Loss of a proteostatic checkpoint in intestinal stem cells contributes to age-related epithelial dysfunction

Abstract: Protein homeostasis (proteostasis) is critical for cell function, and its decline is proposed as a hallmark of aging. Somatic stem cells (SCs) have a unique ability to maintain their proteostatic capacity, yet mechanisms to achieve this remain incompletely understood. Here, we describe and characterize a ‘proteostatic checkpoint’ in Drosophila intestinal SCs (ISCs). Following a breakdown of proteostasis, ISCs coordinate cell cycle arrest with protein aggregate clearance by Atg8-mediated activation of the Nrf2-like transcription factor cap-n-collar C (CncC), which induces the cell cycle inhibitor Dacapo and proteolytic genes. The capacity to engage this checkpoint is lost in ISCs from aging flies, and we show that it can be restored by treating flies with an Nrf2 activator, or by over-expression of CncC or Atg8a. This limits age-related intestinal barrier dysfunction and can result in lifespan extension. Our findings identify a new mechanism by which somatic SCs preserve proteostasis, and highlight potential intervention strategies to maintain regenerative homeostasis. Purpose of Study: To explore the transcriptional program downstream of CncC in Drosophila ISCs in more detail, we performed RNAseq analysis on FACS-purified wild-type or CncC-deficient escargot-positive cells (ISCs/EBs) in which the proteostatic checkpoint was engaged through the expression of HttQ138 or through the knockdown of rpn3. Results: While knockdown of rpn3 resulted in a more pronounced and broader transcriptional response than the expression of HttQ138, 93 of the 200 genes that were induced by HttQ138 expression (using a cut-off of 3-fold induction and a minimum expression of 10 FPKM) were also induced by knockdown of rpn3, illustrating a broad overlap of transcriptional changes elicited by the two conditions. Induction of 167 of those 200 genes was dependent on CncC. GO analysis of either the 93 genes induced in both stress conditions, or of the 167 CncC-dependent genes revealed a significant enrichment of genes encoding proteins involved in proteolysis and protein metabolism. Methods: Intact guts from adult female flies (total of 6 different genotypes, ~ 100 guts per sample) were dissected in cold PBS supplemented with 1% bovine serum albumin and 2% fetal bovine serum. Tissues (~ 100 guts per sample) were trypsinized twice using 0.5% Trypsin EDTA (Gibco Cat.No.15400054) for 30 min on rocker at RT each time, and at each step all cells in suspension were collected. All recovered cells were pooled and washed in cold PBS supplemented with 1% bovine serum albumin and 2% fetal bovine serum. Cells in suspension were passed through the fine mesh of a cell-strainer cap of a 5 ml polystyrene-round bottom tube (Cat. No.Falcon 352235) and then all GFP-positive cells (GFP was driven by escargot promoter expressed in ISCs and EBs) were sorted using FACS. Total RNA was isolated from sorted cells using Trizol reagent, and used as template to generate RNA-seq libraries for Illumina MiSeq sequencing (150bp paired-end reads were obtained). Reads were mapped to the Drosophila genome (release 5.75) using a standard Tuxedo suite pipeline, and FPKM (Fragments Per Kilobase per Million reads) values were recorded for each gene. Analysis of the data was done in Excel, and Gene Ontology analysis was performed using tools at Flymine.org. Grant info used for the project: Heinrich Jasper NIH R01s AG047497 and GM117412 http://grantome.com/grant/NIH/R01-AG047497-04 http://grantome.com/grant/NIH/R01-GM117412-03