mTORC1-independent TFEB activation via Akt inhibition promotes cellular clearance in neurodegenerative storage diseases

Abstract Neurodegenerative diseases characterized by aberrant accumulation of undigested cellular components represent unmet medical conditions for which the identification of actionable targets is urgently needed. Here we identify a pharmacologically actionable pathway that controls cellular clearance via Akt modulation of transcription factor EB (TFEB), a master regulator of lysosomal pathways. We show that Akt phosphorylates TFEB at Ser467 and represses TFEB nuclear translocation independently of mechanistic target of rapamycin complex 1 (mTORC1), a known TFEB inhibitor. The autophagy enhancer trehalose activates TFEB by diminishing Akt activity. Administration of trehalose to a mouse model of Batten disease, a prototypical neurodegenerative disease presenting with intralysosomal storage, enhances clearance of proteolipid aggregates, reduces neuropathology and prolongs survival of diseased mice. Pharmacological inhibition of Akt promotes cellular clearance in cells from patients with a variety of lysosomal diseases, thus suggesting broad applicability of this approach. These findings open new perspectives for the clinical translation of TFEB-mediated enhancement of cellular clearance in neurodegenerative storage diseases.

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Safety profile of the transcription factor EB (TFEB)-based gene therapy through intracranial injection in mice

Translational Neuroscience ◽

10.1515/tnsci-2020-0132 ◽

2020 ◽

Vol 11 (1) ◽

pp. 241-250

Author(s):

Zhenyu Li ◽

Guangqian Ding ◽

Yudi Wang ◽

Zelong Zheng ◽

Jianping Lv

Keyword(s):

Gene Therapy ◽

Transcription Factor ◽

Neurodegenerative Diseases ◽

Brain Tissue ◽

Inflammatory Responses ◽

Tissue Samples ◽

Protein Levels ◽

Virus Serotype ◽

Transcription Factor Eb ◽

The Brain

AbstractTranscription factor EB (TFEB)-based gene therapy is a promising therapeutic strategy in treating neurodegenerative diseases by promoting autophagy/lysosome-mediated degradation and clearance of misfolded proteins that contribute to the pathogenesis of these diseases. However, recent findings have shown that TFEB has proinflammatory properties, raising the safety concerns about its clinical application. To investigate whether TFEB induces significant inflammatory responses in the brain, male C57BL/6 mice were injected with phosphate-buffered saline (PBS), adeno-associated virus serotype 8 (AAV8) vectors overexpressing mouse TFEB (pAAV8-CMV-mTFEB), or AAV8 vectors expressing green fluorescent proteins (GFPs) in the barrel cortex. The brain tissue samples were collected at 2 months after injection. Western blotting and immunofluorescence staining showed that mTFEB protein levels were significantly increased in the brain tissue samples of mice injected with mTFEB-overexpressing vectors compared with those injected with PBS or GFP-overexpressing vectors. pAAV8-CMV-mTFEB injection resulted in significant elevations in the mRNA and protein levels of lysosomal biogenesis indicators in the brain tissue samples. No significant changes were observed in the expressions of GFAP, Iba1, and proinflammation mediators in the pAAV8-CMV-mTFEB-injected brain compared with those in the control groups. Collectively, our results suggest that AAV8 successfully mediates mTFEB overexpression in the mouse brain without inducing apparent local inflammation, supporting the safety of TFEB-based gene therapy in treating neurodegenerative diseases.

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RIP3 impedes transcription factor EB to suppress autophagic degradation in septic acute kidney injury

Cell Death and Disease ◽

10.1038/s41419-021-03865-8 ◽

2021 ◽

Vol 12 (6) ◽

Author(s):

Ruizhao Li ◽

Xingchen Zhao ◽

Shu Zhang ◽

Wei Dong ◽

Li Zhang ◽

...

Keyword(s):

Acute Kidney Injury ◽

Transcription Factor ◽

Nuclear Translocation ◽

Kidney Injury ◽

Septic Acute Kidney Injury ◽

Transcription Factor Eb ◽

Autophagic Degradation ◽

Lps Treatment

AbstractAutophagy is an important renal-protective mechanism in septic acute kidney injury (AKI). Receptor interacting protein kinase 3 (RIP3) has been implicated in the renal tubular injury and renal dysfunction during septic AKI. Here we investigated the role and mechanism of RIP3 on autophagy in septic AKI. We showed an activation of RIP3, accompanied by an accumulation of the autophagosome marker LC3II and the autophagic substrate p62, in the kidneys of lipopolysaccharide (LPS)-induced septic AKI mice and LPS-treated cultured renal proximal tubular epithelial cells (PTECs). The lysosome inhibitor did not further increase the levels of LCII or p62 in LPS-treated PTECs. Moreover, inhibition of RIP3 attenuated the aberrant accumulation of LC3II and p62 under LPS treatment in vivo and in vitro. By utilizing mCherry-GFP-LC3 autophagy reporter mice in vivo and PTECs overexpression mRFP-GFP-LC3 in vitro, we observed that inhibition of RIP3 restored the formation of autolysosomes and eliminated the accumulated autophagosomes under LPS treatment. These results indicated that RIP3 impaired autophagic degradation, contributing to the accumulation of autophagosomes. Mechanistically, the nuclear translocation of transcription factor EB (TFEB), a master regulator of the lysosome and autophagy pathway, was inhibited in LPS-induced mice and LPS-treated PTECs. Inhibition of RIP3 restored the nuclear translocation of TFEB in vivo and in vitro. Co-immunoprecipitation further showed an interaction of RIP3 and TFEB in LPS-treated PTECs. Also, the expression of LAMP1 and cathepsin B, two potential target genes of TFEB involved in lysosome function, were decreased under LPS treatment in vivo and in vitro, and this decrease was rescued by inhibiting RIP3. Finally, overexpression of TFEB restored the autophagic degradation in LPS-treated PTECs. Together, the present study has identified a pivotal role of RIP3 in suppressing autophagic degradation through impeding the TFEB-lysosome pathway in septic AKI, providing potential therapeutic targets for the prevention and treatment of septic AKI.

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The role of phagocytosis-dependent activation of TFEB in the clearance of salmonella

10.32920/ryerson.14655972 ◽

2021 ◽

Author(s):

Erika Ospina Escobar

Keyword(s):

Transcription Factor ◽

Salmonella Typhimurium ◽

Nuclear Translocation ◽

Steady Increase ◽

Phagosome Maturation ◽

Salmonella Infection ◽

Complex Interplay ◽

Transcription Factor Eb ◽

Insight Into

During phagocytosis, macrophages engulf and sequester pathogens into phagosomes. Phagosomes then fuse with acidic and degradative lysosomes to degrade the internalized pathogen. We previously demonstrated that phagocytosis of IgG-opsonized particles and non-opsonized E.coli causes activation of the Transcription Factor EB (TFEB), which enhances the expression of lysosomal genes, increases the degradative capacity of lysosomes and boosts bactericidal activity. However, pathogens like Salmonella typhimurium have evolved mechanisms to evade and/or alter phagosome maturation to promote their own survival. We investigated: i) whether pathogens like Salmonella can alter TFEB activation and ii) whether phagocytosis-dependent activation of TFEB can counteract the pathogenicity of microorganisms. Here, we show that non-viable (heat-killed) S. typhimurium, pathogenic (EHEC and UPEC) and non-pathogenic E.coli (DH5α) all caused TFEB nuclear translocation in RAW macrophages, while strikingly live S. typhimurium maintained TFEB in the cytosol in the first hours post-infection. By contrast, Salmonella mutants for ΔsifA, ΔsopD2, ΔphoP all triggered TFEB activation in the first hour of infection. However, Salmonella infection eventually triggered a steady increase in nuclear TFEB after 4 h of infection, suggesting a more complex interplay between TFEB and Salmonella infection. We dissected the importance of TFEB activation towards Salmonella survivability by pre-activating TFEB before infection within WT macrophages and macrophages with a CRISPR-based deletion of TFEB. Our work suggests that Salmonella actively interferes with TFEB signaling in order to enhance its own survival. These results could provide insight into using TFEB as a target for the clearance of infections.

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Advanced glycation end-products suppress autophagic flux in podocytes by activating mammalian target of rapamycin and inhibiting nuclear translocation of transcription factor EB

The Journal of Pathology ◽

10.1002/path.5077 ◽

2018 ◽

Vol 245 (2) ◽

pp. 235-248 ◽

Cited By ~ 29

Author(s):

Xingchen Zhao ◽

Yuanhan Chen ◽

Xiaofan Tan ◽

Li Zhang ◽

Hong Zhang ◽

...

Keyword(s):

Transcription Factor ◽

Advanced Glycation End Products ◽

Nuclear Translocation ◽

Mammalian Target Of Rapamycin ◽

Target Of Rapamycin ◽

Autophagic Flux ◽

Advanced Glycation ◽

Glycation End Products ◽

End Products ◽

Transcription Factor Eb

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Transcription factor EB: from master coordinator of lysosomal pathways to candidate therapeutic target in degenerative storage diseases

Annals of the New York Academy of Sciences ◽

10.1111/nyas.13131 ◽

2016 ◽

Vol 1371 (1) ◽

pp. 3-14 ◽

Cited By ~ 63

Author(s):

Marco Sardiello

Keyword(s):

Transcription Factor ◽

Therapeutic Target ◽

Storage Diseases ◽

Transcription Factor Eb

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Intracellular Trafficking and Persistence of Acinetobacter baumannii Requires Transcription Factor EB

mSphere ◽

10.1128/msphere.00106-18 ◽

2018 ◽

Vol 3 (2) ◽

Cited By ~ 10

Author(s):

Raquel Parra-Millán ◽

David Guerrero-Gómez ◽

Rafael Ayerbe-Algaba ◽

Maria Eugenia Pachón-Ibáñez ◽

Antonio Miranda-Vizuete ◽

...

Keyword(s):

Transcription Factor ◽

Acinetobacter Baumannii ◽

Intracellular Trafficking ◽

Nuclear Translocation ◽

A549 Cells ◽

Lung Epithelial Cells ◽

Host Cells ◽

Infection Model ◽

Content Type ◽

Transcription Factor Eb

ABSTRACT Acinetobacter baumannii is a significant human pathogen associated with hospital-acquired infections. While adhesion, an initial and important step in A. baumannii infection, is well characterized, the intracellular trafficking of this pathogen inside host cells remains poorly studied. Here, we demonstrate that transcription factor EB (TFEB) is activated after A. baumannii infection of human lung epithelial cells (A549). We also show that TFEB is required for the invasion and persistence inside A549 cells. Consequently, lysosomal biogenesis and autophagy activation were observed after TFEB activation which could increase the death of A549 cells. In addition, using the Caenorhabditis elegans infection model by A. baumannii , the TFEB orthologue HLH-30 was required for survival of the nematode to infection, although nuclear translocation of HLH-30 was not required. These results identify TFEB as a conserved key factor in the pathogenesis of A. baumannii . IMPORTANCE Adhesion is an initial and important step in Acinetobacter baumannii infections. However, the mechanism of entrance and persistence inside host cells is unclear and remains to be understood. In this study, we report that, in addition to its known role in host defense against Gram-positive bacterial infection, TFEB also plays an important role in the intracellular trafficking of A. baumannii in host cells. TFEB was activated shortly after A. baumannii infection and is required for its persistence within host cells. Additionally, using the C. elegans infection model by A. baumannii , the TFEB orthologue HLH-30 was required for survival of the nematode to infection, although nuclear translocation of HLH-30 was not required.

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The Transcription Factor EB Reduces the Intraneuronal Accumulation of the Beta-Secretase-Derived APP Fragment C99 in Cellular and Mouse AD Models

Cells ◽

10.3390/cells9051204 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1204

Author(s):

Anaïs Bécot ◽

Raphaëlle Pardossi-Piquard ◽

Alexandre Bourgeois ◽

Eric Duplan ◽

Qingli Xiao ◽

...

Keyword(s):

Transcription Factor ◽

Mouse Model ◽

Early Stage ◽

Amyloid Β ◽

Newborn Mice ◽

Cellular Models ◽

In Vivo Experiments ◽

Lysosome Biogenesis ◽

Transcription Factor Eb

: Brains that are affected by Alzheimer’s disease (AD) are characterized by the overload of extracellular amyloid β (Aβ) peptides, but recent data from cellular and animal models propose that Aβ deposition is preceded by intraneuronal accumulation of the direct precursor of Aβ, C99. These studies indicate that C99 accumulation firstly occurs within endosomal and lysosomal compartments and that it contributes to early-stage AD-related endosomal-lysosomal-autophagic defects. Our previous work also suggests that C99 accumulation itself could be a consequence of defective lysosomal-autophagic degradation. Thus, in the present study, we analyzed the influence of the overexpression of the transcription factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis, on C99 accumulation occurring in both AD cellular models and in the triple-transgenic mouse model (3xTgAD). In the in vivo experiments, TFEB overexpression was induced via adeno-associated viruses (AAVs), which were injected either into the cerebral ventricles of newborn mice or administrated at later stages (3 months of age) by stereotaxic injection into the subiculum. In both cells and the 3xTgAD mouse model, exogenous TFEB strongly reduced C99 load and concomitantly increased the levels of many lysosomal and autophagic proteins, including cathepsins, key proteases involved in C99 degradation. Our data indicate that TFEB activation is a relevant strategy to prevent the accumulation of this early neurotoxic catabolite.

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PtdIns3P controls mTORC1 signaling through lysosomal positioning

The Journal of Cell Biology ◽

10.1083/jcb.201611073 ◽

2017 ◽

Vol 216 (12) ◽

pp. 4217-4233 ◽

Cited By ~ 55

Author(s):

Zhi Hong ◽

Nina Marie Pedersen ◽

Ling Wang ◽

Maria Lyngaas Torgersen ◽

Harald Stenmark ◽

...

Keyword(s):

Amino Acids ◽

Endoplasmic Reticulum ◽

Transcription Factor ◽

Amino Acid ◽

Cell Periphery ◽

Lipid Kinase ◽

Mechanistic Target Of Rapamycin ◽

Mtorc1 Signaling ◽

Transcription Factor Eb ◽

Mtorc1 Activation

The mechanistic target of rapamycin complex 1 (mTORC1) is a protein kinase complex that localizes to lysosomes to up-regulate anabolic processes and down-regulate autophagy. Although mTORC1 is known to be activated by lysosome positioning and by amino acid–stimulated production of phosphatidylinositol 3-phosphate (PtdIns3P) by the lipid kinase VPS34/PIK3C3, the mechanisms have been elusive. Here we present results that connect these seemingly unrelated pathways for mTORC1 activation. Amino acids stimulate recruitment of the PtdIns3P-binding protein FYCO1 to lysosomes and promote contacts between FYCO1 lysosomes and endoplasmic reticulum that contain the PtdIns3P effector Protrudin. Upon overexpression of Protrudin and FYCO1, mTORC1–positive lysosomes translocate to the cell periphery, thereby facilitating mTORC1 activation. This requires the ability of Protrudin to bind PtdIns3P. Conversely, upon VPS34 inhibition, or depletion of Protrudin or FYCO1, mTORC1-positive lysosomes cluster perinuclearly, accompanied by reduced mTORC1 activity under nutrient-rich conditions. Consequently, the transcription factor EB enters the nucleus, and autophagy is up-regulated. We conclude that PtdIns3P-dependent lysosome translocation to the cell periphery promotes mTORC1 activation.

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