Downregulation of beclin 1 restores arsenite-induced impaired autophagic flux by improving the lysosomal function in cortex

Abstract Abstract Background : Graphene oxide (GO) nanoparticles (NPs) have been widely applied in various fields, especially in biomedical applications. Extensive studies have suggested that GO can pass through the blood-brain barrier (BBB) and induce abnormal autophagy and cytotoxicity in the central nervous system (CNS). However, the effect and specific mechanism of GO on astrocytes, the most abundant cells in the brain still has not been extensively investigated. Results: In this study, we systematically explored the toxicity and mechanism of GO exposure in the rat astroglioma-derived F98 cell line using molecular biological techniques (immunofluorescence staining, flow cytometry and Western blot) at the subcellular level and the signaling pathway level. Cells exposed to GO exhibited decreased cell viability and increased lactate dehydrogenase (LDH) release in a concentration- and time-dependent manner. GO-induced autophagy was evidenced by transmission electron microscopy (TEM) and immunofluorescence staining. Western blots showed that LC3II/I and p62 were upregulated and PI3K/Akt/mTOR was downregulated. Detection of lysosomal acidity and cathepsin B activity assay indicated the impairment of lysosomal function. Annexin V-FITC-PI detection showed the occurrence of apoptosis after GO exposure. The decrease in mitochondrial membrane potential (MMP) with an accompanying upregulation of cleaved caspase-3 and Bax/Bcl-2 further suggested that endogenous signaling pathways were involved in GO-induced apoptosis. Conclusion: The exposure of F98 cells to GO can elicit concentration- and time-dependent toxicological effects. Additionally, increased autophagic response can be triggered after GO treatment and that the blocking of autophagy flux plays a vital role in GO cytotoxicity, which was determined to be related to dysfunction of lysosomal degradation. Importantly, the abnormal accumulation of autophagic substrate p62 protein can induce capase-3-mediated apoptosis. Inhibition of abnormal accumulation of autophagic cargo could alleviate the occurrence of GO-induced apoptosis in F98 cells. Keyword: Graphene oxide; Astrocyte; p62; Autophagy; Apoptosis

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Beclin 1, LC3 and P62 Expression in Equine Sarcoids

Animals ◽

10.3390/ani12010020 ◽

2021 ◽

Vol 12 (1) ◽

pp. 20

Author(s):

Manuela Martano ◽

Gennaro Altamura ◽

Karen Power ◽

Pierluigi Liguori ◽

Brunella Restucci ◽

...

Keyword(s):

Western Blot ◽

Normal Skin ◽

Beclin 1 ◽

Autophagic Flux ◽

Damaged Material ◽

Complex Process ◽

Causative Agents ◽

No Activation ◽

Skin Samples ◽

Selection Of

Background: It is well known that δ-bovine papillomaviruses (BPV-1, BPV-2 and BPV-13) are one of the major causative agents of equine sarcoids, the most common equine skin tumors. Different viruses, including papillomaviruses, evolved ingenious strategies to modulate autophagy, a complex process involved in degradation and recycling of old and damaged material. Methods: The aim of this study was to evaluate, by immunohistochemistry (IHC) and Western blot (WB) analysis, the expression of the main related autophagy proteins (Beclin 1, protein light chain 3 (LC3) and P62), in 35 BPV1/2 positive equine sarcoids and 5 BPV negative normal skin samples. Results: Sarcoid samples showed from strong-to-moderate cytoplasmic immunostaining, respectively, for Beclin 1 and P62 in >60% of neoplastic fibroblasts, while LC3 immunostaining was weak to moderate in ≤60% of neoplastic fibroblasts. Western blot analysis confirmed the specificity of the antibodies and revealed no activation of autophagic flux despite Beclin 1 overexpression in sarcoid samples. Conclusion: Results could suggest the activation of the initial phase of autophagy in equine sarcoids, and its impairment during the following steps. The impairment of autophagy could lead to a selection of a quiescent population of fibroblasts, which survive longer in a hypoxic microenvironment and produced more and/or altered collagen.

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Targeting the potent Beclin 1–UVRAG coiled-coil interaction with designed peptides enhances autophagy and endolysosomal trafficking

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1721173115 ◽

2018 ◽

Vol 115 (25) ◽

pp. E5669-E5678 ◽

Cited By ~ 14

Author(s):

Shuai Wu ◽

Yunjiao He ◽

Xianxiu Qiu ◽

Wenchao Yang ◽

Wenchao Liu ◽

...

Keyword(s):

Egf Receptor ◽

Coiled Coil ◽

Transport Processes ◽

Beclin 1 ◽

Autophagic Flux ◽

Endosomal Trafficking ◽

Class Iii ◽

Coiled Coil Domain ◽

Complementary Interactions ◽

Self Association

The Beclin 1–Vps34 complex, known as “mammalian class III PI3K,” plays essential roles in membrane-mediated transport processes including autophagy and endosomal trafficking. Beclin 1 acts as a scaffolding molecule for the complex and readily transits from its metastable homodimeric state to interact with key modulators such as Atg14L or UVRAG and form functionally distinct Atg14L/UVRAG-containing Beclin 1–Vps34 subcomplexes. The Beclin 1–Atg14L/UVRAG interaction relies critically on their coiled-coil domains, but the molecular mechanism remains poorly understood. We determined the crystal structure of Beclin 1–UVRAG coiled-coil complex and identified a strengthened interface with both hydrophobic pairings and electrostatically complementary interactions. This structure explains why the Beclin 1–UVRAG interaction is more potent than the metastable Beclin 1 homodimer. Potent Beclin 1–UVRAG interaction is functionally significant because it renders UVRAG more competitive than Atg14L in Beclin 1 binding and is critical for promoting endolysosomal trafficking. UVRAG coiled-coil mutants with weakened Beclin 1 binding do not outcompete Atg14L and fail to promote endolysosomal degradation of the EGF receptor (EGFR). We designed all-hydrocarbon stapled peptides that specifically targeted the C-terminal part of the Beclin 1 coiled-coil domain to interfere with its homodimerization. One such peptide reduced Beclin 1 self-association, promoted Beclin 1–Atg14L/UVRAG interaction, increased autophagic flux, and enhanced EGFR degradation. Our results demonstrate that the targeting Beclin 1 coiled-coil domain with designed peptides to induce the redistribution of Beclin 1 among its self-associated form or Atg14L/UVRAG-containing complexes enhances both autophagy and endolysosomal trafficking.

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PIKfyve inhibition reveals a novel role for Inpp4b in the regulation of PtdIns(3)P and lysosome dynamics

10.1101/2021.12.13.472440 ◽

2021 ◽

Author(s):

Golam T. Saffi ◽

Emily Mangialardi ◽

Jean Vacher ◽

Roberto J. Botelho ◽

LEONARDO SALMENA

Keyword(s):

Autophagic Flux ◽

Contributing Factors ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Lipid Kinase ◽

Lysosomal Function ◽

Cargo Delivery ◽

Simultaneous Inhibition ◽

And Function ◽

Underlying Processes

Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that co-ordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the action of diverse PtdIns kinases and phosphatases. Specific roles for PtdIns in lysosomal function and dynamics are currently unclear and require further investigation. PIKfyve, a lipid kinase which synthesizes PtdIns(3,5)P2 from PtdIns(3)P, controls lysosome fusion-fission cycles, autophagosome turnover and endocytic cargo delivery. We have recently characterized a role for INPP4B, a PtdIns phosphatase which hydrolyses PtdIns(3,4)P2 to form PtdIns(3)P, in the regulation of lysosomal biogenesis and function. To gain a better understanding of PtdIns homeostasis on lysosomes, we investigated the consequence of disrupting PIKfyve in Inpp4b-deficient mouse embryonic fibroblasts. Surprisingly, simultaneous inhibition of Inpp4b and PIKfyve functions impair lysosome fission and exacerbate lysosome enlargement and inhibit autophagic flux. Further examination into the underlying processes that may explain exaggerated lysosome enlargement revealed elevated levels of lysosome-associated PtdIns(3)P as contributing factors that control lysosome morphology in cells where Inpp4b and PIKfyve are disrupted. Overall, our study suggests that lysosomal functions are regulated by Inpp4b, through a paradoxical role in suppressing the induction of PtdIns(3)P production.

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Abstract 250: Differential Role of Autophagy in Idiopathic versus Ischemic Heart Failure in Humans

Circulation Research ◽

10.1161/res.119.suppl_1.250 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Antoinette Bugyei-Twum ◽

Krishna K Singh ◽

Filio Billia ◽

Kim A Connelly

Keyword(s):

Heart Failure ◽

Ischemia Reperfusion ◽

Fold Increase ◽

Left Ventricular ◽

Beclin 1 ◽

Ischemic Heart ◽

Autophagic Flux ◽

Ischemic Heart Failure ◽

Autophagic Activity

Background: Autophagy is an evolutionary conserved process that plays a key role in a variety of physiological and pathological processes. Despite its beneficial role, excessive/insufficient autophagic activity is known to contribute to the pathogenesis of cardiovascular disorders, including ischemia/reperfusion injury and heart failure. However, the differential role of autophagy in idiopathic versus ischemic heart failure remains unknown. Methods: To investigate the role of autophagy and associated apoptosis in idiopathic versus ischemic heart failure, we obtained LV myocardium biopsies from healthy controls (via 3 commercial sources), and from 10 patients with idiopathic and ischemic end-stage heart failure before the insertion of a left ventricular assist devise. The expression of inducers/markers of autophagy (mTOR, phospho-mTOR, LC3-I/II, p62, Beclin-1, autophagy-related genes ATG4B/ATG5) and apoptosis (Bcl-2 and caspase-3) were assessed at the transcript and protein level using quantitative RT-PCR and Western blotting. Results: Autophagy was activated in both idiopathic and ischemic heart failure in comparison to control, as confirmed by a significant reduction in mTOR expression/activation and a 3.4-fold and 2.2-fold increase in LC3 II/I ratio, respectively. An increase in apoptosis, marked by increased caspase3 and Bcl2 expression, was also observed in both groups in comparison to control. Interestingly, autophagy activity—marked by decreased mTOR expression/activation, increased ATG4B, ATG5, and Beclin-1—was significantly higher in idiopathic heart failure, when compared to ischemic heart failure. While we observed increased autophagic activity in idiopathic heart failure, p62 expression was also significantly increased in this group (2.8-fold increase; p<0.05), demonstrating an impairment of autophagic flux in idiopathic versus ischemic heart failure. Conclusions: For the first time, we provide a direct comparision of autophagy and apoptosis in idiopathic versus ischemic heart failure. Our data, demonstrating an excessive yet insufficient autophagic activity in idiopathic heart failure, suggests a differential role of autophagy apoptosis in idiopathic versus ischemia-related heart failure.

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