Fig 2: Activation of HSF1 by epoxyazadiradione. (A) Treatment of HEK293T cells with Tau and epoxyazadiradione exhibited no changes of HSF1 in western blotting analysis and its quantification. (B) Extracellular Tau aggregates reduced the cellular HSF1 level and epoxyazadiradione increased HSF1 level in cytosol and nucleus. Tau aggregates were found to adhere in the cell periphery whereas the intracellular Tau level was negligible in HEK293T cells. (C,D) IF quantification showed a significant increase in intracellular HSF1 and Tau aggregates were observed on the cellular periphery. Quantification was carried out by Zen 2.3 software (https://www.zeiss.com/microscopy/int/products/light-microscopes/axio-observer-for-biology.html).

Fig 3: Hsp90 Inhibitors and the Transcriptional Landscape of Geroprotective Drugs(A) Heatmap clustering analysis of monorden-, LY-294002-, and rapamycin-induced transcriptomic changes (CMap data). Lines represent transcripts. Only those changing at least 1.5-fold are shown. Five main transcript clusters emerge, indicated in different colors: blue, purple, light green, dark green, and yellow. See Table S6 for the genes present in the transcript clusters.(B) The ten most enriched GO terms within the five clusters from (A). See Table S6 for all enrichments.(C) Heatmap clustering analysis of the transcriptomic changes induced by all CMap compounds that are listed as lifespan-extending in the DrugAge database, including previously undocumented compounds discovered by our study (monorden, tanespimycin, and felbinac). The bottom two rows indicate candidate compounds described in this study (black tiles) and any known Hsp90 inhibitors (green tiles). Lines represent transcripts; only transcripts changing at least 2-fold in any given drug were used for clustering and are shown. See Table S6 for all the transcripts used in the clustering analysis. The yellow box indicates a cluster of HSPs upregulated by monorden, tanespimycin, geldanamycin, and celastrol, all of which are known Hsp90 inhibitors. See Table S6 for the transcripts comprising and the GO term enrichments within the yellow cluster.(D) Heat shock survival assay performed at 35°C. See Table S4 for worm numbers and statistics.(E) Lifespan analysis for solvent control- and monorden-treated N2 and hsf-1(sy441) animals. See Table S4 for worm numbers and statistics.(F) Model of how Hsp90 inhibitors such as monorden and tanespimycin may confer their geroprotective effects. Hsp90 inhibitors lead to release of the transcription factor HSF1 from sequestration by Hsp90 proteins, allowing HSF1 to trimerize and activate transcription of its target genes in the nucleus, most importantly HSPs/chaperones. These HSPs/chaperones help to ensure the correct folding of proteins and prevent their aggregation with age, promoting protein homeostasis and cytoprotection, which lead to improved health and longevity.

Fig 4: Proposed mechanism for HSF1-mediated clearance of Tau. In normal conditions HSF1 is present as an inactive monomer in cytosol. HSF1 in inactive state forms complex with Hsp70. Triggering of Tau-induced stress or treatment of cells with limonoids leads to dissociation of Hsp70 from HSF1. HSF1 monomer is now subjected to phosphorylation and is transported into nucleus. In nucleus, HSF1 trimerizes and interacts with heat shock element (HSE) present in the upstream of Hsp70 promoter. This leads to increase in expression of Hsp70 and thus enhancing the cytosolic levels of Hsp70.