion may be detrimental to oncogenic HRas-transformed cells. Additionally, NPRL2 depletion reduced heat shock factor 1/heat shock element- and NRF2/antioxidant response element-directed luciferase reporter activities in Ras-AI-T2 cells, indicating that NPRL2 depletion led to the suppression of two key cytoprotective processes in oncogenic HRas-transformed cells. Overall, our data suggest that oncogenic HRas-transduced and malignantly transformed cells may depend on NPRL2 for survival and proliferation, and depletion of NPRL2 also induces a stressed state in these cells.
PubMed: 38915098
DOI: 10.1186/s13008-024-00126-w