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Potentiation of rifampin activity in a mouse model of tuberculosis by activation of host transcription factor EB.

ABSTRACT: Efforts at host-directed therapy of tuberculosis have produced little control of the disease in experimental animals to date. This is not surprising, given that few specific host targets have been validated, and reciprocally, many of the compounds tested potentially impact multiple targets with both beneficial and detrimental consequences. This puts a premium on identifying appropriate molecular targets and subjecting them to more selective modulation. We discovered an aminopyrimidine small molecule, 2062, that had no direct antimycobacterial activity, but synergized with rifampin to reduce bacterial burden in Mtb infected macrophages and mice and also dampened lung immunopathology. We used 2062 and its inactive congeners as tool compounds to identify host targets. By biochemical, pharmacologic, transcriptomic and genetic approaches, we found that 2062's beneficial effects on Mtb control and clearance in macrophages and in mice are associated with activation of transcription factor EB via an organellar stress response. 2062-dependent TFEB activation led to improved autophagy, lysosomal acidification and lysosomal degradation, promoting bacterial clearance in macrophages. Deletion of TFEB resulted in the loss of IFN?-dependent control of Mtb replication in macrophages. 2062 also targeted multiple kinases, such as PIKfyve, VPS34, JAKs and Tyk2, whose inhibition likely limited 2062's efficacy in vivo. These findings support a search for selective activators of TFEB for HDT of TB.

SUBMITTER: Bryk R

PROVIDER: S-EPMC7337396 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Potentiation of rifampin activity in a mouse model of tuberculosis by activation of host transcription factor EB.

Bryk Ruslana R Mundhra Shashirekha S Jiang Xiuju X Wood Madeleine M Pfau Daniel D Weber Elaina E Park Suna S Zhang Li L Wilson Colin C Van der Westhuyzen Renier R Street Leslie L Chibale Kelly K Zimmerman Matthew M Dartois Véronique V Pastore Nunzia N Ballabio Andrea A Hawryluk Natalie N Canan Stacie S Khetani Vikram V Camardo Joseph J Nathan Carl C

PLoS pathogens 20200623 6

Efforts at host-directed therapy of tuberculosis have produced little control of the disease in experimental animals to date. This is not surprising, given that few specific host targets have been validated, and reciprocally, many of the compounds tested potentially impact multiple targets with both beneficial and detrimental consequences. This puts a premium on identifying appropriate molecular targets and subjecting them to more selective modulation. We discovered an aminopyrimidine small mole ...[more]

PMID: 32574211

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Project description:Background Mutations in ?B-crystallin result in proteotoxic cardiomyopathy with desmin mislocalization to protein aggregates. Intermittent fasting ( IF ) is a novel approach to activate transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in the myocardium. We tested whether TFEB activation can be harnessed to treat advanced proteotoxic cardiomyopathy. Methods and Results Mice overexpressing the R120G mutant of ?B-crystallin in cardiomyocytes ( Myh6-Cry ABR 120G) were subjected to IF or ad-lib feeding, or transduced with adeno-associated virus- TFEB or adeno-associated virus-green fluorescent protein after development of advanced proteotoxic cardiomyopathy. Adeno-associated virus-short hairpin RNA-mediated knockdown of TFEB and HSPB 8 was performed simultaneously with IF . Myh6-Cry ABR 120G mice demonstrated impaired autophagic flux, reduced lysosome abundance, and mammalian target of rapamycin activation in the myocardium. IF resulted in mammalian target of rapamycin inhibition and nuclear translocation of TFEB with restored lysosome abundance and autophagic flux; and reduced aggregates with normalized desmin localization. IF also attenuated left ventricular dilation and myocardial hypertrophy, increased percentage fractional shortening, and increased survival. Adeno-associated virus- TFEB transduction was sufficient to rescue cardiomyopathic manifestations, and resulted in reduced aggregates and normalized desmin localization in Myh6-Cry ABR 120G mice. Cry ABR 120G-expressing hearts demonstrated increased interaction of desmin with ?B-crystallin and reduced interaction with chaperone protein, HSPB 8, compared with wild type, which was reversed by both IF and TFEB transduction. TFEB stimulated autophagic flux to remove protein aggregates and transcriptionally upregulated HSPB 8, to restore normal desmin localization in Cry ABR 120G-expressing cardiomyocytes. Short hairpin RNA-mediated knockdown of TFEB and HSPB 8 abrogated IF effects, in vivo. Conclusions IF and TFEB activation are clinically relevant therapeutic strategies to rescue advanced R120G ?B-crystallin mutant-induced cardiomyopathy by normalizing desmin localization via autophagy-dependent and autophagy-independent mechanisms.

Project description:The stimulation of autophagy or lysosomes has been considered therapeutic for neurodegenerative disorders because the accumulation of misfolded proteins is commonly observed in the brains of individuals with these diseases. Although zinc is known to play critical roles in the functions of lysosomes and autophagy, the mechanism behind this regulatory relationship remains unclear. Therefore, in this study, we examined which mechanism is involved in zinc-mediated activation of autophagy and lysosome. Exposure to zinc at a sub-lethal concentration activated autophagy in a concentration-dependent manner in mRFP-GFP-LC3-expressing H4 glioma cells. Zinc also rescued the blocking of autophagic flux arrested by pharmaceutical de-acidification. Co-treatment with zinc attenuated the chloroquine (CQ)-induced increase in the number and size of mRFP-GFP-LC3 puncta in H4 cells and accumulation of p62 by CQ or ammonium chloride in both H4 and mouse cerebrocortical cultures. Zinc rapidly induced the expression of cathepsin B (CTSB) and cathepsin D (CTSD), representative lysosomal proteases in neurons, which appeared likely to be mediated by transcription factor EB (TFEB). We observed the translocation of TFEB from neurite to nucleus and the dephosphorylation of TFEB by zinc. The addition of cycloheximide, a chemical inhibitor of protein synthesis, inhibited the activity of CTSB and CTSD at 8 h after zinc exposure but not at 1 h, indicating that only late lysosomal activation was dependent on the synthesis of CTSB and CTSD proteins. At the very early time point, the activation of cathepsins was mediated by an increased assembly of V-ATPase on lysosomes and resultant lysosomal acidification. Finally, considering that P301L mutation in tau protein causes frontotemporal dementia through aggressive tau accumulation, we investigated whether zinc reduces the accumulation of protein aggregates in SK-N-BE(2)-C neuroblastoma cells expressing wild-type tau or mutant P301L-tau. Zinc markedly attenuated the levels of phosphorylated tau and total tau as well as p62 in both wild-type and mutant tau-overexpressing cells. We also observed that zinc was more effective than rapamycin at inducing TFEB-dependent CTSB and CTSD expression and V-ATPase-dependent lysosomal acidification and CTSB/CTSD activation. These results suggest that the regulation of zinc homeostasis could be a new approach for developing treatments for neurodegenerative diseases, including Alzheimer's and Parkinson's.

Project description:RationalePostischemic angiogenesis is critical to limit the ischemic tissue damage and improve the blood flow recovery. The regulation and the underlying molecular mechanisms of postischemic angiogenesis are not fully unraveled. TFEB (transcription factor EB) is emerging as a master gene for autophagy and lysosome biogenesis. However, the role of TFEB in vascular disease is less understood.ObjectiveWe aimed to determine the role of endothelial TFEB in postischemic angiogenesis and its underlying molecular mechanism.Methods and resultsIn primary human endothelial cells (ECs), serum starvation induced TFEB nuclear translocation. VEGF (vascular endothelial growth factor) increased TFEB expression level and nuclear translocation. Utilizing genetically engineered EC-specific TFEB transgenic and KO (knockout) mice, we investigated the role of TFEB in postischemic angiogenesis in the mouse hindlimb ischemia model. We observed improved blood perfusion and increased capillary density in the EC-specific TFEB transgenic mice compared with the wild-type littermates. Furthermore, blood flow recovery was attenuated in EC-TFEB KO mice compared with control mice. In aortic ring cultures, the TFEB transgene significantly increased vessel sprouting, whereas TFEB deficiency impaired the vessel sprouting. In vitro, adenovirus-mediated TFEB overexpression promoted EC tube formation, migration, and survival, whereas siRNA-mediated TFEB knockdown had the opposite effect. Mechanistically, TFEB activated AMPK (AMP-activated protein kinase)-α signaling and upregulated autophagy. Through inactivation of AMPKα or inhibition of autophagy, we demonstrated that the AMPKα and autophagy are necessary for TFEB to regulate angiogenesis in ECs. Finally, the positive effect of TFEB on AMPKα activation and EC tube formation was mediated by TFEB-dependent transcriptional upregulation of MCOLN1 (mucolipin-1).ConclusionsIn summary, our data demonstrate that TFEB is a positive regulator of angiogenesis through activation of AMPKα and autophagy, suggesting that TFEB constitutes a novel molecular target for ischemic vascular disease.

Dataset Information

Potentiation of rifampin activity in a mouse model of tuberculosis by activation of host transcription factor EB.

Publications

Potentiation of rifampin activity in a mouse model of tuberculosis by activation of host transcription factor EB.

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