Topoisomerase inhibitors demonstrate geroprotective effects

In order to identify small molecules capable of mimicking the transcriptional signature of AKT1 knockdown, we designed an in silico and in vivo drug discovery pipeline, ultimately resulting in the identification of amonafide as a novel geroprotector (Fig. 1A). The approach consisted of three main sections: (a) drug screening based on public databases and computational analysis, (b) assessment of candidates for geroprotective effects in C. elegans, and (c) validation of the longevity mechanism of the lead candidate, amonafide (Fig. 1A). Using the AKT1 knockdown transcriptional signature available in the Library of Integrated Network-based Cellular Signatures (LINCS) database [24, 25], we used the same drug screening method as previously used by our lab [18, 19] to identify compounds that induce effects akin to AKT1 knockdown. A ranked list of small molecules possessing transcriptomics signatures most similar to the transcriptomic signature of AKT1 inhibition across various cell strains was generated using the publicly available data and user interface of the LINCS database [24, 25]. We further refined this list by searching for drug classes that were enriched among the highest-ranked small molecules (Fig. 1B). This analysis allowed us to identify prevalent drug classes in our list relative to the entire dataset. Notably, AKT inhibitors, mTOR inhibitors, and PI3 kinase inhibitors all emerged as strongly enriched drug classes, aligning with the AKT1 inhibition transcriptional signature (Fig. 1B). Given that AKT inhibitors, mTOR inhibitors, and PI3 kinase inhibitors are recognized for their ability to extend lifespan and operate within the PI3K-AKT longevity pathway [26,27,28], our findings suggest that this compound screening approach effectively identified compounds mimicking AKT1 inhibition. Several AKT1 inhibitors revealed as hits in our screen included A-443644, staurosporine, and hexamethylenebisacetamide. Notably, topoisomerase inhibitors emerged as the most prominent class of compounds exhibiting a transcriptomic profile closely resembling AKT1 inhibition (Fig. 1B). Topoisomerase inhibitors, typically used as chemotherapeutic agents, interfere with the topoisomerase enzymes (topoisomerase I and II), governing changes in DNA structure [29]. Despite their known chemotherapeutic role, there is currently no established link between topoisomerase inhibitors and longevity regulation, drawing our attention to further investigate this drug class.

Fig. 1

Topoisomerase inhibitors exhibit geroprotective effects in C. elegans. A Diagram shows how this study identified amonafide as a geroprotective compound, using in silico and in vivo methods. B Results of drug classes enriched as mimicking the transcriptional signature of AKT1 knockdown in cells. Drug classes are plotted against the number of hits (X-axis) and the p-value significance of enrichment (Y-axis, with an adjusted p-value threshold of < 0.05 for inclusion). The size of each dot correlates with the percentage of hits within that drug class. C Violin plot representing the mobility of C. elegans (N2) under vehicle (water) and suramin treatment. D Violin plot representing the mobility of C. elegans (N2) under the vehicle (DMSO) and etoposide treatment. E Violin plot representing the mobility of C. elegans (N2) under the vehicle (DMSO) and amonafide treatment. C–E Y-axis shows the average moving speed of C. elegans at day 13. The bar in the center of the violin plot represents the median value of mobility. The statistical analysis was performed using a one-way ANOVA test followed by Tukey post hoc test; groups were compared to vehicle. *** represents p-value < 0.001, and “ns” represents not significant. F Lifespan curves of C. elegans (N2) treated with vehicle (water) or suramin. G Lifespan curves of C. elegans (N2) treated with vehicle (DMSO) or etoposide. H Lifespan curves of C. elegans (N2) treated with vehicle (DMSO) or amonafide. F–H p-values represent a comparison with the controls calculated using a log-rank test. **** represents p-value < 0.0001, and ns represents not significant

We focused our study on three distinct topoisomerase inhibitors: suramin [30], etoposide [31], and amonafide [32]. Suramin, a hexasulfated naphthylurea that inhibits topoisomerase II, has been used in the treatment of trypanosomiasis [30]. Etoposide, which targets topoisomerase II, is a commonly used anti-cancer agent [31]. Amonafide is a DNA intercalating agent that interferes with topoisomerase II activity and is a promising anti-cancer compound [32] (Table S1). Healthspan and lifespan were assessed at two different doses for each topoisomerase inhibitor (Fig. 1C–E). The concentrations of the low doses were set at either 10 μM (suramin) or 50 μM (etoposide and amonafide), relative to the dosing most generally used in cell culture, and the high dose was set at 100 μM for all three compounds. Healthspan was evaluated by measuring the mobility of C. elegans treated with either vehicle or each respective topoisomerase inhibitor. Mobility assays conducted on day 13 of adulthood revealed a significant improvement in the average movement of worms at 10 μM for suramin; however, this effect was not present at 100 μM (Fig. 1C). Both the 50 μM and 100 μM of etoposide significantly increased the mobility of worms (Fig. 1D). A similar beneficial effect was observed with amonafide at both 50 μM and 100 μM doses (Fig. 1E). Further lifespan tests for these beneficial doses of each compound revealed that, despite the healthspan increase observed in certain doses of suramin and etoposide, the lifespan curves showed no significant effects for these two topoisomerase inhibitors (Fig. 1F–G). However, we observed a 25% lifespan extension in worms treated with 100 μM amonafide compared to vehicle controls (Fig. 1H). To further investigate the effects of suramin, etoposide, and amonafide on worm development, we measured the body size of treated worms. Our analysis revealed that worms treated with suramin and amonafide displayed a slightly larger body size compared to the control group, whereas worms treated with etoposide showed a body size similar to that of the controls (S1A Fig). Taken together, these results suggest that the tested topoisomerase inhibitors may have geroprotective properties, with all three tested topoisomerase inhibitors enhancing healthspan in C. elegans. Furthermore, demonstrates potential in promoting both healthspan and longevity.

The roles of top-2/TOP2 and daf-16/FOXO in amonafide’s healthspan and lifespan extension

Given the pronounced effect of amonafide on lifespan extension and healthspan improvement, we further investigated its impact on healthspan at various time points (day 7, day 10, and day 13). Mobility assays revealed a consistent increase in mobility at all tested time points with 100-μM amonafide treatment (Fig. 2A). The lower dose of amonafide, 50 μM, did not lead to evident improvement of mobility at earlier time points, but improved mobility at day 13 (Fig. 2A). We also observed a trend indicating that amonafide’s positive impact on mobility became more pronounced as the worms aged (Fig. 2A, S1B Fig). In addition to assessing healthspan, we conducted lifespan measurements on worms subjected to different doses (50, 100, and 200 μM) of amonafide. The lifespan curves demonstrated a remarkable extension in lifespan across all three administered doses compared to the control (Fig. 2B). Among the tested dosages, 100 μM amonafide exhibited the most substantial extension of lifespan, while the lifespan curve for 200 μM amonafide overlapped with that of 50 μM amonafide. This observation suggests that elevating the dosage to 200 μM does not confer additional benefits to lifespan (Fig. 2B). To determine whether the beneficial effects of amonafide were mediated through bacterial metabolism, we tested its impact on worms maintained on bacteria killed using UV cross-linking to eliminate bacterial metabolism. Significant lifespan extension was still observed in worms cultured on inactivated bacteria and treated with amonafide compared to the control (Fig. 2C). This result suggests that the lifespan-extending effects of amonafide are likely attributable to direct interactions with the worms rather than reliance on bacterial metabolism.

Fig. 2

Amonafide improves healthspan and extends lifespan. A Violin plot representing the healthspan of C. elegans (N2) under vehicle (DMSO) or amonafide treatment in different doses as specified. Mobility of worms was measured at three time points as specified. The statistical analysis was performed using a one-way ANOVA test; groups were compared to vehicle. **** represents p-value < 0.0001, * represents p-value < 0.05, and ns represents not significant. B Lifespan curves of C. elegans (N2) treated with vehicle (DMSO) or amonafide in different doses as specified. p-values were calculated using the log-rank test for comparisons with the control group. *** represents p-value < 0.001. C Lifespan curves of C. elegans (N2) maintained on dead E. coli OP50. Worms were treated with vehicle (DMSO) or 100 μM amonafide as indicated. p-values were calculated using the log-rank test for comparisons with the control group. **** represents p-value < 0.0001. D Violin plot representing the mobility of C. elegans (N2) under the same conditions as shown in C at day 13. The statistical analysis was performed using a one-way ANOVA test followed by Tukey post hoc test; groups were compared to control (HT115). ** represents p-value < 0.01, * represents p-value < 0.05, and ns represents not significant. E Lifespan curves of C. elegans (N2) treated with control (HT115), 5% top-2 RNAi, and 10% top-2 RNAi. Percentage represents the concentration of top-2 RNAi bacteria. p-values were calculated using the log-rank test for comparisons with the control group. * represents p-value < 0.05, and ns represents not significant. F Violin plot representing the mobility of daf-16(mu86) under treatment of vehicle or 50 μM amonafide. The statistical analysis was performed using a wilcox.test. ** represents p-value < 0.01. G Lifespan curves of daf-16(mu86) treated with vehicle or 50 μM amonafide. The p-value represents the comparison with the controls calculated using the log-rank test. H Hypothesis of mechanisms underlying the longevity effect of amonafide

Amonafide, a naphthalimide derivative, has been investigated as an anticancer agent. Its inhibitory effect on topoisomerase was identified in the late 1980s [33, 34]. Topoisomerases, comprising two major classes, type I (TOP1) and type II (TOP2), play a crucial role in altering the topological properties of genetic material [29]. Amonafide functions as a DNA intercalating agent, disrupting the activity of TOP2 [33]. We considered the possibility that the observed improvement in healthspan and lifespan with amonafide treatment may be linked to its inhibition of TOP2. To verify this connection, we measured the healthspan of C. elegans treated with top-2 RNAi bacteria. We first assessed the efficiency of top-2 knockdown using qPCR. The qPCR results revealed a clear pattern, demonstrating a dose-dependent reduction in top-2 expression in worms as the concentration of top-2 RNAi bacteria increased (S1C Fig). We observed a dose-dependent increase in average mobility in worms treated with lower concentrations of top-2 RNAi bacteria (2.5%, 5%, 10%, Fig. 2D). Conversely, the mobility of worms exhibited a dose-dependent decrease when the concentration of top-2 RNAi bacteria exceeded 10% (Fig. 2D). Strikingly, the lifespan of worms treated with 5% top-2 RNAi overlapped with the control, while worms treated with 10% top-2 RNAi lived shorter than the control (Fig. 2E). Therefore, despite both 5% and 10% top-2 RNAi treatments exhibiting a beneficial effect on mobility in worms, these did not translate into improvements in lifespan (Fig. 2D–E). To further investigate the role of top-2 in the lifespan-extending effects of amonafide, we conducted lifespan assays under top-2 knockdown conditions. Our data indicate that worms treated with top-2 RNAi alone exhibited a significantly shortened lifespan compared to the control group, confirming that top-2 is essential for the normal health and longevity of C. elegans (S1D Fig). When worms were treated with a combination of top-2 RNAi and amonafide, we observed a lifespan extension compared to worms treated with top-2 RNAi alone (S1D Fig). However, this extended lifespan was notably shorter than that observed in control worms treated with amonafide alone. These results suggest that amonafide retains some lifespan-extending effects even in the context of top-2 RNAi treatment, and if beneficial, rather than harmful, to lifespan. Notably however, we detected residual top-2 mRNA expression in worms cultured on 100% top-2 RNAi bacteria, indicating that top-2 was not fully depleted in our study (S1C Fig). This partial knockdown suggests that residual top-2 activity may contribute to the observed effects of amonafide, and makes it difficult to ascertain conclusively that amonafide operates fully independently of top-2. An experiment with a full knock-out of top-2 would be required to ascertain if amonafide operates fully independently of top-2. Nonetheless, our experimental evidence on healthspan and lifespan is highly suggestive that the impact of amonafide on healthspan may not be exclusively attributed to its inhibition of top-2 and the benefit of amonafide treatment to lifespan is independent of top-2.

It is well-documented that AKT-1 phosphorylates DAF-16, retaining it in the cytoplasm and preventing its entry into the nucleus, thereby inhibiting its transcriptional activity. This inactivation of DAF-16 by AKT-1 results in reduced lifespan and lowered resistance to stress, linking AKT-1 activity to aging processes [35]. Given that our compound screen aimed to mimic AKT1 inhibition, we next investigated the role of daf-16/FOXO in amonafide-induced lifespan extension, as daf-16/FOXO is a downstream mediator of lifespan extension from reduced AKT1 levels [36]. To address this, we turned to daf-16(mu86) mutant strain of worms which lack a function daf-16/FOXO transcription factor. In comparison to the vehicle, amonafide was still able to significantly increase the mobility of daf-16(mu86) worms (Fig. 2F). Furthermore, we also observed an extension in the lifespan of daf-16(mu86) worms treated with 50 μM amonafide, although the observed beneficial effect was not as pronounced as in the wild type (Fig. 2G). Taken together, we concluded that the mechanism underlying the longevity effect of amonafide differs in part from AKT1 inhibition, as it is not fully blocked by daf-16 inhibition. Additionally, mild top-2 RNAi, which mimics the effect of top-2 inhibition, increased mobility but not lifespan. This also implies that pathways other than the canonical targets of amonafide are involved in promoting the longevity effect of amonafide treatment (Fig. 2H).

The transcriptome altered by amonafide exhibits predicted ages of a younger state and activated defense responses

To untangle the mechanism by which amonafide benefits healthspan and lifespan, we conducted RNA sequencing on total RNA isolated from N2 worms treated with or without amonafide. Principal component analysis (PCA) demonstrated that amonafide treatment had a pronounced impact on the transcriptome compared to vehicle (DMSO)-treated worms (Fig. 3A). To further explore the effects resulting from amonafide treatment, we performed differential expression analysis, where we found that compared to the vehicle, 2727 genes were downregulated and 1313 genes were upregulated upon amonafide treatment (adjusted p-value < 0.05, Fig. 3B). In line with the longevity effect and the fact that our screen was based on transcriptionally mimicking AKT1 knockdown, we found that akt-1 was transcriptionally downregulated following amonafide treatment (S1E Fig).

Fig. 3

The transcriptome altered by amonafide exhibits predicted ages of a younger state and activated defense responses. A PCA plot of the transcriptomic analysis. Samples from amonafide clustered separately from vehicle (DMSO) samples. B Volcano plot of genes in amonafide vs. vehicle (adjusted p-value < 0.05, absolute log2FC > 1, y-axis values exceeding 300 were capped at 300). C Predicted biological age of worms treated with amonafide or vehicle (DMSO). ** represents a two-tailed t-test p-value < 0.01. D Histogram representing the gene length (log2 scale) of DEGs (using adjust p-value < 0.05, absolute log fold change > 0.5). Shorter genes are significantly more likely to be downregulated in amonafide-treated worms than longer genes (p-value < 0.0001; unpaired t-test, dash line represents the median gene length in each group). E Top 10 GSEA enriched gene sets in significantly upregulated genes (adjust p-value < 0.05, log fold change > 0.5) in amonafide vs vehicle. F Top 5 over-representation enriched Biological Process gene sets in significantly upregulated genes (adjust p-value < 0.05, log fold change > 0.5) in amonafide vs vehicle. G Graph representation of the top enriched KEGG sets in genes upregulated by amonafide

To better understand how the amonafide-altered transcriptome related to longevity, we explored our RNA-seq data using two recent tools and discoveries that are able to characterize a youthful transcriptome. These included (1) a recently developed binarized transcriptomic aging (BiT age) clock designed to predict the biological age of C. elegans based on RNA-seq data [37] and (2) the observation that in global assessment of young and old RNA-seq data, younger transcriptomes possess larger amounts of transcripts from longer genes [38]. We applied the BiT age clock to our RNAseq-dataset, and in line with amonafide’s lifespan extension effects, found a reduction in the predicted biological age of worms treated with amonafide compared to the vehicle (Fig. 3C). Furthermore, assessing the lengths of differentially expressed genes, we observed a significant tendency for longer genes to be upregulated by amonafide, in line with the observation that younger transcriptomes are more likely to express longer genes (Fig. 3D). In summary, the global alterations in the transcriptome aligned with the observed beneficial effects on healthspan and lifespan of amonafide (Fig. 2A, B).

Next, to explore the potential function of genes regulated by amonafide and uncover the mechanism explaining how amonafide improved healthspan and lifespan, we performed Gene Sets Enrichment Analysis (GSEA). We found that the top positively enriched gene sets were “xenobiotic metabolic process,” “cellular response to exobiotic stimulus,” and “xenobiotic catabolic response” (Fig. 3E). Further over-representation analysis on genes upregulated by amonafide revealed top Biological Process gene sets enriched in “response to biotic stimulus,” “biological process involved in interspecies,” and “defense response” (Fig. 3F). Moreover, top KEGG sets enriched in genes upregulated by amonafide included pathways related to “drug metabolism cytostome P450,” “glutathione metabolism,” “retinol metabolism,” and “lysosome,” most of which are involved in responses to biotic stimuli (Fig. 3G). Collectively, these results suggest that amonafide activates key pathways associated with cellular defense mechanisms.

Amonafide activates gene and protein expression levels in mitochondria-, pathogen-, and xenobiotic-associated responses

To further dissect the stress responses induced by amonafide treatment, we examined the transcriptional changes in specific key stress response pathways associated with longevity. These pathways include p38 MAPK pathway, skn-1-mediated oxidative stress response, daf-16-mediated antioxidant activity, zip-2-mediated pathogen defense response, atf-4-mediated integrated stress response (ISR), and the atfs-1-mediated mitochondrial unfolded protein response (UPRmt). The p38 MAPK pathway, known for promoting pathogen resistance, contributes to the longevity observed in daf-2 mutants [39]. skn-1-mediated oxidative stress response plays a central role in various genetic and pharmacologic interventions that promote longevity in C. elegans [40]. daf-16 is essential for the longevity induced by akt-1 inhibition [41]. The zip-2-mediated infection defense response is critical for survival following pathogen infection [42]. Meanwhile, atf-4, the transcriptional effector of ISR, is required for longevity induced by the global protein synthesis stress [43]. Additionally, atfs-1-mediated UPRmt is necessary for lifespan extension induced by mitonuclear protein imbalance and the mitochondrial unfolded protein response [44]. Collectively, these transcription factors and pathways play crucial roles in regulating defense responses, lifespan, and aging in C. elegans, and may therefore be integral for lifespan or healthspan extension resulting from amonafide treatment (Fig. 4A).

Fig. 4

Amonafide activates defense pathways: impact on mRNA and protein levels in pathogen- and xenobiotic-associated responses. A Schematic of key longevity-related defense processes in C. elegans. The numbered boxes on the right correspond to gene clusters utilized for assessing these defense responses; (1) oxidative stress response, (2) pathogen defense response, (3) integrated stress response, (4) mitochondrial unfolded protein response. B Heatmap of key effectors in skn-1 mediated oxidative stress response in worms treated with vehicle or amonafide. All presented genes were significantly differentially expressed, adjusted p-value less than 0.05. C Representative fluorescence images of irg-1p::GFP reporter worms under vehicle or amonafide (50 μM and 100 μM) on day 1. Scale bar = 500 μm. D Quantification of relative fluorescence intensity of irg-1p::GFP reporter worms under vehicle or amonafide (50 μM and 100 μM) on day 1. E Boxplot of gene expression of hsp-16.2 under vehicle or amonafide. **** represents a two-tailed t-test p-value < 0.0001. F Heatmap of key effectors of UPR.mt in worms treated with vehicle or amonafide. All presented genes were significantly differentially expressed, adjusted p-value less than 0.05. G Representative fluorescence images of hsp-6p::GFP reporter worms under vehicle or amonafide (50 μM and 100 μM) on day 1. Scale bar = 500 μm. H Quantification of relative fluorescence intensity of hsp-6p::GFP reporter worms under vehicle or amonafide (50 μM and 100 μM) on day 1. D, H Each dot represents a biological replicate. The statistical analysis was performed using a two-tailed t-test. **** represents p-value < 0.0001, *** represents p-value < 0.001, and ** represents p-value < 0.01

First, we investigated the expression of p38 MAPK pathway, observing that expression of key components in the p38 MAPK pathway (pmk-1, nsy-1, and sek-1) under amonafide treatment remained comparable between amonafide treatment and the vehicle (S1F Fig). Next, we explored differentially expressed genes related to the skn-1-mediated oxidative stress response. Notably, the expression of key effectors of skn-1, such as gcs-1, gst-4, gst-5, and gst-7, was significantly upregulated following amonafide treatment (Fig. 4B). A similar pattern was also observed in several daf-16 targeted genes, including sod-3 and mtl-1 [45] (S1G Fig). Proceeding to evaluate the expression of the zip-2-mediated infection defense response, we turned to examine the infection response gene irg-1. This gene serves as a reporter for zip-2 activity, being activated by P. aeruginosa infection and cadmium poisoning in a zip-2-dependent manner [42]. The irg-1::GFP worms are a commonly used fluorescent reporter strain [42]. We further observed that the expression of irg-1::GFP significantly increased in a dose-dependent manner under amonafide treatment (Fig. 4C, D). We then evaluated the chaperone genes including hsp-70, hsp-16.2, and hsp-12.3 that are activated through a variety of stress responses including the atf-4-mediated ISR [43, 46]. Upon treatment with amonafide, we observed an increase in hsp-16.2 levels, while the expression of hsp-70 and hsp-12.3 decreased (Fig. 4E, S1H Fig). Finally, to assess the UPRmt, we investigated the levels of UPRmt-related heat shock proteins 6 and 60 (hsp-6 and hsp-60) and other UPRmt targets, which exhibited a significant increase upon amonafide treatment (Fig. 4F). Further evaluation using an hsp-6::GFP tagged reporter revealed an activation of UPRmt in a dose-dependent manner following amonafide treatment (Fig. 4G, H). Taken together, amonafide not only activated the mRNA expression of pathways associated with mitochondria-, pathogen-, and xenobiotic-associated defense response but also elevated the protein level of effectors of zip-2-mediated pathogen defense and the UPRmt.

atfs-1 is required for healthspan and lifespan improvement by amonafide

Given the observed activation of defense responses induced by amonafide, we next asked if these were required for the healthspan or lifespan benefits. To do this, we assessed the mobility of worm mutant strains in which defense responses were blocked, with and without amonafide treatment. These included administering amonafide to worms with mutations in skn-1, atf-4, atfs-1, and zip-2. Specifically, a dose of 50 μM was used for all mutant lifespan assays. This dose was chosen because it was the lowest dose that demonstrated a lifespan benefit in N2 worms, minimizing potential toxic effects while maximizing the likelihood of observing lifespan extension in mutants. Notably, amonafide exhibited a significant increase in the mobility of skn-1(mg570) worms (Fig. 5A). However, the mobility of zip-2(ok3730), atf-4(ok576), and atfs-1(gk3094) worms remained similar in both control and amonafide treatment (Fig. 5B–D). This demonstrated that healthspan benefits of amonafide were dependent on zip-2, atf-4, and atfs-1.

Fig. 5

atfs-1 is required in mediating healthspan improvement and lifespan extension promoted by amonafide. A–D Violin plot representing the mobility of different defense response deficiency worms including skn-1(mg570), atf-4(ok576), zip-2(ok3730), and atfs-1(gk3094) under treatment of vehicle or 50 μM amonafide. Y-axis shows the average moving speed of C. elegans. The bar in the center of the violin plot represents the median value of mobility. The statistical analysis was performed using a wilcox.test. *** represents p-value < 0.001, * represents p-value < 0.05, and ns represents not significant. E Lifespan curves of skn-1(mg570) treated with vehicle or 50 μM amonafide. F Lifespan curves of atf-4(ok576) treated with vehicle or 50 μM amonafide. G Lifespan curves of zip-2(ok3730) treated with vehicle or 50 μM amonafide. H Lifespan curves of atfs-1(gk3094) treated with vehicle or 50 μM amonafide. E–H p-value represents the comparison with the controls calculated using log-rank test. I–J The violin plot displays the thrashing frequency and crawling speed of control worms (UM9) and PD worms (Parkinson’s disease worm, UM10) under vehicle (DMSO) or amonafide 50-μM treatment. The statistical analysis was performed using a two-tailed t-test. ** represents p-value < 0.01 and ns represents not significant. K The diagram illustrates the study’s findings: TOP II inhibitors and mild RNAi top-2 enhance mobility, showing promise for promoting longevity. Amonafide exhibits geroprotective effects, relying on zip-2, atf-4, and atfs-1 pathways for mobility and atfs-1 for lifespan

We next investigated if lifespan extension following amonafide treatment also depended on these same regulators. Consistent with the mobility assay, amonafide significantly increased the lifespan of skn-1(mg570) worms (Fig. 5E). The lifespan of zip-2(ok3730) and atf-4(ok576) worms also increased upon amonafide treatment, contrary to the findings that the mobility of these worm strains did not change with amonafide treatment (Fig. 5F, G). Noticeably, the lifespan of atfs-1(gk3094) worms did not exhibit significant changes with the treatment of amonafide, aligning with our observations in the healthspan assay, where the average mobility of atfs-1(gk3094) worms remained similar between amonafide treatment and control conditions (Fig. 5D, H). This demonstrated that lifespan extension resulting from amonafide treatment was dependent on atfs-1.

Due to the essential role of the UPRmt activator atfs-1 in mediating both healthspan and lifespan effects in amonafide treatment, we next aimed to investigate the therapeutic potential of amonafide on a disease model where activation of the UPRmt can play a protective role, namely neurodegeneration [47]. Specifically, we looked at Parkinson’s disease (PD), the second most prevalent age-related neurodegenerative disorder, with aging being the foremost risk factor for the development of idiopathic PD [48]. To evaluate the potential of amonafide in alleviating this age-related disease, we assessed its effects in a PD worm model. Using the UM10 worm model with pathological α-synuclein accumulation [49], we measured thrashing and crawling behavior to represent the disease state. Remarkably, we found that the thrashing frequency