Suppression rather than activation of the integrated stress response (GCN2-ATF4) pathway extends lifespan in the fly.

Stress response pathways are emerging as conserved modulators of lifespan. The prevailing hypothesis is that activation of stress-responsive pathways, including the amino acid deprivation arm of the integrated stress response (ISR; the GCN2-ATF4 pathway), is prolongevity. Activation of <i>ATF4</i> orthologs extends lifespan in <i><i>Saccharomyces cerevisiae</i></i> and <i><i>Caenorhabditis elegans</i></i>, but its role in other longer-lived organisms remains unclear. We comprehensively tested the role of the GCN2-ATF4 pathway in longevity in the fly (<i><i>Drosophila melanogaster</i></i>) for the first time. We used conditional genetic manipulation of <i>dGCN2</i> and its downstream effector <i>Drosophila ATF4</i> (<i>crc</i>; <i>dATF4</i>). In contrast to previous studies, we show that overexpression of <i>dGCN2</i> and <i>dATF4</i> significantly reduces lifespan, while knockdown (in vivo RNAi) of <i>dATF4</i> extends lifespan. We confirmed that dATF4 activity was successfully modulated using a fluorescent <i>dATF4</i> activation reporter. Borrelidin, a tRNA synthetase inhibitor, significantly reduced lifespan in a both <i>dATF4</i> and diet-dependent manner, independent of microbial load, showing our modulation of <i>dATF4</i> altered nutrient to ISR signaling. We further conducted long-read RNA sequencing and found that our manipulation of <i>dATF4</i> changed global transcription in opposite directions, including known <i>ATF4</i> target genes. Enrichment analysis revealed that <i>dATF4</i> overexpression may drive metabolic stress, while <i>dATF4</i> knockdown may upregulate proteostasis(definition) and DNA repair pathways. Our work reveals that <i>ATF4</i> may exhibit a dual, dose-, and context-dependent role in aging. Chronic <i>dATF4</i> activation is detrimental in flies, while chronic suppression is prolongevity. The GCN2-ATF4 pathway thus qualifies as a modifiable control of lifespan with cross-species relevance.