-
BioRxiv : the Preprint Server For... Jun 2024Cell corpses must be cleared in an efficient manner to maintain tissue homeostasis and regulate immune responses. Ubiquitin-like Atg8/LC3 family proteins promote the...
Cell corpses must be cleared in an efficient manner to maintain tissue homeostasis and regulate immune responses. Ubiquitin-like Atg8/LC3 family proteins promote the degradation of membranes and internal cargo during both macroautophagy and corpse clearance, raising the question how macroautophagy contributes to corpse clearance. Studying the clearance of non-apoptotic dying polar bodies in embryos, we show that the LC3 ortholog LGG-2 is enriched in the polar body phagolysosome independent of membrane association or autophagosome formation. We demonstrate that ATG-16.1 and ATG-16.2, which promote membrane association of lipidated Atg8/LC3 proteins, redundantly promote polar body membrane breakdown in phagolysosomes independent of their role in macroautophagy. We also show that the lipid scramblase ATG-9 is needed for autophagosome formation in early embryos but is dispensable for timely polar body membrane breakdown or protein cargo degradation. These findings demonstrate that macroautophagy is not required to promote polar body degradation, in contrast to recent findings with apoptotic corpse clearance in embryos. Determining how membrane association of Atg8/LC3 promotes the breakdown of different types of cell corpses in distinct cell types or metabolic states is likely to give insights into the mechanisms of immunoregulation during normal development, physiology, and disease.
PubMed: 38948720
DOI: 10.1101/2024.06.19.599770 -
Theranostics 2024Autophagy dysregulation is known to be a mechanism of doxorubicin (DOX)-induced cardiotoxicity (DIC). Mitochondrial-Endoplasmic Reticulum Contacts (MERCs) are where...
Autophagy dysregulation is known to be a mechanism of doxorubicin (DOX)-induced cardiotoxicity (DIC). Mitochondrial-Endoplasmic Reticulum Contacts (MERCs) are where autophagy initiates and autophagosomes form. However, the role of MERCs in autophagy dysregulation in DIC remains elusive. FUNDC1 is a tethering protein of MERCs. We aim to investigate the effect of DOX on MERCs in cardiomyocytes and explore whether it is involved in the dysregulated autophagy in DIC. We employed confocal microscopy and transmission electron microscopy to assess MERCs structure. Autophagic flux was analyzed using the mCherry-EGFP-LC3B fluorescence assay and western blotting for LC3BII. Mitophagy was studied through the mCherry-EGFP-FIS1 fluorescence assay and colocalization analysis between LC3B and mitochondria. A total dose of 18 mg/kg of doxorubicin was administrated in mice to construct a DIC model . Additionally, we used adeno-associated virus (AAV) to cardiac-specifically overexpress FUNDC1. Cardiac function and remodeling were evaluated by echocardiography and Masson's trichrome staining, respectively. DOX blocked autophagic flux by inhibiting autophagosome biogenesis, which could be attributed to the downregulation of FUNDC1 and disruption of MERCs structures. FUNDC1 overexpression restored the blocked autophagosome biogenesis by maintaining MERCs structure and facilitating ATG5-ATG12/ATG16L1 complex formation without altering mitophagy. Furthermore, FUNDC1 alleviated DOX-induced oxidative stress and cardiomyocytes deaths in an autophagy-dependent manner. Notably, cardiac-specific overexpression of FUNDC1 protected DOX-treated mice against adverse cardiac remodeling and improved cardiac function. : In summary, our study identified that FUNDC1-meditated MERCs exerted a cardioprotective effect against DIC by restoring the blocked autophagosome biogenesis. Importantly, this research reveals a novel role of FUNDC1 in enhancing macroautophagy via restoring MERCs structure and autophagosome biogenesis in the DIC model, beyond its previously known regulatory role as an mitophagy receptor.
PubMed: 38948070
DOI: 10.7150/thno.92771 -
MedRxiv : the Preprint Server For... Jun 2024Blood-based biomarkers are gaining grounds for Alzheimer's disease (AD) detection. However, two key obstacles need to be addressed: the lack of methods for multi-analyte...
BACKGROUND
Blood-based biomarkers are gaining grounds for Alzheimer's disease (AD) detection. However, two key obstacles need to be addressed: the lack of methods for multi-analyte assessments and the need for markers of neuroinflammation, vascular, and synaptic dysfunction. Here, we evaluated a novel multi-analyte biomarker platform, NULISAseq CNS disease panel, a multiplex NUcleic acid-linked Immuno-Sandwich Assay (NULISA) targeting ∼120 analytes, including classical AD biomarkers and key proteins defining various disease hallmarks.
METHODS
The NULISAseq panel was applied to 176 plasma samples from the MYHAT-NI cohort of cognitively normal participants from an economically underserved region in Western Pennsylvania. Classical AD biomarkers, including p-tau181, p-tau217, p-tau231, GFAP, NEFL, Aβ40, and Aβ42, were also measured using Single Molecule Array (Simoa). Amyloid pathology, tau pathology, and neurodegeneration were evaluated with [11C] PiB PET, [18F]AV-1451 PET, and MRI, respectively. Linear mixed models were used to examine cross-sectional and Wilcoxon rank sum tests for longitudinal associations between NULISA biomarkers and AD pathologies. Spearman correlations were used to compare NULISA and Simoa.
RESULTS
NULISA concurrently measured 116 plasma biomarkers with good technical performance, and good correlation with Simoa measures. Cross-sectionally, p-tau217 was the top hit to identify Aβ pathology, with age, sex, and genotype-adjusted AUC of 0.930 (95%CI: 0.878-0.983). Fourteen markers were significantly decreased in Aβ-PET+ participants, including TIMP3, which regulates brain Aβ production, the neurotrophic factor BDNF, the energy metabolism marker MDH1, and several cytokines. Longitudinally, FGF2, IL4, and IL9 exhibited Aβ PET-dependent yearly increases in Aβ-PET+ participants. Markers with tau PET-dependent longitudinal changes included the microglial activation marker CHIT1, the reactive astrogliosis marker CHI3L1, the synaptic protein NPTX1, and the cerebrovascular markers PGF, PDGFRB, and VEFGA; all previously linked to AD but only reliably measured in cerebrospinal fluid. SQSTM1, the autophagosome cargo protein, exhibited a significant association with neurodegeneration status after adjusting age, sex, and ε4 genotype.
CONCLUSIONS
Together, our results demonstrate the feasibility and potential of immunoassay-based multiplexing to provide a comprehensive view of AD-associated proteomic changes. Further validation of the identified inflammation, synaptic, and vascular markers will be important for establishing disease state markers in asymptomatic AD.
PubMed: 38947065
DOI: 10.1101/2024.06.15.24308975 -
Journal of Molecular Biology Jun 2024Autophagy is a cellular degradation pathway where double-membrane autophagosomes form de novo to engulf cytoplasmic material destined for lysosomal degradation. This... (Review)
Review
Autophagy is a cellular degradation pathway where double-membrane autophagosomes form de novo to engulf cytoplasmic material destined for lysosomal degradation. This process requires regulated membrane remodeling, beginning with the initial autophagosomal precursor and progressing to its elongation and maturation into a fully enclosed, fusion-capable vesicle. While the core protein machinery involved in autophagosome formation has been extensively studied over the past two decades, the role of phospholipids in this process has only recently been studied. This review focuses on the phospholipid composition of the phagophore membrane and the mechanisms that supply lipids to expand this unique organelle.
PubMed: 38944336
DOI: 10.1016/j.jmb.2024.168691 -
Cellular Signalling Jun 2024Silicosis, one of the occupational health illnesses is caused by inhalation of crystalline silica. Deposition of extracellular matrix and fibroblast proliferation in...
BACKGROUND AND OBJECTIVES
Silicosis, one of the occupational health illnesses is caused by inhalation of crystalline silica. Deposition of extracellular matrix and fibroblast proliferation in lungs are linked to silicosis development. Mitochondrial dysfunction plays critical role in some diseases, but how these processes progress and regulated in silicosis, remains limited. Detailed study of silica induced pulmonary fibrosis in mouse model, its progression and severity may be helpful in designing future therapeutic strategies.
METHODS
In present study, mice model of silicosis has been developed after repeated silica exposures which may closely resemble clinical symptoms of silicosis in human. In addition to efficiently mimicking the acute/chronic transformation processes of silicosis, this is practical and efficient in terms of time and output, which avoids mechanical injury to the upper respiratory tract due to surgical interventions. Sonicated sterile silica suspension (120 mg/kg) was administered through intranasal route thrice a week at regular intervals (21, 28 and 35 days).
RESULTS
Presence of minute to larger silicotic nodules in H&E-stained lung sections were observed in all silica induced model groups. Enhanced ECM deposition was noted in MT stained lung sections of silica exposure groups as compared to control which were confirmed by significantly higher MMP9 expression levels and hydroxyproline content in silica 35 days group. Increase in Reactive oxygen species (ROS), inflammatory cell recruitment mainly, neutrophils and macrophage were observed in all three silica exposure groups. Transmission electron microscopic analysis has confirmed presence of many aberrant shaped mitochondria (swollen, round shape) in 35 days model where autophagosomes were minimum. Western blot analysis of mitophagy and autophagy markers such as Pink1, Parkin, Cytochrome c, SQSTM1/p62, the ratio of light chain LC3B II/LC3B I was found higher in 21 and 28 days which were significantly reduced in 35 days silica model.
CONCLUSIONS
Higher MMP9 activity and MMP9 /TIMP1 ratio demonstrate excessive extracellular matrix damage and deposition in 35 days model. Significantly reduced expressions of autophagy and mitophagy markers have also confirmed progression in fibrosis severity and its association with repeated silica exposures in 35 days model group.
PubMed: 38944258
DOI: 10.1016/j.cellsig.2024.111272 -
Cell Death & Disease Jun 2024High basal autophagy and enhanced mitochondrial fission in triple-negative breast cancer (TNBC) cells support cell migration and promote plasticity of cancer cell...
High basal autophagy and enhanced mitochondrial fission in triple-negative breast cancer (TNBC) cells support cell migration and promote plasticity of cancer cell metabolism. Here, we suggest a novel combination therapy approach for the treatment of TNBC that targets Drp1-mediated mitochondrial fission and autophagy pathways. Hydrogen sulfide (HS) mediates a myriad of biological processes, including autophagy and mitochondrial function. In this study, we demonstrated that 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), one of the most widely utilized sustained-release HS donors, effectively suppresses metastasis of TNBC cells in the absence of proliferation inhibition in vitro and in vivo. ADT-OH treatment ameliorated autophagy flux by suppressing autophagosome formation and induced mitochondrial elongation through decreasing expression of dynamin-related protein 1 (Drp1) and increasing expression of mitochondrial fusion protein (Mfn2). At the same time, ADT-OH downregulated mitophagy flux and inhibited mitochondrial function, eventually leading to the inhibition of migration and invasion in TNBC cells. In vivo, intraperitoneal administration of ADT-OH revealed a potent anti-metastatic activity in three different animal models, the MDA-MB-231 orthotopic xenograft model, the 4T1-Luci orthotopic model and the 4T1-Luci tail vein metastasis model. However, ADT-OH has an extremely low water solubility, which is a significant barrier to its effectiveness. Thus, we demonstrated that the solubility of ADT-OH in water can be improved significantly by absorption with hydroxypropyl-β-cyclodextrin (CD). Remarkably, the obtained CD-ADT-OH demonstrated superior anti-cancer effect to ADT-OH in vivo. Altogether, this study describes a novel regulator of mammalian mitochondrial fission and autophagy, with potential utility as an experimental therapeutic agent for metastatic TNBC.
Topics: Triple Negative Breast Neoplasms; Mitochondrial Dynamics; Humans; Animals; Autophagy; Female; Cell Line, Tumor; Mice; Cell Movement; Mice, Nude; Thiones; Xenograft Model Antitumor Assays; Mice, Inbred BALB C; Mitochondria; Cell Proliferation; Neoplasm Metastasis; Hydrogen Sulfide; Dynamins; Thiophenes
PubMed: 38942765
DOI: 10.1038/s41419-024-06829-w -
Tissue & Cell Jun 2024Exposure to the neonicotinoid insecticide, imidacloprid (IMI), causes reproductive toxicity in mammals and reptiles. However, reports on the effects of IMI on the gonads...
Exposure to the neonicotinoid insecticide, imidacloprid (IMI), causes reproductive toxicity in mammals and reptiles. However, reports on the effects of IMI on the gonads in birds are grossly lacking. Therefore, this study investigated the effects of pubertal exposure to IMI on the histology, ultrastructure, as well as the cytoskeletal proteins, desmin, smooth muscle actin and vimentin, of the gonads of Japanese quail (Coturnix coturnix japonica). Quails were randomly divided into four groups at 5 weeks of age. The control group was given only distilled water, whereas, the other three experimental groups, IMI was administered by oral gavage at 1.55, 3.1, and 6.2 mg/kg, twice per week for 4 weeks. Exposure to IMI doses of 3.1 and 6.2 mg/kg caused dose-dependent histopathological changes in the ovary and testis. In the ovary, accumulation of lymphocytes, degenerative changes, and necrosis with granulocyte infiltrations were observed, while in the testis, distorted seminiferous tubules, germ cell sloughing, vacuolisations, apoptotic bodies, autophagosomes, and mitochondrial damage were detected. These changes were accompanied by a decreased number of primary follicles (P ≤ 0.05) in the ovary and a decrease (P ≤ 0.05) in the epithelial height, luminal, and tubular diameters of seminiferous tubules at the two higher dosages. In addition, IMI had a negative effect on the immunostaining intensity of desmin, smooth muscle actin, and vimentin in the ovarian and testicular tissue. In conclusion, exposure to IMI during puberty can lead to a range of histopathological alterations in the gonads of Japanese quails, which may ultimately result in infertility.
PubMed: 38941762
DOI: 10.1016/j.tice.2024.102450 -
Biochemistry Jun 2024Protein advanced glycation end products (AGEs) can be formed via nonenzymatic glycation and accumulated intracellularly to disrupt cellular homeostasis for protein...
Protein advanced glycation end products (AGEs) can be formed via nonenzymatic glycation and accumulated intracellularly to disrupt cellular homeostasis for protein clearance. Here, we investigated the formation particulars of intracellular protein AGEs and sought to elucidate the molecular events implicated in the impact of cellular clearance systems. The formation and accumulation of intracellular protein AGEs increased protein aggregation and protease resistance, potentially overwhelming the ubiquitin-proteasome system (UPS). At high levels of protein AGEs, the abundance of many E3 ligases decreased and the overall ubiquitination level was reduced, all of which indicated decreased UPS activity. On the other hand, autophagy activity was stimulated, as evidenced by the upregulation of autophagy marker LC3II and important proteins in autophagosome and autolysosome formation, as well as downregulation of mTOR. Understanding the functional impacts of intracellular protein AGEs on the UPS and autophagy could pave the way for the future development of pharmaceutical agents targeting AGE-related diseases.
PubMed: 38941592
DOI: 10.1021/acs.biochem.4c00250 -
Sheng Li Xue Bao : [Acta Physiologica... Jun 2024The purpose of the study was to investigate the mechanism of TFEB activator 1 (TA1) improving the autophagic degradation of oligomeric amyloid-β (oAβ) in microglia,...
The purpose of the study was to investigate the mechanism of TFEB activator 1 (TA1) improving the autophagic degradation of oligomeric amyloid-β (oAβ) in microglia, and to explore the therapeutic effect of TA1 on an in vitro model of microglia in Alzheimer's disease (AD). Primary microglia were exposed to 1 μmol/L oAβ for 0, 3, 12, and 24 h respectively to construct the in vitro model of microglia in AD. In order to explore the therapeutic effect of TA1, primary microglia were co-treated with 1 μmol/L oAβ and 1 μmol/L TA1 for 12 h. To determine the autophagy flux, the above cells were further treated with 100 nmol/L Bafilomycin A1 for 1 h before fixation. Fluorescent probes were used to detect the endocytosis or degradation of oAβ by microglia. The autophagic flux was determined by infection of lentivirus mCherry-EGFP-LC3. The nuclear TFEB intensity, the autophagosomes number, and the colocalization ratio of oAβ with lysosome-associated membrane protein 1 (LAMP1) or microtubule-associated protein light chain 3 (LC3), were detected by immunofluorescence assay. Expressions of autophagy-related-genes, including Lamp1, Atg5, and Map1lc3b, were detected by qRT-PCR. Results showed that prolonged oAβ exposure inhibited the endocytosis and degradation of oAβ by microglia. Meanwhile, the number of autophagosomes and autophagy flux in microglia decreased after 12 h of oAβ treatment. We further found that the nuclear expression of autophagy regulator TFEB decreased after 12 h of oAβ exposure, resulting in the decrease of autophagy genes, thus leading to the damage of autophagic degradation of oAβ. Therefore, long-term oAβ exposure was considered to construct the in vitro model of microglia in AD. After TA1 treatment, the nuclear expression of TFEB in cells was obviously upregulated. TA1 treatment upregulated the expressions of autophagy-related genes, leading to the recovery of autophagy flux. TA1 also recovered the endocytosis and degradation of oAβ by microglia. In conclusion, TA1 could improve oAβ clearance by microglia in AD by upregulating microglial TFEB-mediated autophagy, suggesting TA1 as a potential therapeutic drug for AD.
Topics: Microglia; Amyloid beta-Peptides; Autophagy; Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Alzheimer Disease; Cells, Cultured; Mice
PubMed: 38939931
DOI: No ID Found -
Molecular Therapy : the Journal of the... Jun 2024Galactosyl-ceramidase (GALC) is a ubiquitous lysosomal enzyme crucial for the correct myelination of the mammalian nervous system during early postnatal development....
Deficiency of galactosylceramidase in adult oligodendrocytes worsens the neurological deficits and shortens the survival during chronic experimental allergic encephalomyelitis.
Galactosyl-ceramidase (GALC) is a ubiquitous lysosomal enzyme crucial for the correct myelination of the mammalian nervous system during early postnatal development. However, the physiological consequence of GALC deficiency in the adult brain remains unknown. In this study, we found that mice with conditional ablation of GALC activity in post-myelinating oligodendrocytes were lethally sensitized when challenged with chronic experimental allergic encephalomyelitis (EAE), in contrast to the non-lethal dysmyelination observed in GALC-ablated mice without the EAE challenge. Mechanistically, we found a strong inflammatory demyelination without remyelination and an impaired fusion of lysosomes and autophagosomes with accumulation of myelin debris following a TFEB-dependent increase in the lysosomal autophagosome flux. These results indicate that the physiological impact of GALC deficiency is highly influenced by the cell context (oligodendroglial vs global expression), the presence of inflammation, and the developmental time when it happens (pre-myelination vs post-myelination). We conclude that GALC expression in adult oligodendrocytes is crucial for the maintenance of adult central myelin and to reduce vulnerability to additional demyelinating insults.
PubMed: 38937968
DOI: 10.1016/j.ymthe.2024.06.035