Transcriptomic and functional profiling reveal autophagy inhibition and persistent bioenergetic collapse following parallel photodamage to lysosomes and mitochondria

Photodynamic therapy (PDT) using 1,9-dimethyl methylene blue (DMMB) induces coordinated mitochondrial and lysosomal damage and results in strong cellular death induction. However, the underlying transcriptional regulation in response to DMMB remains elusive. We compared the transcriptome response of photoactivated DMMB (paDMMB) to the gene signature triggered by autophagy-modulating agents: rapamycin (an autophagy activator) and bafilomycin A1 (an autophagy inhibitor). Transcriptome analysis revealed a pronounced transcriptomic response to paDMMB, with 884 differentially expressed genes (DEGs), compared to 291 for bafilomycin and 154 for rapamycin. paDMMB treatment upregulated genes associated with autophagy, mitochondrial stress responses, and proteostasis, while downregulating genes involved in miRNA processing and lipid catabolism. Rapamycin treatment downregulated amino acid biosynthesis pathways, while upregulating processes associated with nutrient starvation. Conversely, bafilomycin treatment upregulated genes related to lipid metabolism, while suppressing cytoskeletal programs. We observed that approximately 80% of bafilomycin DEGs also changed in paDMMB-treated cells, and about 96% of these shared genes showed concordant regulation. This suggests that the paDMMB molecular signature is consistent with the inhibition of autophagic flux. Among the several biological processes affected by paDMMB, mitochondrial-related processes were enriched. To determine whether the acute transcriptome changes caused by paDMMB led to persistent functional effects, we stimulated cells with DMMB and assessed mitochondrial respiration after a recovery period. paDMMB reduced basal respiration, ATP production, proton leak, and maximal respiration. These effects were not further altered by bafilomycin co-treatment but were markedly exacerbated by rapamycin. Collectively, we show that paDMMB leads to a transcriptome rewiring, closely resembling autophagy inhibition with a sustained mitochondrial dysfunction. These findings provide a valuable resource to understand the interplay between DMMB-induced lysosomal stress, transcriptional regulation, and PDT. • DMMB induces a broad transcriptional response, impacting genes involved in autophagy, proteostasis, and mitochondrial respiration. • The DMMB transcriptional signature resembles that of autophagy-inducing drug (Bafilomycin). • DMMB treatment causes persistent mitochondrial dysfunction, leading to impaired respiration. • Activation of autophagy by Rapamycin exacerbates mitochondrial collapse. • Dual lysosomal and mitochondrial photodamage emerges the underlying mechanism of DMMB.