-
Molecular Cell May 2024Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases,...
Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.
Topics: Peptides; Autophagy; Humans; Huntingtin Protein; Autophagosomes; Sequestosome-1 Protein; Amyloid; Huntington Disease; Protein Aggregates; Cryoelectron Microscopy; Animals; Protein Aggregation, Pathological
PubMed: 38759629
DOI: 10.1016/j.molcel.2024.04.018 -
Cellular and Molecular Life Sciences :... May 2024The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along...
The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2β and p150. We found that the mTORC1 inhibitor rapamycin enhanced p150-AP-2β complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150-AP-2β interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150-AP-2β interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150-AP-2β interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150-AP-2β interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport.
Topics: Animals; Humans; Mice; Adaptor Protein Complex 2; Autophagosomes; Autophagy; Dynactin Complex; Lysosomal-Associated Membrane Protein 1; Lysosomes; Mechanistic Target of Rapamycin Complex 1; Neurons; Protein Binding; Sirolimus
PubMed: 38758395
DOI: 10.1007/s00018-024-05256-6 -
Clinical and Experimental Reproductive... May 2024Autophagy is a major intracellular catabolic pathway governed by the sequential actions of proteins encoded by autophagy-related genes (Atg). ATG9, the only...
OBJECTIVE
Autophagy is a major intracellular catabolic pathway governed by the sequential actions of proteins encoded by autophagy-related genes (Atg). ATG9, the only transmembrane protein involved in this process, regulates phospholipid translocation to autophagosomes during the early phases of autophagy. In mammals, two Atg9 isoforms have been reported: Atg9a and Atg9b. In this study, we examined whether the molecular and cellular characteristics of these two isoforms differed in mice.
METHODS
Whole uteri were collected on days 1, 4, and 8 of pregnancy and from ovariectomized mice injected with vehicle, progesterone, or 17β-estradiol. Cells from reproductive tissues, such as granulosa cells, uterine epithelial cells (UECs), uterine stromal cells (USCs), and oocytes were collected. Two human uterine cell lines were also used in this analysis. Reverse transcription-polymerase chain reaction tests, Western blotting, and immunofluorescence staining were performed. Serum starvation conditions were used to induce autophagy in primary cells.
RESULTS
Atg9a and Atg9b were expressed in multiple mouse tissues and reproductive cells. Neither Atg9A nor Atg9B significantly changed in response to steroid hormones. Immunofluorescence staining of the UECs and USCs showed that ATG9A was distributed in a punctate-like pattern, whereas ATG9B exhibited a pattern of elongated tubular shapes in the cytoplasm. In human cancer cell lines, ATG9B was undetectable, whereas ATG9A was found in all cell types examined.
CONCLUSION
The Atg9 isoforms exhibited distinct subcellular localizations in UECs and may play different roles in autophagy. Notably, human uterine cells exhibited reduced ATG9B expression, suggesting that this suppression may be due to epigenetic regulation.
PubMed: 38757275
DOI: 10.5653/cerm.2023.06737 -
Journal of Dairy Science May 2024Excessive concentrations of free fatty acids (FFA) are the main factors causing immune dysfunction and inflammation in dairy cows with ketosis. Polarization of...
Fatty acids promote M1 polarization of monocyte-derived macrophages in healthy or ketotic dairy cows and a bovine macrophage cell line via impairing mTOR-mediated autophagy.
Excessive concentrations of free fatty acids (FFA) are the main factors causing immune dysfunction and inflammation in dairy cows with ketosis. Polarization of macrophages (the process of macrophages freely switching from one phenotype to another) into M1 or M2 phenotypes is an important event during inflammation induced by environmental stimuli. In non-ruminants, mammalian target of rapamycin (mTOR)-mediated autophagy (a major waste degradation process) regulates macrophage polarization. Thus, the objective was to unravel the role of mTOR-mediated autophagy on macrophage polarization in ketotic dairy cows. Four experiments were performed as follows: (1) In vitro differentiated monocyte-derived macrophages from healthy dairy cows or dairy cows with clinical ketosis (CK) were treated with 100 ng/mL lipopolysaccharide (LPS) and 100 ng/mL interferon-γ (IFN-γ) or 10 ng/mL interleukin-4 (IL4) and 10 ng/mL interleukin-10 (IL10) for 24 h; (2) Immortalized bovine macrophages were treated with 0, 0.3, 0.6, 1.2 mM FFA and LPS and IFN-γ or IL4 and IL10 for 24 h; (3) Macrophages were pretreated with 2 μM 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine (MHY1485) for 30 min before treatment with LPS and IFN-γ or IL4 and IL10; (4) Macrophages were pretreated with 100 nM rapamycin (RAPA) for 2 h before treatment with LPS and IFN-γ or IL4 and IL10. Compared with healthy cows, cows with CK had a greater mean fluorescence intensity (MFI) of CD86, but lower MFI of CD206 and lower number of autophagosomes and autolysosomes in macrophages. Exogenous FFA treatment upregulated protein abundance of inducible nitric oxide synthase (iNOS) and mean fluorescence intensity of CD86, whereas it downregulated the protein abundance of arginase 1 (ARG1) and mean fluorescence intensity of CD206. In addition, FFA increased the p-p65/p65 protein abundance and tumor necrosis factor α (TNFA), interleukin-1B (IL1B), and interleukin-6 (IL6) mRNA abundance, but decreased LC3-phosphatidylethanolamine conjugate (LC3-II) protein abundance and autophagosomes and autolysosomes number. Pretreatment with MHY1485 promoted macrophage M1 polarization and inhibited macrophage M2 polarization via decreased mTOR-mediated autophagy. Activation of mTOR-mediated autophagy by pretreatment with RAPA attenuated the upregulation of inflammation in M1 macrophages that was induced by FFA. These data revealed that high concentrations of FFA promote macrophage M1 polarization in ketotic dairy cows via impairing mTOR-mediated autophagy.
PubMed: 38754818
DOI: 10.3168/jds.2023-24357 -
Journal of Cell Science May 2024Peroxisomes are highly plastic organelles that are involved in several metabolic processes, including fatty acid oxidation, ether lipid synthesis and redox homeostasis.... (Review)
Review
Peroxisomes are highly plastic organelles that are involved in several metabolic processes, including fatty acid oxidation, ether lipid synthesis and redox homeostasis. Their abundance and activity are dynamically regulated in response to nutrient availability and cellular stress. Damaged or superfluous peroxisomes are removed mainly by pexophagy, the selective autophagy of peroxisomes induced by ubiquitylation of peroxisomal membrane proteins or ubiquitin-independent processes. Dysregulated pexophagy impairs peroxisome homeostasis and has been linked to the development of various human diseases. Despite many recent insights into mammalian pexophagy, our understanding of this process is still limited compared to our understanding of pexophagy in yeast. In this Cell Science at a Glance article and the accompanying poster, we summarize current knowledge on the control of mammalian pexophagy and highlight which aspects require further attention. We also discuss the role of ubiquitylation in pexophagy and describe the ubiquitin machinery involved in regulating signals for the recruitment of phagophores to peroxisomes.
Topics: Peroxisomes; Humans; Animals; Ubiquitination; Autophagy; Macroautophagy; Mammals; Membrane Proteins
PubMed: 38752931
DOI: 10.1242/jcs.259775 -
Aging May 2024Mangiferin, a naturally occurring potent glucosylxanthone, is mainly isolated from the plant and shows potential pharmacological properties, including anti-bacterial,...
Mangiferin, a naturally occurring potent glucosylxanthone, is mainly isolated from the plant and shows potential pharmacological properties, including anti-bacterial, anti-inflammation, and antioxidant in sepsis-induced lung and kidney injury. However, there was a puzzle as to whether mangiferin had a protective effect on sepsis-associated encephalopathy. To answer this question, we established an cell model of sepsis-associated encephalopathy and investigated the neuroprotective effects of mangiferin in primary cultured hippocampal neurons challenged with lipopolysaccharide (LPS). Neurons treated with 20 μmol/L or 40 μmol/L mangiferin for 48 h can significantly reverse cell injuries induced by LPS treatment, including improved cell viability, decreased inflammatory cytokines secretion, relief of microtubule-associated light chain 3 expression levels and several autophagosomes, as well as attenuated cell apoptosis. Furthermore, mangiferin eliminated pathogenic proteins and elevated neuroprotective factors at both the mRNA and protein levels, showing strong neuroprotective effects of mangiferin, including anti-inflammatory, anti-autophagy, and anti-apoptotic effects on neurons .
Topics: Xanthones; Animals; Neurons; Lipopolysaccharides; Hippocampus; Neuroprotective Agents; Cells, Cultured; Apoptosis; Cell Survival; Autophagy; Rats; Cytokines
PubMed: 38752883
DOI: 10.18632/aging.205830 -
Cell Communication and Signaling : CCS May 2024The pathway involving PTEN-induced putative kinase 1 (PINK1) and PARKIN plays a crucial role in mitophagy, a process activated by artesunate (ART). We propose that...
BACKGROUND
The pathway involving PTEN-induced putative kinase 1 (PINK1) and PARKIN plays a crucial role in mitophagy, a process activated by artesunate (ART). We propose that patients with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis exhibit insufficient mitophagy, and ART enhances mitophagy via the PINK1/PARKIN pathway, thereby providing neuroprotection.
METHODS
Adult female mice aged 8-10 weeks were selected to create a passive transfer model of anti-NMDAR encephalitis. We conducted behavioral tests on these mice within a set timeframe. Techniques such as immunohistochemistry, immunofluorescence, and western blotting were employed to assess markers including PINK1, PARKIN, LC3B, p62, caspase3, and cleaved caspase3. The TUNEL assay was utilized to detect neuronal apoptosis, while transmission electron microscopy (TEM) was used to examine mitochondrial autophagosomes. Primary hippocampal neurons were cultured, treated, and then analyzed through immunofluorescence for mtDNA, mtROS, TMRM.
RESULTS
In comparison to the control group, mitophagy levels in the experimental group were not significantly altered, yet there was a notable increase in apoptotic neurons. Furthermore, markers indicative of mitochondrial leakage and damage were found to be elevated in the experimental group compared to the control group, but these markers showed improvement following ART treatment. ART was effective in activating the PINK1/PARKIN pathway, enhancing mitophagy, and diminishing neuronal apoptosis. Behavioral assessments revealed that ART ameliorated symptoms in mice with anti-NMDAR encephalitis in the passive transfer model (PTM). The knockdown of PINK1 led to a reduction in mitophagy levels, and subsequent ART intervention did not alleviate symptoms in the anti-NMDAR encephalitis PTM mice, indicating that ART's therapeutic efficacy is mediated through the activation of the PINK1/PARKIN pathway.
CONCLUSIONS
At the onset of anti-NMDAR encephalitis, mitochondrial damage is observed; however, this damage is mitigated by the activation of mitophagy via the PINK1/PARKIN pathway. This regulatory feedback mechanism facilitates the removal of damaged mitochondria, prevents neuronal apoptosis, and consequently safeguards neural tissue. ART activates the PINK1/PARKIN pathway to enhance mitophagy, thereby exerting neuroprotective effects and may achieve therapeutic goals in treating anti-NMDAR encephalitis.
Topics: Animals; Artesunate; Mice; Female; Neuroprotective Agents; Disease Models, Animal; Anti-N-Methyl-D-Aspartate Receptor Encephalitis; Protein Kinases; Neurons; Microscopy, Electron, Transmission; Mitophagy; Apoptosis; Ubiquitin-Protein Ligases; Mitochondria; Hippocampus
PubMed: 38745240
DOI: 10.1186/s12964-024-01652-4 -
Journal of Translational Medicine May 2024Interstitial lung disease (ILD) represents a significant complication of rheumatoid arthritis (RA) that lacks effective treatment options. This study aimed to...
BACKGROUND AND PURPOSE
Interstitial lung disease (ILD) represents a significant complication of rheumatoid arthritis (RA) that lacks effective treatment options. This study aimed to investigate the intrinsic mechanism by which resveratrol attenuates rheumatoid arthritis complicated with interstitial lung disease through the AKT/TMEM175 pathway.
METHODS
We established an arthritis model by combining chicken type II collagen and complete Freund's adjuvant. Resveratrol treatment was administered via tube feeding for 10 days. Pathological changes in both the joints and lungs were evaluated using HE and Masson staining techniques. Protein expression of TGF-β1, AKT, and TMEM175 was examined in lung tissue. MRC-5 cells were stimulated using IL-1β in combination with TGF-β1 as an in vitro model of RA-ILD, and agonists of AKT, metabolic inhibitors, and SiRNA of TMEM175 were used to explore the regulation and mechanism of action of resveratrol RA-ILD.
RESULTS
Resveratrol mitigates fibrosis in rheumatoid arthritis-associated interstitial lung disease and reduces oxidative stress and inflammation in RA-ILD. Furthermore, resveratrol restored cellular autophagy. When combined with the in vitro model, it was further demonstrated that resveratrol could suppress TGF-β1 expression, and reduce AKT metamorphic activation, consequently inhibiting the opening of AKT/MEM175 ion channels. This, in turn, lowers lysosomal pH and enhances the fusion of autophagosomes with lysosomes, ultimately ameliorating the progression of RA-ILD.
CONCLUSION
In this study, we demonstrated that resveratrol restores autophagic flux through the AKT/MEM175 pathway to attenuate inflammation as well as fibrosis in RA-ILD by combining in vivo and in vitro experiments. It further provides a theoretical basis for the selection of therapeutic targets for RA-ILD.
Topics: Resveratrol; Arthritis, Rheumatoid; Proto-Oncogene Proteins c-akt; Animals; Lung Diseases, Interstitial; Humans; Inflammation; Signal Transduction; Fibrosis; Transforming Growth Factor beta1; Membrane Proteins; Autophagy; Oxidative Stress; Cell Line; Lung; Male
PubMed: 38745204
DOI: 10.1186/s12967-024-05228-1 -
Cellular & Molecular Biology Letters May 2024Genetic abnormalities in the FGFR signalling occur in 40% of breast cancer (BCa) patients resistant to anti-ER therapy, which emphasizes the potential of FGFR-targeting...
BACKGROUND
Genetic abnormalities in the FGFR signalling occur in 40% of breast cancer (BCa) patients resistant to anti-ER therapy, which emphasizes the potential of FGFR-targeting strategies. Recent findings indicate that not only mutated FGFR is a driver of tumour progression but co-mutational landscapes and other markers should be also investigated. Autophagy has been recognized as one of the major mechanisms underlying the role of tumour microenvironment in promotion of cancer cell survival, and resistance to anti-ER drugs. The selective autophagy receptor p62/SQSTM1 promotes Nrf-2 activation by Keap1/Nrf-2 complex dissociation. Herein, we have analysed whether the negative effect of FGFR2 on BCa cell response to anti-ER treatment involves the autophagy process and/or p62/Keap1/Nrf-2 axis.
METHODS
The activity of autophagy in ER-positive MCF7 and T47D BCa cell lines was determined by analysis of expression level of autophagy markers (p62 and LC3B) and monitoring of autophagosomes' maturation. Western blot, qPCR and proximity ligation assay were used to determine the Keap1/Nrf-2 interaction and Nrf-2 activation. Analysis of 3D cell growth in Matrigel® was used to assess BCa cell response to applied treatments. In silico gene expression analysis was performed to determine FGFR2/Nrf-2 prognostic value.
RESULTS
We have found that FGFR2 signalling induced autophagy in AMPKα/ULK1-dependent manner. FGFR2 activity promoted dissociation of Keap1/Nrf-2 complex and activation of Nrf-2. Both, FGFR2-dependent autophagy and activation of Nrf-2 were found to counteract the effect of anti-ER drugs on BCa cell growth. Moreover, in silico analysis showed that high expression of NFE2L2 (gene encoding Nrf-2) combined with high FGFR2 expression was associated with poor relapse-free survival (RFS) of ER+ BCa patients.
CONCLUSIONS
This study revealed the unknown role of FGFR2 signalling in activation of autophagy and regulation of the p62/Keap1/Nrf-2 interdependence, which has a negative impact on the response of ER+ BCa cells to anti-ER therapies. The data from in silico analyses suggest that expression of Nrf-2 could act as a marker indicating potential benefits of implementation of anti-FGFR therapy in patients with ER+ BCa, in particular, when used in combination with anti-ER drugs.
Topics: Humans; Autophagy; NF-E2-Related Factor 2; Breast Neoplasms; Receptor, Fibroblast Growth Factor, Type 2; Female; Kelch-Like ECH-Associated Protein 1; Cell Line, Tumor; MCF-7 Cells; Signal Transduction; Sequestosome-1 Protein
PubMed: 38745155
DOI: 10.1186/s11658-024-00586-6 -
International Journal of Molecular... Apr 2024(1) Autophagy plays a significant role in development and cell proliferation. This process is mainly accomplished by the LC3 protein, which, after maturation, builds the...
(1) Autophagy plays a significant role in development and cell proliferation. This process is mainly accomplished by the LC3 protein, which, after maturation, builds the nascent autophagosomes. The inhibition of LC3 maturation results in the interference of autophagy activation. (2) In this study, starting from the structure of a known LC3B binder (LIR2-RavZ peptide), we identified new LC3B ligands by applying an in silico drug design strategy. The most promising peptides were synthesized, biophysically assayed, and biologically evaluated to ascertain their potential antiproliferative activity on five humans cell lines. (3) A cyclic peptide (named Pep6), endowed with high conformational stability (due to the presence of a disulfide bridge), displayed a K value on LC3B in the nanomolar range. Assays accomplished on PC3, MCF-7, and A549 cancer cell lines proved that Pep6 exhibited cytotoxic effects comparable to those of the peptide LIR2-RavZ, a reference LC3B ligand. Furthermore, it was ineffective on both normal prostatic epithelium PNT2 and autophagy-defective prostate cancer DU145 cells. (4) Pep6 can be considered a new autophagy inhibitor that can be employed as a pharmacological tool or even as a template for the rational design of new small molecules endowed with autophagy inhibitory activity.
Topics: Humans; Autophagy; Peptides, Cyclic; Drug Design; Cell Line, Tumor; Cell Proliferation; Antineoplastic Agents; Microtubule-Associated Proteins; Molecular Docking Simulation; A549 Cells; MCF-7 Cells
PubMed: 38731842
DOI: 10.3390/ijms25094622