TFEB‐dependent lysosome biogenesis is required for senescence

Abstract The accumulation of senescent cells is recognised as a driver of tissue and organismal ageing. One of the gold‐standard hallmarks of a senescent cell is an increase in lysosomal content, as measured by senescence‐associated β‐galactosidase (Senβ‐Gal) activity. The lysosome plays a central role in integrating mitogenic and stress cues to control cell metabolism, which is known to be dysregulated in senescence. Despite this, little is known about the cause and consequence of lysosomal biogenesis in senescence. We find here that lysosomes in senescent cells are dysfunctional; they have higher pH, increased evidence of membrane damage and reduced proteolytic capacity. The significant increase in lysosomal content is however sufficient to maintain degradative capacity of the cell to a level comparable to proliferating control cells. We demonstrate that increased nuclear TFEB/TFE3 supports lysosome biogenesis, is a hallmark of multiple forms of senescence and is required for senescent cell survival. TFEB/TFE3 are hypo‐phosphorylated and show constitutive nuclear localisation in senescence. Evidence suggests that several pathways may contribute to TFEB/TFE3 dysregulation in senescence.

. Targeting lysosomes in senescence promotes cell death.
A EtOH and 4OHT-treated fibroblasts were fixed, immunostained for Galectin 1 (Gal1) and Lamp1. The number of Gal1 + puncta was quantified. Scale bar: 10 lm (n = 3 independent experimental repeats (at least 40 cells analysed from at least four fields of view per repeat)). B Proliferating and IR fibroblasts were fixed, immunostained for Galectin 1 (Gal1) and Lamp1. The number of Gal1 + puncta was quantified. Scale bar: 20 lm (n = 3 independent experimental repeats (at least 40 cells analysed from at least four fields of view per repeat)). C EtOH and 4OHT-treated fibroblasts were incubated in the presence/absence of LLoMe for 2 h, immunostained with antibodies against Gal1 and Lamp1, and the number of Gal1 + puncta was quantified. Scale bar: 20 lm (n = 4 independent experimental repeats (at least 40 cells analysed from at least four fields of view per repeat)). D Proliferating and IR fibroblasts were incubated in the presence/absence of LloMe for 2 h, immunostained with antibodies against Gal1 and Lamp1, and the number of Gal1 + puncta was quantified. Scale bar: 20 lm (n = 3 independent experimental repeats (at least 40 cells analysed from at least four fields of view per repeat)). E Cells as in (C) were incubated with cell-permeable live/dead dye, imaged and cell death quantified. Scale bar: 100 lm (n = 3 independent experimental repeats (at least 300 cells analysed from at least six fields of view per repeat)). F Senescent fibroblasts were incubated with LloMe and incubated with cell-permeable live/dead dyes. Scale bar: 100 lm (n = 2 independent experimental repeats (at least 200 cells analysed from at least four fields of view per repeat)).
Data information: All graphs show individual data points, mean and error bars represent standard deviation. All data (where n = 3) are analysed by 2-tailed, nonpaired Student's t-test (***< 0.001) except (E) which was analysed by one-way ANOVA with Tukey's multiple comparison test (***< 0.001).

Prol.
Prol. A Quantification of nuclear TFE3 in EtOH and 4OHT-treated fibroblasts (n = 3 independent experimental repeats). B Representative images and quantification of nuclear TFE3 in proliferating and replicative senescent (RS) fibroblasts. Scale bar: 20 lm (n = 3 independent experimental repeats (at least 100 cells analysed from at least five fields of view per repeat)). C Representative images and quantification of nuclear TFE3 in proliferating versus etoposide or doxorubicin-induced senescent fibroblasts. Scale bar: 20 lm (n = 3 independent experimental repeats (at least 100 cells analysed from at least five fields of view per repeat)). D Representative images and quantification of nuclear TFE3 in proliferating versus CDK4i-induced senescent fibroblasts. Scale bar: 20 lm (n = 3 independent experimental repeats (at least 100 cells analysed from at least five fields of view per repeat)). E Quantification of nuclear TFE3 in proliferating and IR-induced senescent fibroblasts (n = 3 independent experimental repeats (at least 100 cells analysed from at least five fields of view per repeat)). F Proliferating and IR-induced senescent fibroblasts immunostained for endogenous TFE3 in the conditions indicated; starvation indicates serum starvation overnight and 1-h amino acid starvation; refeeding was with full nutrient medium (including FCS) for 2 h. Cells were incubated with leptomycin B (LMB) for 3 h and where indicated, wash out involved replacement of the media for a further 2 h. Scale bar: 20 lm. G Quantification of (F); % cells with nuclear TFE3 was quantified (n = 3 independent experimental repeats (at least 150 cells analysed from at least five fields of view per repeat)).

A
Fibroblasts were transduced with TFEB and TFE3 shRNA before induction of senescence by IR. Cells were fixed and stained with crystal violet (n = 1). B Fibroblasts were transduced with TFEB and TFE3 shRNA before induction of senescence by CDK4i. Cells were fixed and stained with crystal violet (n = 1). C Fibroblasts were transduced with TFEB and TFE3 shRNA immediately following induction of senescence by IR (or incubated with proliferating controls) and analysed by Western blot 10 days later. D-F Quantification of blots shown in (C) as indicated (n = 3 independent experimental repeats). G Cells treated as in (C) were fixed and immunostained for Lamp2 and p62. Scale bar: 20 lm. H Quantification of p62-Lamp2 colocalisation (Mander's coefficient) from (G) (n = 3 independent experimental repeats (at least 40 cells analysed from at least four fields of view per repeat)).
Data information: All graphs show individual data points, mean and error bars represent standard deviation. All data were analysed by one-way ANOVA with Tukey's multiple comparison test (*< 0.05, **< 0.01). Source data are available online for this figure.
A Fibroblasts treated with EtOH or 4OHT were fixed and immunostained for Lamp2 and RagC. Scale bar: 20 lm. B Quantification of (B) (Mander's coefficient between Lamp2 and RagC) (n = 2 independent repeats (at least 40 cells analysed from at least four fields of view per repeat)). C Cells as in (A) were lysed and subject to Western blot. D Quantification of (C); expression of RagA, RagB and RagC, normalised to tubulin and relative to EtOH control (n = 2 independent experimental repeats). E Fibroblasts stably expressing GFP or GFP-RagC 75L were treated with EtOH or 4OHT as indicated, fixed and immunostained for endogenous TFE3. Scale bar: 20 lm. F Quantification of (E) (n = 2 independent experimental repeats (at least 50 cells analysed in each condition from at least five fields of view per repeat)). G Fibroblasts stably expressing GFP or GFP-RagC 75L and FLAG-TFEB were treated with EtOH or 4OHT as indicated. Cells were lysed and subject to Western blotting. H Quantification of (G) (n = 4 independent experimental repeats). I Quantification of (G) (n = 4 independent experimental repeats). J Fibroblasts were transduced with p16 shRNA simultaneously with induction of senescence by 4OHT. Cells were subject to starvation (serum-free media overnight, 1 h amino acid-free media) or starved and refed (starvation protocol as above, refeeding with full nutrient media for 2 h). Cells were fixed and immunostained for endogenous TFE3. Scale bar: 100 lm. K Quantification of (J) (n = 2 independent repeats (at least 100 cells analysed in each condition from at least five fields of view per repeat)).
Data information: All graphs show individual data points, and error bars represent standard deviation. Data analysed by one-way ANOVA with Tukey's multiple comparison test (***< 0.001). Source data are available online for this figure.

Rachel Curnock et al
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