Metcalf ISCIENCE-D-23-07458R1_N2_forward

Description

Glia are the protectors of the nervous system, providing neurons with support and protection from cytotoxic insults. We previously discovered that four astrocyte-like glia can regulate organismal proteostasis and longevity in C. elegans. Expression of the UPRER transcription factor, XBP-1s, in these glia increases stress resistance, longevity, and activates the UPRER in intestinal cells via neuropeptides. Autophagy, a key regulator of metabolism and aging, has been described as a cell autonomous process. Surprisingly, we find that glial XBP-1s enhances proteostasis and longevity by cell non-autonomously reprogramming organismal lipid metabolism and activating autophagy. Glial XBP-1s regulates the activation of another transcription factor, HLH-30/TFEB, in the intestine. HLH-30 activates intestinal autophagy, increases intestinal lipid catabolism, and upregulates a robust transcriptional program. Our study reveals a novel role for glia in regulating peripheral lipid metabolism, autophagy, and organellar health through peripheral activation of HLH-30 and autophagy

Steps to reproduce

Animals were bleach synchronized and grown to day 2 of adulthood on large NGM plates spotted with 1mL of OP50. Around 1,000 animals per condition and replicate were collected by washing off with M9 buffer. Each condition had a total of 5 biological replicates. Animals were spun down at 1,000 RCF for 30 seconds, M9 removed and washed again for a total of 3 washes. M9 was then replaced with 1 mL of Trizol and animals were subsequently frozen, via liquid nitrogen, and thawed, in a 37°C water bath, 3 times. After the final thaw, chloroform was added at a ratio of 1:5 (chloroform:trizol) for aqueous separation of RNA using heavy gel phase-lock tubes (VWR, 10847-802). Library preparation and RNA-sequencing was performed by Genewiz (Azenta Life Sciences). Library preparation was performed with Poly A selection and HiSeq xSequencing using NEBNext Ultra RNA Library Prep Kit for Illumina. Sequencing libraries were clustered into 1 lane of a flow cell, loaded on Illumina HiSeq instrument (4000 or equivalent) and samples were sequenced using 2x150bp Paired End configuration. Base calling was conducted on HiSeq Control Software. For RNA-sequencing analysis, raw sequencing data were uploaded to the Galaxy project web platform and the public server at usegalaxy.org was used to analyze the data. Paired end reads were aligned using the Kallisto quant tool (Version 0.48.0) with WBcel235 as the reference genome. Fold changes and statistics were generated using the DESeq2 tool with Kallisto quant count files as the input. The fold change and adjusted-p values generated by the DESeq2 analysis were used to plot the data using GraphPad Prism 9.

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