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Molecular Biology of the Cell Mar 2024Synaptic plasticity is a process that shapes neuronal connections during neurodevelopment and learning and memory. Autophagy is a mechanism that allows the cell to...
Synaptic plasticity is a process that shapes neuronal connections during neurodevelopment and learning and memory. Autophagy is a mechanism that allows the cell to degrade its unnecessary or dysfunctional components. Autophagosomes appear at dendritic spines in response to plasticity-inducing stimuli. Autophagy defects contribute to altered dendritic spine development, autistic-like behavior in mice, and neurological disease. While several studies have explored the involvement of autophagy in synaptic plasticity, the initial steps of the emergence of autophagosomes at the postsynapse remain unknown. Here, we demonstrate a postsynaptic association of autophagy-related protein 9A (Atg9A), known to be involved in the early stages of autophagosome formation, with Rab11, a small GTPase that regulates endosomal trafficking. Rab11 activity was necessary to maintain Atg9A-positive structures at dendritic spines. Inhibition of mTOR increased Rab11 and Atg9A interaction and increased the emergence of LC3 positive vesicles, an autophagosome membrane-associated protein marker, in dendritic spines when coupled to NMDA receptor stimulation. Dendritic spines with newly formed LC3+ vesicles were more resistant to NMDA-induced morphologic change. Rab11 DN overexpression suppressed appearance of LC3+ vesicles. Collectively, these results suggest that initiation of autophagy in dendritic spines depends on neuronal activity and Rab11a-dependent Atg9A interaction that is regulated by mTOR activity.
Topics: Animals; Mice; Autophagosomes; Autophagy; Dendritic Spines; N-Methylaspartate; TOR Serine-Threonine Kinases
PubMed: 38294869
DOI: 10.1091/mbc.E23-02-0060 -
Communications Biology Nov 2023Polphylipoprotein (PLP) is a recently developed nanoparticle with high biocompatibility and tumor selectivity, and which has demonstrated unprecedentedly high...
Polphylipoprotein (PLP) is a recently developed nanoparticle with high biocompatibility and tumor selectivity, and which has demonstrated unprecedentedly high performance photosensitizer in photodynamic therapy (PDT) and photodynamic diagnosis. On the basis of these discoveries, PLP is anticipated to have a very high potential for PDT. However, the mechanism by which PLP kills cancer cells effectively has not been sufficiently clarified. To comprehensively understand the PLP-induced PDT processes, we conduct multifaceted experiments using both normal cells and cancer cells originating from the same sources, namely, RGM1, a rat gastric epithelial cell line, and RGK1, a rat gastric mucosa-derived cancer-like mutant. We reveal that PLP enables highly effective cancer treatment through PDT by employing a unique mechanism that utilizes the process of autophagy. The dynamics of PLP-accumulated phagosomes immediately after light irradiation are found to be completely different between normal cells and cancer cells, and it becomes clear that this difference results in the manifestation of the characteristic effect of PDT when using PLP. Since PLP is originally developed as a drug delivery agent, this study also suggests the potential for intracellular drug delivery processes through PLP-induced autophagy.
Topics: Rats; Animals; Photochemotherapy; Cell Line, Tumor; Photosensitizing Agents; Autophagy; Nanoparticles
PubMed: 38017279
DOI: 10.1038/s42003-023-05598-0 -
American Journal of Respiratory Cell... Jun 2024
Topics: TRPV Cation Channels; Humans; Animals; Phagosomes; Mice
PubMed: 38502902
DOI: 10.1165/rcmb.2024-0070ED -
Molecular Medicine (Cambridge, Mass.) Jan 2024Autophagic defects are involved in Methamphetamine (Meth)-induced neurotoxicity. Syntaxin 17 (Stx17), a member of the SNARE protein family, participating in several...
BACKGROUND
Autophagic defects are involved in Methamphetamine (Meth)-induced neurotoxicity. Syntaxin 17 (Stx17), a member of the SNARE protein family, participating in several stages of autophagy, including autophagosome-late endosome/lysosome fusion. However, the role of Stx17 and potential mechanisms in autophagic defects induced by Meth remain poorly understood.
METHODS
To address the mechanism of Meth-induced cognitive impairment, the adenovirus (AV) and adeno-associated virus (AAV) were injected into the hippocampus for stereotaxis to overexpress Stx17 in vivo to examine the cognitive ability via morris water maze and novel object recognition. In molecular level, the synaptic injury and autophagic defects were evaluated. To address the Meth induced neuronal damage, the epidermal growth factor receptor (EGFR) degradation assay was performed to evaluate the degradability of the "cargos" mediated by Meth, and mechanistically, the maturation of the vesicles, including autophagosomes and endosomes, were validated by the Co-IP and the GTP-agarose affinity isolation assays.
RESULTS
Overexpression of Stx17 in the hippocampus markedly rescued the Meth-induced cognitive impairment and synaptic loss. For endosomes, Meth exposure upregulated Rab5 expression and its guanine-nucleotide exchange factor (GEF) (immature endosome), with a commensurate decreased active form of Rab7 (Rab7-GTP) and impeded the binding of Rab7 to CCZ1 (mature endosome); for autophagosomes, Meth treatment elicited a dramatic reduction in the overlap between Stx17 and autophagosomes but increased the colocalization of ATG5 and autophagosomes (immature autophagosomes). After Stx17 overexpression, the Rab7-GTP levels in purified late endosomes were substantially increased in parallel with the elevated mature autophagosomes, facilitating cargo (Aβ42, p-tau, and EGFR) degradation in the vesicles, which finally ameliorated Meth-induced synaptic loss and memory deficits in mice.
CONCLUSION
Stx17 decrease mediated by Meth contributes to vesicle fusion defects which may ascribe to the immature autophagosomes and endosomes, leading to autophagic dysfunction and finalizes neuronal damage and cognitive impairments. Therefore, targeting Stx17 may be a novel therapeutic strategy for Meth-induced neuronal injury.
Topics: Animals; Mice; Autophagosomes; Autophagy; Endosomes; ErbB Receptors; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate
PubMed: 38172666
DOI: 10.1186/s10020-023-00765-9 -
Autophagy Aug 2023The precursors to mammalian autophagosomes originate from preexisting membranes contributed by a number of sources, and subsequently enlarge through intermembrane lipid...
The precursors to mammalian autophagosomes originate from preexisting membranes contributed by a number of sources, and subsequently enlarge through intermembrane lipid transfer, then close to sequester the cargo, and merge with lysosomes to degrade the cargo. Using cellular and in vitro membrane fusion analyses coupled with proteomic and biochemical studies we show that autophagosomes are formed from a hybrid membrane compartment referred to as a prophagophore or HyPAS (hybrid preautophagosomal structure). HyPAS is initially LC3-negative and subsequently becomes an LC3-positive phagophore. The prophagophore emerges through fusion of RB1CC1/FIP200-containing vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes. A specialized Ca-responsive apparatus controls prophagophore biogenesis and can be modulated by pharmacological agents such as SIGMAR1 agonists and antagonists including chloroquine. Autophagic prophagophore formation is inhibited during SARS-CoV-2 infection and is recapitulated by expression of SARS-CoV-2 nsp6. These findings show that mammalian autophagosomal prophagophores emerge via the convergence of secretory and endosomal pathways in a process that is targeted by microbial factors including coronaviral membrane proteins. CLEM, correlative light and electron microscopy; CQ, chloroquine; HyPAS, hybrid preautophagosomal; strcuture/prophagophore; LC3, microtubule associated protein 1 light chain 3; RUPEX, a combination of RUSH and APEX2 systems; SARS-CoV-2, SARS-CoV-2 virus, causative agent of COVID19.
Topics: Humans; Animals; Autophagosomes; Autophagy; Proteomics; COVID-19; SARS-CoV-2; Mammals
PubMed: 36571474
DOI: 10.1080/15548627.2022.2161728 -
Cells Aug 2023The putative phospholipase Atg15 is required for the intravacuolar lysis of autophagic bodies and MVB vesicles. Intracellular membrane lysis is a highly sophisticated...
The putative phospholipase Atg15 is required for the intravacuolar lysis of autophagic bodies and MVB vesicles. Intracellular membrane lysis is a highly sophisticated mechanism that is not fully understood. The amino-terminal transmembrane domain of Atg15 contains the sorting signal for entry into the MVB pathway. By replacing this domain, we generated chimeras located in the cytosol, the vacuole membrane, and the lumen. The variants at the vacuole membrane and in the lumen were highly active. Together with the absence of Atg15 from the phagophore and autophagic bodies, this suggests that, within the vacuole, Atg15 can lyse vesicles where it is not embedded. In-depth topological analyses showed that Atg15 is a single membrane-spanning protein with the amino-terminus in the cytosol and the rest, including the active site motif, in the ER lumen. Remarkably, only membrane-embedded Atg15 variants affected growth when overexpressed. The growth defects depended on its active site serine 332, showing that it was linked to the enzymatic activity of Atg15. Interestingly, the growth defects were independent of vacuolar proteinase A and vacuolar acidification.
Topics: Saccharomyces cerevisiae; Autophagy; Autophagosomes; Cell Death; Cell Movement; Fungal Proteins; Membrane Proteins
PubMed: 37626866
DOI: 10.3390/cells12162056 -
Autophagy Sep 2023Endoplasmic reticulum (ER) membrane contacts play a central role in regulating autophagosome formation in yeast and mammals. However, a direct functional linkage between...
Endoplasmic reticulum (ER) membrane contacts play a central role in regulating autophagosome formation in yeast and mammals. However, a direct functional linkage between the ER and autophagosomes in plants remains elusive. We have recently identified and characterized a plant-unique protein complex consisting of AT4G22540/OSBP2A/ORP2A (oxysterol binding protein-related protein 2A), the ER resident protein AT3G60600/VAP27-1 (vesicle-associated protein 27-1) and AT2G45170/ATG8e (autophagy related 8e) that mediate the ER-autophagosome membrane contact site (EACS) in the model plant . Knockdown (KD) of ORP2A affects autophagosome formation and seedling development, whereas ORP2A KD root cells show accumulation of the macroautophagic/autophagic core machinery and PtdIns3P in enlarged ER membranes under autophagy conditions. This study reveals the molecular architecture and functions of a distinct plant EACS in regulating autophagosome formation via lipid redistribution.
Topics: Animals; Autophagosomes; Autophagy; Endoplasmic Reticulum; Mitochondrial Membranes; Arabidopsis; Mammals
PubMed: 36730606
DOI: 10.1080/15548627.2023.2176028 -
The Journal of Cell Biology Jul 2024Autophagy serves as a stress response pathway by mediating the degradation of cellular material within lysosomes. In autophagy, this material is encapsulated in...
Autophagy serves as a stress response pathway by mediating the degradation of cellular material within lysosomes. In autophagy, this material is encapsulated in double-membrane vesicles termed autophagosomes, which form from precursors referred to as phagophores. Phagophores grow by lipid influx from the endoplasmic reticulum into Atg9-positive compartments and local lipid synthesis provides lipids for their expansion. How phagophore nucleation and expansion are coordinated with lipid synthesis is unclear. Here, we show that Faa1, an enzyme activating fatty acids, is recruited to Atg9 vesicles by directly binding to negatively charged membranes with a preference for phosphoinositides such as PI3P and PI4P. We define the membrane-binding surface of Faa1 and show that its direct interaction with the membrane is required for its recruitment to phagophores. Furthermore, the physiological localization of Faa1 is key for its efficient catalysis and promotes phagophore expansion. Our results suggest a positive feedback loop coupling phagophore nucleation and expansion to lipid synthesis.
Topics: Autophagosomes; Autophagy; Fatty Acids; Feedback; Macroautophagy; Saccharomyces cerevisiae
PubMed: 38573225
DOI: 10.1083/jcb.202309057 -
Clinical and Translational Science Dec 2023Aberrant autophagic activity is observed in osteoarthritic joints. Vitamin D was shown to alleviate not only osteoarthritis severity, but also autophagy process....
Aberrant autophagic activity is observed in osteoarthritic joints. Vitamin D was shown to alleviate not only osteoarthritis severity, but also autophagy process. However, the influence of vitamin D on autophagy in knee osteoarthritis (KOA) remains ambiguous. This study aimed to determine the effect of vitamin D on serum levels of autophagosome protein LC3A in patients with KOA and whether LC3A levels were correlated with serum 25-hydroxyvitamin D (25(OH)D) and clinical outcomes of patients with KOA. A total of 165 patients with KOA and 25 healthy controls were recruited. Vitamin D (ergocalciferol) was administered to patients with KOA at a weekly dosage of 40,000 IU. Serum LC3A, knee pain and functional scores, muscle strength, physical performance, and biochemical parameters were examined before and after 6 months of vitamin D supplementation. Serum LC3A levels were significantly higher in patients with KOA than healthy controls. In patients with KOA, vitamin D supplementation significantly decreased serum LC3A levels. Furthermore, baseline levels of serum LC3A were significantly associated with radiographic severity, pain and functional scores, total cholesterol, hs-CRP, IL-6, protein carbonyl, and serum 25(OH)D. After adjusting for established confounders, independent relationships among serum LC3A and radiographic severity, pain and functional scores, total cholesterol, hs-CRP, IL-6, protein carbonyl, and serum 25(OH)D were also observed. Vitamin D supplementation was shown to not only decrease serum levels of LC3A, inflammatory markers, as well as oxidative stress, but also improve muscle strength and physical performance in patients with KOA.
Topics: Humans; Osteoarthritis, Knee; C-Reactive Protein; Vitamin D Deficiency; Autophagosomes; Interleukin-6; Vitamin D; Inflammation; Pain; Dietary Supplements; Physical Functional Performance; Cholesterol
PubMed: 37749758
DOI: 10.1111/cts.13646 -
Infection and Immunity Dec 2023Pulmonary host defense is critical for the control of lung infection and inflammation. An increased expression and activity of Toll-like receptor 4 (TLR4) induce...
Pulmonary host defense is critical for the control of lung infection and inflammation. An increased expression and activity of Toll-like receptor 4 (TLR4) induce phagocytic uptake/clearance and inflammation against Gram-negative bacteria. In this study, we addressed the mechanistic aspect of the immunomodulatory activity of the TLR4-interacting SPA4 peptide (amino acid sequence GDFRYSDGTPVNYTNWYRGE) against . Binding of the SPA4 peptide to bacteria and direct anti-bacterial effects were investigated using flow cytometric, microscopic, and bacteriological methods. The bacterial uptake and inflammatory cytokine response were studied in dendritic cells expressing endogenous basal level of TLR4 or overexpressing TLR4. The subcellular distribution and co-localization of TLR4 and bacteria were investigated by immunocytochemistry. Furthermore, we studied the cellular expression and co-localization of endoplasmic reticulum (ER) molecules (calnexin and ER membrane protein complex subunit 1; EMC1) with lysosomal-associated membrane protein 1 (LAMP1) in cells infected with and treated with the SPA4 peptide. Simultaneously, the expression of histone H2A protein was quantitated by immunoblotting. Our results demonstrate no binding or direct killing of the bacteria by SPA4 peptide. Instead, it induces the uptake and localization of in the phagolysosomes for lysis and simultaneously suppresses the secreted levels of TNF-α. Overexpression of TLR4 further augments the pro-phagocytic and anti-inflammatory activity of SPA4 peptide. A time-dependent change in subcellular distribution of TLR4 and an increased co-localization of TLR4 with in SPA4 peptide-treated cells suggest an enhanced recognition and internalization of bacteria in conjugation with TLR4. Furthermore, an increased co-localization of calnexin and EMC1 with LAMP1 indicates the involvement of ER in pro-phagocytic activity of SPA4 peptide. Simultaneous reduction in secreted amounts of TNF-α coincides with suppressed histone H2A protein expression in the SPA4 peptide-treated cells. These results provide initial insights into the plausible role of ER and histones in the TLR4-immunomodulatory activity of SPA4 peptide against Gram-negative bacteria.
Topics: Humans; Toll-Like Receptor 4; Escherichia coli; Histones; Tumor Necrosis Factor-alpha; Calnexin; Inflammation; Endoplasmic Reticulum; Lipopolysaccharides
PubMed: 37909750
DOI: 10.1128/iai.00311-23