Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Amyloid β induces hormetic-like effects through major stress pathways in a C. elegans model of Alzheimer’s Disease

  • James D. Lichty,

    Roles Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing

    Affiliation Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, United States of America

    ⨯
  • Hrishikesh Mane,

    Roles Investigation, Visualization

    Affiliation Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, United States of America

    ⨯
  • Victoria R. Yarmey,

    Roles Investigation, Visualization, Writing – review & editing

    Affiliations Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, United States of America, Joint Department of Biomedical Engineering, North Carolina State University, University of North Carolina at Chapel Hill, Raleigh, North Carolina, United States of America

    ⨯
  • Adriana San Miguel

    Roles Conceptualization, Funding acquisition, Investigation, Project administration, Supervision, Visualization, Writing – review & editing

    asanmig@ncsu.edu

    Affiliation Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, United States of America

    ⨯

Abstract

Amyloid β (Aβ) is a peptide known for its characteristic aggregates in Alzheimer’s Disease and its ability to induce a wide range of detrimental effects in various model systems. However, Aβ has also been shown to induce some beneficial effects, such as antimicrobial properties against pathogens. In this work, we explore the influence of Aβ in stress resistance in a C. elegans model of Alzheimer’s Disease. We found that C. elegans that express human Aβ exhibit increased resistance to heat and anoxia, but not to oxidative stress. This beneficial effect of Aβ was driven from Aβ in neurons, where the level of induction of Aβ expression correlated with stress resistance levels. Transcriptomic analysis revealed that this selective stress resistance was mediated by the Heat Shock Protein (HSPs) family of genes. Furthermore, neuropeptide signaling was necessary for Aβ to induce stress resistance, suggesting neuroendocrine signaling plays a major role in activating organismal stress response pathways. These results highlight the potential beneficial role of Aβ in cellular function, as well as its complex effects on cellular and organismal physiology that must be considered when using C. elegans as a model for Alzheimer’s Disease.

Citation: Lichty JD, Mane H, Yarmey VR, Miguel AS (2025) Amyloid β induces hormetic-like effects through major stress pathways in a C. elegans model of Alzheimer’s Disease. PLoS ONE 20(4): e0315810. https://doi.org/10.1371/journal.pone.0315810

Editor: Nektarios Tavernarakis, Foundation for Research and Technology-Hellas, GREECE

Received: August 30, 2024; Accepted: December 2, 2024; Published: April 24, 2025

Copyright: © 2025 Lichty et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: Data is available in a dryad repository, link: https://doi.org/10.5061/dryad.p5hqbzkzk

Funding: This work was supported by the following grants: 1) Department of Defense W81XWH-18-1-0701 (ASM); 2) National Institutes of Health R00AG046911 (ASM); 3) National Science Foundation 2039226 (ASM); and 4) some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist

Introduction

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that causes loss of cognitive function in patients, typically starting at an advanced age. AD is characterized by several features: the formation of amyloid β (Aβ) aggregates and micro-tubule associated protein tau (tau) tangles, immune activation, inflammation, oxidative stress, and neuron loss [1]. In addition to the plaques and aggregates observed in the brain of AD patients, mutations in Amyloid Precursor Protein (APP) that lead to altered levels of Aβ are associated with familial AD, while Aβ expression also induces AD-like symptoms in cell and animal models [2–5]. These lines of evidence have made Aβ a priority target in AD research. Interestingly, some studies have also indicated that Aβ can potentially act as an antimicrobial peptide, suggesting a protective role for Aβ [6,7]. Extensive work seeking to identify how Aβ becomes toxic in the brain has pointed to certain Aβ isoforms, small oligomer intermediates, and Aβ localization as key determinants in toxicity [8–10]. Despite these discoveries, the role of Aβ in AD is still unclear, creating a need for well-characterized, robust models.

The nematode C. elegans has been previously used as a model for AD [11–13]. C. elegans boasts many benefits as a model for neurodegenerative diseases: it is transparent, allowing for in vivo imaging; has high homology with the human genome and proteome, including several stress-related pathways; and shares many neuron subtypes with humans [14]. Current models of AD in C. elegans focus on intracellular expression of human Aβ either pan-neuronally or pan-muscularly [11–13]. These models exhibit several pathological features such as severe paralysis, behavioral and motor defects, and reduced lifespan [11–13]. Aβ expression in worms has also been linked to loss of function and stability in mitochondria, leading to oxidative and metabolic stress [15]. Conversely, worms expressing Aβ have also been shown to exhibit increased pathogen resistance, indicating a possible beneficial effect against environmental stressors [6]. Aβ shares some characteristics and behaviors with antimicrobial peptides, such as pathogen cell surface adhesion and pore formation, which may be the cause of this interaction [6,7]. The relationships between Aβ and other environmental insults have yet to be examined despite several stressors, like oxidative stress, being associated with higher rates of AD [16,17]. Investigating these interactions may improve our understanding of the role of Aβ in AD.

There is significant overlap between stress pathways in animals, including C. elegans and humans. The C. elegans gene daf-16, an ortholog for the human forkhead transcription factor (FOXO), has been highlighted for its ability to extend lifespan and increase stress resistance [18]. Another C. elegans gene, hsf-1, is involved in the heat stress response and is an ortholog for the human HSF1 [19]. The hif-1 gene (ortholog of human HIF1A) regulates many hypoxia response genes, including vhl-1 and egl-9 [20]. The skn-1 gene acts as a master regulator of the oxidative stress response in worms and its human orthologs, the Nrf/CNC proteins, are also regulators of oxidative stress [21]. All four of these transcription factor pathways have important roles in aging and aging-related diseases in humans and have been implicated in AD [22–25]. Due to the substantial overlap between human and C. elegans stress response pathways, analysis of interactions between stress and Aβ in C. elegans can shed light on the effects of Aβ in humans.

In this work, we used several C. elegans models of AD to examine whether interactions exist between environmental stressors and the effects of Aβ expression and identified underlying pathways behind any changes in Aβ-driven resistance to stress. We assessed the resistance of Aβ-expressing worms to oxidative stress, heat stress, and anoxia. Our results indicate that Aβ expression selectively increases resistance to heat stress and anoxia while reducing resistance to oxidative stress. Despite this, Aβ’s ability to increase heat and anoxia resistance do not appear to be driven by reactive oxygen species (ROS) hormesis, as antioxidant supplementation with n-acetyl cysteine (NAC) did not affect stress resistance. Additionally, we found that neuronal expression of Aβ affected stress resistance in a dose-dependent manner. Using nCounter gene expression analysis, we identified several stress genes upregulated by Aβ in both unstressed and stressed worms. Several of these genes act downstream of the major stress response pathways modulated by daf-16, hif-1, hsf-1, and skn-1, suggesting Aβ activates these transcription factors either directly or by inducing cellular stress. Since stress response genes are mainly activated in the intestine, we sought to determine how neuronal Aβ was influencing other tissues to induce stress resistance. Using RNAi to knockdown two well-known genes required for neuropeptide signaling, unc-31 and egl-3 [26,27], stress resistance returned to wild-type (WT) levels, pointing to neuropeptide signaling as the source of communication from neurons to other tissues for Aβ-induced stress resistance. These factors taken together highlight the complex effects of Aβ in cells and at the organismal level. Aβ expression has some beneficial effect for the animals in some contexts and thus results of studies of Aβ in C. elegans should consider possible interactions with environmental conditions. These results suggest that Aβ may play a substantial role in stress response, either through directly activating stress pathways or acting as a hormetic stressor.

Materials and methods

Strains

The following strains were used in this study: N2 Bristol (wild-type); JKM2 (Is [rgef-1p::Signalpeptide-Abeta(1−42)::hsp-3(IRES)::wrmScarlet-Abeta(1−42)::unc-54(3’UTR) + rps-0p::HygroR]); JKM3 (Is [rgef-1p::wrmScarlet::unc-54(3’UTR) + rps-0p::HygroR]); CL2355 (dvIs50 [pCL45 (snb-1::Abeta 1–42::3’ UTR(long) + mtl-2::GFP] I); CL4176 (dvIs27 [myo-3p::A-Beta (1–42)::let-851 3’UTR) + rol-6(su1006)] X); OH438 (otIs117 [unc-33p::GFP + unc-4(+)]); NL2099 (rrf-3(pk1426) II); ASM35: cross of JKM2 x NL2099; MF190 (hmIs4 [des-2::GFP + rol-6(su1006)]).

C. elegans growth and maintenance

Nematodes were grown and maintained at 20 °C on nematode growth medium (NGM) seeded with E. coli OP50 as a food source, unless otherwise specified, according to standard protocols [28]. Age-synchronization for experiments was performed by washing plates with 1mL of a solution of M9 media supplemented with 0.01% v/v TX-100 (M9TX). Worms were allowed to settle, and supernatant was removed and replaced with 1mL of 1:2:1 mixture of bleach, 1M NaOH, and water. Once eggs were released, they were washed 3 times with 1mL M9TX, and transferred to fresh plates.

C. elegans crossing

The JKM2 strain naturally produces a higher proportion of males than N2, so no extra procedures were needed to obtain males for the cross. Young adult NL2099 hermaphrodites were isolated onto a plate with JKM2 males in a 1:10 hermaphrodite to male ratio. After several days, single F1 offspring were isolated and allowed to self-fertilize. The F2 progeny was then analyzed to check for homozygosity in the reporter and mutation. Presence of the JKM2 transgene was checked using fluorescence microscopy. To check for the mutation, genomic DNA was collected from at least 100 worms using standard methods. PCR was performed using primers flanking the rrf-3(pk1426) deletion site obtained from the Wormbase online resource. The pk1426 deletion was identified through gel electrophoresis and Sanger sequencing.

Oxidative stress assay

Animals were age-synchronized by bleaching and cultured to the L4 stage at 20 °C. After another 24 hrs. of growth, approximately 30 worms were picked to a fresh plate containing 50mM paraquat (added during plate production after media was cooled to 50 °C) per cohort. These plates were transferred to a 20 °C incubator for 24 hrs.. then scored for survival by prodding with a platinum wire worm pick to check for movement, where animals that did not respond were marked as dead.

Heat stress assay

Animals were age-synchronized by bleaching and cultured to the L4 stage at 20 °C. After another 24 hrs. of growth, approximately 30 worms were picked to a fresh plate per cohort at room temperature (20 °C). These were transferred to an incubator set at 37 °C for the specified time (4 hrs. or 5.5 hrs.), before being returned to the 20 °C growth incubator for overnight recovery. For the 25 °C upshift experiments, these plates were instead first transferred to a 25 °C incubator for the specified time before the 37 °C stress. Animals were scored for survival by prodding with a platinum wire worm pick to check for movement, where animals that did not respond were marked as dead.

Anoxia assay

Animals were age-synchronized by bleaching and cultured to the L4 stage at 20 °C. After another 24 hrs. of growth, approximately 30 worms were picked to a fresh plate per cohort. These plates were put in a GasPak chamber with 3 satchels to lower the oxygen concentration to below 0.1%. After 48 hrs., the chamber was opened and the worms were allowed to recover overnight before scoring. Animals were scored for survival by prodding with a platinum wire worm pick to check for movement, where animals that did not respond were marked as dead.

RNA extraction and gene expression profiling

Animals were age-synchronized by bleaching and cultured to the L4 stage at 20 °C. After another 24 hrs. of growth, worms were split into three treatment groups: unstressed, heat stressed, or anoxia stressed. Unstressed worms were allowed to grow for another 24 hrs. before RNA was extracted. Heat stressed worms were stressed the next day at 37 °C for 2.5 hrs. Anoxia stressed worms were stressed for 24 hrs. as described above. Following treatment, RNA was extracted according to the Direct-zol RNA Miniprep extraction kit (Cat# R2051 Zymo Research). During the TRI Reagent step, a motorized pestle was used for 1 min to help break the worm cuticle. RNA samples were then provided to the UNC Respiratory TRACTS Core, who performed the NanoString analysis (nCounter® Elements). For each combination of strain and condition, three biological replicates were pooled together.

RNAi plate preparation and suppression assay

Bacteria and plates were prepared according to a modified protocol from Timmons et al., [29]. In summary, bacteria was cultured from the Ahringer RNAi Library [30] in fresh LB media supplemented with 50 µg/mL ampicillin and 12.5 µg/mL tetracycline. After overnight growth (16–18 hrs.), the culture was centrifuged and resuspended in fresh LB media supplemented with 50 µg/mL ampicillin and 1mM Isopropyl-β-d-thiogalactopyranoside (IPTG) to OD600=0.5. After another 4 hrs. of growth, bacteria was seeded onto NGM plates supplemented with 50 µg/mL ampicillin and 1mM IPTG. Animals were age-synchronized by bleaching and cultured to the L4 stage at 20 °C on plates containing the relevant RNAi E. coli strain (control, unc-31, egl-3). After another 24 hrs. of growth, approximately 30 worms were picked to a fresh plate per cohort containing their respective RNAi E. coli strain. The heat stress assay was then performed as described above.

Statistical analysis

Statistical analysis (ANOVA and t-test) was performed using MATLAB. P-values were considered significant when: p<0.05(*), p<0.01(**), and p<0.001(***).

Results and discussion

Aβ induces selective stress resistance in C. elegans

To determine whether Aβ modulates responses to environmental stress, we analyzed the resistance of worms expressing Aβ to various stressors. We chose three strains for exposure: a wildtype N2 (WT); JKM2 (Aβ+), which co-expresses Aβ and Aβ fused to wrmScarlet pan-neuronally; and JKM3 (Aβ-), which expresses wrmScarlet pan-neuronally [11]. The Aβ+ strain carries a construct where Aβ is driven by a pan-neuronal promoter while Aβ::wrmScarlet is driven by an IRES (internal ribosome entry site) and thus the tagged Aβ is expressed at a lower level to limit wrmScarlet’s impact on Aβ aggregation [11]. Notably, the JKM3 stain expresses wrmScarlet under the pan-neuronal promoter rgef-1p and is thus expected to have higher levels of the fluorescent protein than JKM2. To assess worms’ resistance to oxidative stress, we exposed them to a 50 mM paraquat solution for 24 hrs. (Fig 1A). As expected, this toxic dose of paraquat [31] resulted in reduced survival in the Aβ+ strain (37%) as compared to the N2 wild-type (92%) and the Aβ- strain (69%) (Fig 1B). Aβ overexpression leads to elevated levels of oxidative stress [12,32], possibly increasing their susceptibility to external oxidative stress and thus reduced resistance to paraquat. The Aβ- strain exhibited a less severe decrease in survival, which could stem from the presence of foreign wrmScarlet. As mentioned above, the Aβ+ strain also expresses Aβ-wrmScarlet, but at a sub-stoichiometric ratio, and thus free wrmScarlet abundance is expected to be significantly higher in the Aβ- than the Aβ-wrmScarlet fusion in the Aβ+ strain. Moreover, this effect does not completely account for the discrepancy in survival between the two strains.

thumbnail
Download:
Fig 1. Aβ provides selective resistance to severe heat and hypoxic stress, but not paraquat-induced oxidative stress.

(A) Overview of experimental setup. Animals were age-synchronized to L4 larvae and then allowed to grow for another 24 hrs. before exposure to stressors. (B) Survival rate after 24 hrs. exposure to 50mM paraquat stress. (C) Survival rate after 4 hrs. 37 °C heat stress exposure and overnight recovery at 20 °C. (D) Survival rate after 48 hrs. anoxia (< 0.1% O2) exposure and overnight recovery. WT is wild-type N2 Bristol, Aβ+ is JKM2, Aβ- is JKM3. N = 9 (12 for anoxia stress) replicates per strain. Each dot represents a replicate of approx. 30 worms. Statistical analysis was performed using ANOVA. * is p-value < 0.05, ** is p-value < 0.01, *** is p-value < 0.005.

https://doi.org/10.1371/journal.pone.0315810.g001

We next expanded our assay to include two additional common environmental stressors: heat stress and anoxia. We exposed the worms to a severe heat stress of 37 °C for 4 hrs. and then allowed them to recover overnight at 20 °C (Fig 1A). Unexpectedly, the Aβ+ strain exhibited increased heat stress resistance, surviving at an average rate of 90%, compared to 42% and 62% in WT and Aβ-, respectively (Fig 1C). Similarly, when exposed to anoxic conditions (<0.1% O2) for 48 hrs. followed by overnight recovery (Fig 1A), the Aβ+ strain had increased anoxia resistance, with an average survival rate of 82%, compared to 22% and 65% in WT and Aβ-, respectively (Fig 1D). We attributed this increased resistance to a possible hormesis-like effect. Notably, the Aβ+ strain does not display increased lifespan, which is often a hallmark of hormesis [11]. The Aβ- control strain also demonstrated a higher tolerance to these two stressors when compared to WT, although not as high as the Aβ+ strain. This effect could stem from the wrmScarlet protein, as in the oxidative stress assay (Fig 1B). However, the substantial survival difference between the Aβ+ and the Aβ- indicate that wrmScarlet alone is not driving these differences. Aβ and wrmScarlet could induce similar protective effects, but the Aβ- strain lacks several other physiological deficits shown in the Aβ+ strain such as decreased lifespan, brood size, and locomotion [11].

Aβ-induced stress resistance is dependent on Aβ levels and localization

To validate that Aβ induces heat stress resistance, we tested the effects of increasing doses of Aβ in strain CL2355, referred to as the nAβ strain here. The nAβ strain expresses Aβ pan-neuronally upon upshift to 25 °C due to a smg-1 temperature-sensitive mutation in the nonsense-mediated decay pathway [13]. In strain CL4176, which uses the same system for Aβ expression in muscle, Aβ abundance is influenced by the time spent at 25 °C [12]. We upshifted the nAβ strain to 25 °C for increasing amounts of time and then exposed it to heat stress (37 °C for 5.5 hrs.) followed by overnight recovery at 20 °C (Fig 2A-B) to test heat stress resistance. To account for hormetic effects of exposure to 25 °C, we also upshifted the WT strain for equivalent amounts of time. The nAβ strain exhibited increased heat stress resistance when compared to the WT strain, which increased with time at 25 °C. Notably, the 12 hr. timepoint showed the greatest difference in resistance between strains, with the nAβ strain exhibiting an average survival rate of 60% compared to the WT strain’s 33% average survival rate. We also noted that the upshift to 25 °C produces an increase in heat stress resistance in the WT strain, which results in insignificant survival rate differences between strains at 24 and 48 hrs. The trend of 25 °C-induced heat stress resistance in wild-type animals is consistent with prior reports [33,34]. Despite this effect, our results suggest that Aβ expression induces an additional protective effect, since a significant difference in heat stress survival is observed between the two strains at 12 hrs.

thumbnail
Download:
Fig 2. Aβ-induced heat stress resistance is dependent on Aβ expression levels and neuronal localization.

(A) Overview of experimental setup. Temperature upshift to 25 °C was staggered such that stress exposure occurred at the same time for each condition. (B) Survival rate after 5.5 hr. at 37 °C heat stress exposure and overnight recovery at 20 °C. Time at 25 °C correlates to levels of Aβ in the neurons in nAβ strain. (C) Survival rate after 5.5 hr. 37 °C heat stress exposure and overnight recovery at 20 °C. Time at 25 °C correlates to levels of Aβ in the neurons in nAβ strain and in muscles in mAβ strain. In C, statistical values represent whether each strain is affected by temperature, comparisons between strains are not made. WT is wild-type N2 Bristol, nAβ is CL2355, mAβ is CL4176. N=9 replicates per condition. Each dot represents a replicate of approx. 30 worms. Statistical analysis was performed using two-sample t-test and two-way ANOVA. * is p-value < 0.05, ** is p-value < 0.01, *** is p-value < 0.005.

https://doi.org/10.1371/journal.pone.0315810.g002

Since both the Aβ+ and the nAβ strains express Aβ in neurons, we next tested whether the increased stress resistance is specific to Aβ expression in this tissue. We expanded the heat stress assay to include strain CL4176 (referred to as mAβ) (Fig 2C). The mAβ strain also expresses Aβ upon upshift to 25 °C [12], but in muscles instead of neurons. Rather than showing a dose-dependent heat stress resistance as we expected, the mAβ strain maintained a survival rate of approximately 64% regardless of the time spent at 25 °C (Fig 2C). One possible explanation for this result is that this strain exhibits leaky Aβ expression even at low temperatures (Wormbase), which might be sufficient to induce a hormetic effect. While the nAβ strain contains a fluorescence marker, the mAβ has a rol-6(su1006) marker that deforms the cuticle and results in a roller phenotype [35], which could also explain the enhanced heat stress resistance. To test this hypothesis, we measured heat stress resistance in strain MF190, which carries the same roller mutation, but does not express Aβ. Our results indicate that the rol-6 mutation leads to reduced heat stress resistance, and thus we conclude that the increased heat stress resistance in the mAβ strain is not explained by the cuticle defect alone (S1 Fig). These results indicate that Aβ induces hormetic effects when expressed in neurons, while the potential influence of Aβ in muscles is less clear. Thus, we opted to further explore how neuronal Aβ drives increased stress resistance.

Foreign protein expression only partially accounts for increased stress resistance

As noted in our initial stress assays, wrmScarlet in the Aβ- strain induces some level of stress resistance, suggesting that the effects observed are not specific to Aβ, but that expression of any foreign protein might lead to activation of pathways that result in increased stress resistance. To test if foreign protein expression drives this effect, we tested strain OH435, which expresses GFP pan-neuronally (referred to as nGFP) (Fig 3A) [36]. The nGFP strain exhibited increased heat stress resistance similar to the Aβ- strain, with an average survival rate of 53%. This result strengthens the idea that the hormesis-like effect stems at least partially from foreign protein expression in the neurons. Yet, the Aβ+ strain had significantly higher survival than the Aβ- strain (Fig 1), implying that while the protective effects are not attributable to Aβ alone, Aβ induces a substantially stronger protective effects against stress.

thumbnail
Download:
Fig 3. Heat stress resistance is partially replicated with other neuronally expressed proteins and is not eliminated with NAC treatment.

(A) Survival rate after 4 hr. 37 °C heat stress exposure and overnight 20 °C recovery in pan-neuronal GFP strain. (B) Survival rate after 4 hr. 37 °C heat stress exposure and overnight recovery in worms grown on control and 5 mM NAC plates for 24 hrs. before heat stress exposure. WT is wild-type N2 Bristol, nGFP is OH438, Aβ+ is JKM2, Aβ- is JKM3. N=9 replicates per condition. Each dot represents a replicate of approx. 30 worms. Statistical analysis was performed using two-sample t-test and two-way ANOVA. * is p-value < 0.05, ** is p-value < 0.01, *** is p-value < 0.005.

https://doi.org/10.1371/journal.pone.0315810.g003

Antioxidant exposure does not suppress the protective effect of Aβ

Since Aβ has been shown to induce oxidative stress in C. elegans [32], we hypothesized that the protective effect induced by Aβ could be the result of oxidative stress-driven hormesis [37,38]. We reasoned that if Aβ drives increased heat stress resistance due to an increased ROS load, inhibiting ROS accumulation should suppress the protective effect of Aβ. To test this theory, we exposed worms to 5 mM NAC for 24 hrs., which has been shown to reduce oxidative stress in C. elegans [39,40]. After NAC exposure, animals were heat stressed at 37 °C for 4 hrs. and allowed to recover overnight. NAC had an insignificant effect on the Aβ+ and Aβ- strains, which can indicate that reducing oxidative stress does not suppress the protective effects of Aβ+. In the presence of NAC, the Aβ+ strain still exhibits higher resistance to heat than the WT strain. This suggests that Aβ-induced heat stress resistance is independent of any NAC-induced effects. Notably, NAC supplementation also increased WT resistance (Fig 3B), recapitulating previous findings [39,40]. However, the beneficial effects of NAC on the WT strain are not hormesis-dependent. Taken together, these results suggest that while Aβ suppresses the protective effect of NAC on stress resistance, the specific mechanism by which Aβ drives stress resistance is independent of oxidative stress. It is also possible that the NAC dosage used, while sufficient for WT, does not sufficiently counteract the natively high ROS load in the Aβ+ strain.

Aβ upregulates several stress response pathways

To elucidate how Aβ induces stress resistance, we probed the expression level of several stress resistance pathways using NanoString nCounter analysis [41]. We used a predefined panel of targets that included several genes modulated by daf-16, hsf-1, hif-1, and skn-1; central regulators of the main stress response pathways. RNA samples from 3 biological replicates of each of the WT, Aβ+ and Aβ- strains were pooled for unstressed, heat stressed (2.5 hrs. at 37 °C), and anoxic (<0.1% O2 for 24 hrs.) conditions (Fig 4A-C). This analysis revealed several stress resistance genes that were upregulated in the Aβ+ strain in both unstressed and stressed conditions, including several HSPs. HSPs are associated with protein misfolding and molecular chaperones involved in the stress response, specifically heat stress, anoxia, and oxidative stress [19,42]. During stress, HSPs are typically upregulated in the neurons and intestine as a part of the stress response [43]. Aβ+ worms exhibited elevated levels of four HSPs under all conditions: hsp-16.1, hsp-16.2, hsp-16.49, and hsp-70 (Fig 4A-C). This may indicate why Aβ induces more stress resistance than wrmScarlet, as the Aβ- strain only showed increased expression of these genes under anoxia (Fig 4C). The HSPs are co-regulated by the daf-16, hsf-1, hif-1, and skn-1 transcription factors [19,44–46], making it unclear which pathway or pathways Aβ is inducing. Surprisingly, Aβ expression does not upregulate any of the tested hypoxia or autophagy genes when compared to the controls, even under stressed conditions, suggesting the increased resistance is mediated mainly by the HSPs. Some of the main oxidative stress response genes, sod-3 and gst-4 [47,48], are slightly upregulated despite the Aβ+ strain exhibiting lowered oxidative stress resistance, which is likely explained by Aβ-induced oxidative stress. Interestingly, the ttr-33 gene, another oxidative stress response gene responsible for paraquat and hydrogen peroxide resistance [49], is highly upregulated in unstressed worms, but this does not translate to increased paraquat resistance when exposed (Fig 1B). Overall, this gene expression analysis suggests that Aβ induces stress resistance through upregulation of HSPs.

thumbnail
Download:
Fig 4. nCounter expression analysis indicates several resistance genes upregulated in both stressed and unstressed worms and RNAi suppression suggests resistance is communicated through neuropeptide signaling. (A-C) nCounter analysis on unstressed (A), heat stressed (B), and hypoxic stressed (C) worms. Genes are grouped by type and box color indicates expression changes. 3 biological replicates were pooled per sample. (D) Survival rate after 4 hr. 37 °C heat stress exposure in RNAi suppressed worms. RNAi control was HT115 with empty vector. WT is wild-type N2 Bristol, Aβ+ is JKM2, Aβ- is JKM3, rrf-3(-) is RNAi-sensitive strain NL2099, Aβ+rrf-3(-) is ASM35, a cross between JKM2 and NL2099. N=9 replicates per condition. Each dot represents a replicate of approx. 30 worms. Statistical analysis was performed using two-sample t-test and two-way ANOVA. * is p-value < 0.05, ** is p-value < 0.01, *** is p-value < 0.005.

https://doi.org/10.1371/journal.pone.0315810.g004

Aβ-induced stress resistance requires neuropeptide signaling

Stress resistance in C. elegans is a cell nonautonomous response mediated by communication between several tissues, most commonly the intestine and neurons [43,50–52]. Stress leads to upregulation of transcription factors like hif-1 in neurons, which can activate serotonin signaling and other pathways in a cell nonautonomous manner [19,43,50–52]. Neuroendocrine communication to the intestine through neuropeptide signaling can drive stress resistance through the activation of transcription factors in the intestine like daf-16, skn-1, and hsf-1 [19,43,50–52]. The intestine can also act as a sensor for stress, such as detecting dietary restriction through food intake and daf-16 activation, oxidative stress through skn-1 activation, and heat stress through hsf-1 activation [19,43,50–52]. The intestine can then communicate these signals back to the neurons to trigger an organism-level stress response [19,43,50–52]. Therefore, we next focused on whether neuronal Aβ could activate stress resistance pathways in the intestine through neuroendocrine communication. It has been previously shown that Aβ can spread from neurons to other tissues in aged C. elegans [11]. In our experiments, we evaluated young worms, before the age where Aβ dispersal has been identified [11], thus ensuring Aβ is restricted to neurons. To understand how neuronal Aβ influences organism-level stress resistance, we assessed whether disrupting neuropeptide signaling between the neurons and other tissues had an effect on stress resistance. To knockdown neuropeptide signaling, we used RNAi by feeding [29], targeting two key neuropeptide signaling genes, unc-31 and egl-3. unc-31 regulates neuropeptide release from dense core vesicles [26] and egl-3 is necessary for neuropeptide maturation [27]; knocking out either severely limits neuropeptide signaling. To ensure silencing was effective in neurons, we used an RNAi sensitive strain, NL2099 (rrf-3(-)) [53], as our control. This strain was crossed with the Aβ+ strain to generate ASM35 (Aβ+;rrf-3(-)). Upon knockdown of either gene (Fig 4D), Aβ-induced stress resistance in Aβ+;rrf-3(-) worms dropped to similar levels as rrf-3(-). This result suggests that Aβ in neurons upregulates stress resistance pathways in other tissues through neuropeptide signaling, thus driving increased organismal resistance to stress. It is unclear if Aβ is directly initiating neuropeptide signaling, or if Aβ induces neuronal stress that may be responsible for the activation of the stress response at the organismal level.

Conclusions

In summary, this study found that Aβ induces resistance to heat and anoxia but not oxidative stress by activating key stress response genes. These genes include several members of the HSP family, which were significantly induced in the Aβ-expressing strain under both unstressed and stressed conditions. In neurons, Aβ abundance appears to influence the levels of stress resistance induced. While strains expressing fluorescent proteins in neurons also exhibited increased resistance to heat, the effects from Aβ are significantly stronger. Thus, while foreign protein expression modulates stress resistance pathways, Aβ drives this effect more strongly. It is unclear if protein aggregation plays a role in this protective effect, as both Aβ and wrmScarlet-tagged Aβ aggregate [11], but aggregation levels were not assessed in this work. Gene expression analysis indicates that Aβ activates some combination of the daf-16, hsf-1, hif-1, and skn-1 pathways to induce stress resistance, but further work is needed to determine if all four pathways participate in this effect. The HSP family of genes falls under numerous transcriptional regulators, and expanding the breadth of genes covered by this kind of screen may provide more insight into which pathways are activated. RNAi knockdown of unc-31 and egl-3 shows that to induce stress resistance, Aβ activates neuropeptide signaling to communicate with other tissues. More work is needed to examine exactly how Aβ activates neuropeptide signaling, whether that be directly or indirectly by inducing cellular stress. This study highlights the need to more fully characterize the effect of Aβ on C. elegans, and to consider its complex organismal effects when using it as a model for AD. If the increased stress resistance is not consistent across species, there may be more limitations of C. elegans as a model for AD and more factors to consider before its use as such. Knowing these limitations and interactions will make interpretations of results more meaningful. The results of this work also imply that Aβ may have beneficial effects for the host outside of AD. The role of Aβ in healthy humans is still unclear, but several theories regarding its function include pathogen defense, injury recovery, and regulation of synaptic function [54]. Inducing stress resistance pathways and acting as an early signal for stress may be another role it plays in human biology. It will be crucial to further characterize the role of Aβ in C. elegans and other model organisms to better understand the mechanisms by which Aβ induces physiological changes in AD and other human diseases.

Supporting information

S1 Fig. Animals carrying a transgene with rol-6(su1006) exhibit reduced resistance to heat stress.

Survival rate after 4 hrs. at 37 °C heat stress exposure and overnight recovery at 20 °C. MF190 non-rollers do not exhibit a roller phenotype, while MF190 exhibit a roller phenotype. MF190 drives GFP in the PVD neuron by the des-2 promoter, and also contains a rol-6(su-1006) rescue.

https://doi.org/10.1371/journal.pone.0315810.s001

(TIF)

Acknowledgments

Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We thank Janine Kirstein for sharing strains JKM2 and JKM3 and for useful insights on strain culture and handling.

References

  1. 1. Mucke L. Neuroscience: alzheimer’s disease. Nature. 2009;461(7266):895–7. pmid:19829367
  2. 2. Alvarez J, Alvarez-Illera P, Santo-Domingo J, Fonteriz RI, Montero M. Modeling alzheimer’s disease in caenorhabditis elegans. Biomedicines. 2022;10(2):288. pmid:35203497
  3. 3. Blanchard JW, Victor MB, Tsai L-H. Dissecting the complexities of Alzheimer disease with in vitro models of the human brain. Nat Rev Neurol. 2022;18(1):25–39. pmid:34750588
  4. 4. De Jonghe C, Esselens C, Kumar-Singh S, Craessaerts K, Serneels S, Checler F, et al. Pathogenic APP mutations near the gamma-secretase cleavage site differentially affect Abeta secretion and APP C-terminal fragment stability. Hum Mol Genet. 2001;10(16):1665–71. pmid:11487570
  5. 5. Yokoyama M, Kobayashi H, Tatsumi L, Tomita T. Mouse models of alzheimer’s disease. Front Mol Neurosci. 2022;15:912995. pmid:35799899
  6. 6. Kumar DKV, Choi SH, Washicosky KJ, Eimer WA, Tucker S, Ghofrani J, et al. Amyloid-β peptide protects against microbial infection in mouse and worm models of Alzheimer’s disease. Sci Transl Med. 2016;8(340):340ra72. pmid:27225182
  7. 7. Pastore A, Raimondi F, Rajendran L, Temussi PA. Why does the Aβ peptide of Alzheimer share structural similarity with antimicrobial peptides?. Commun Biol. 2020;3(1):135. pmid:32193491
  8. 8. Busch L, Eggert S, Endres K, Bufe B. The hidden role of non-canonical amyloid β isoforms in alzheimer’s disease. Cells. 2022;11(21):3421. pmid:36359817
  9. 9. Gallego Villarejo L, Bachmann L, Marks D, Brachthäuser M, Geidies A, Müller T. Role of intracellular amyloid β as pathway modulator, biomarker, and therapy target. Int J Mol Sci. 2022;23(9):4656. pmid:35563046
  10. 10. Rosenblum WI. Structure and location of amyloid beta peptide chains and arrays in Alzheimer’s disease: new findings require reevaluation of the amyloid hypothesis and of tests of the hypothesis. Neurobiol Aging. 2002;23(2):225–30. pmid:11804706
  11. 11. Gallrein C, Iburg M, Michelberger T, Koçak A, Puchkov D, Liu F, et al. Novel amyloid-beta pathology C. elegans model reveals distinct neurons as seeds of pathogenicity. Prog Neurobiol. 2021;198:101907. pmid:32926945
  12. 12. Link CD, Taft A, Kapulkin V, Duke K, Kim S, Fei Q, et al. Gene expression analysis in a transgenic Caenorhabditis elegans Alzheimer’s disease model. Neurobiol Aging. 2003;24(3):397–413. pmid:12600716
  13. 13. Wu Y, Wu Z, Butko P, Christen Y, Lambert MP, Klein WL, et al. Amyloid-beta-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans. J Neurosci. 2006;26(50):13102–13. pmid:17167099
  14. 14. Corsi AK, Wightman B, Chalfie M. A Transparent window into biology: a primer on Caenorhabditis elegans. WormBook. 2015:1–31. pmid:26087236
  15. 15. Teo E, Ravi S, Barardo D, Kim H-S, Fong S, Cazenave-Gassiot A, et al. Metabolic stress is a primary pathogenic event in transgenic Caenorhabditis elegans expressing pan-neuronal human amyloid beta. Elife. 2019;8:e50069. pmid:31610847
  16. 16. Bisht K, Sharma K, Tremblay M-È. Chronic stress as a risk factor for Alzheimer’s disease: roles of microglia-mediated synaptic remodeling, inflammation, and oxidative stress. Neurobiol Stress. 2018;9:9–21. pmid:29992181
  17. 17. Ionescu-Tucker A, Cotman CW. Emerging roles of oxidative stress in brain aging and Alzheimer’s disease. Neurobiol Aging. 2021;107:86–95. pmid:34416493
  18. 18. Tissenbaum HA. DAF-16: FOXO in the context of C. elegans. Curr Top Dev Biol. 2018;127:1–21. pmid:29433733
  19. 19. Kyriakou E, Taouktsi E, Syntichaki P. The thermal stress coping network of the nematode caenorhabditis elegans. IJMS. 2022;23(23):14907.
  20. 20. Powell-Coffman JA. Hypoxia signaling and resistance in C. elegans. Trends Endocrinol Metab. 2010;21(7):435–40. pmid:20335046
  21. 21. Blackwell TK, Steinbaugh MJ, Hourihan JM, Ewald CY, Isik M. SKN-1/Nrf, stress responses, and aging in Caenorhabditis elegans. Free Radic Biol Med. 2015;88(Pt B):290–301. pmid:26232625
  22. 22. Calderwood SK, Murshid A. Molecular chaperone accumulation in cancer and decrease in alzheimer’s disease: the potential roles of HSF1. Front Neurosci. 2017;11:192. pmid:28484363
  23. 23. Du S, Zheng H. Role of FoxO transcription factors in aging and age-related metabolic and neurodegenerative diseases. Cell Biosci. 2021;11(1):188. pmid:34727995
  24. 24. Hassan H, Chen R. Hypoxia in Alzheimer’s disease: effects of hypoxia inducible factors. Neural Regen Res. 2021;16(2):310–1. pmid:32859789
  25. 25. Yuan J, Zhang S, Zhang Y. Nrf1 is paved as a new strategic avenue to prevent and treat cancer, neurodegenerative and other diseases. Toxicol Appl Pharmacol. 2018;360:273–83. pmid:30267745
  26. 26. Cornell R, Cao W, Liu J, Pocock R. Conditional degradation of UNC-31/CAPS enables spatiotemporal analysis of neuropeptide function. J Neurosci. 2022;42(46):8599–607. pmid:36302635
  27. 27. Salem JB, Nkambeu B, Arvanitis DN, Beaudry F. Deciphering the role of EGL-3 for neuropeptides processing in caenorhabditis elegans using high-resolution quadrupole-orbitrap mass spectrometry. Neurochem Res. 2018;43(11):2121–31. pmid:30229400
  28. 28. Wood W. The nematode Caenorhabditis elegans. Cold Spring Harb Monogr Arch. 1988;17.
  29. 29. Timmons L, Court DL, Fire A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene. 2001;263(1–2):103–12. pmid:11223248
  30. 30. Kamath RS, Ahringer J. Genome-wide RNAi screening in Caenorhabditis elegans. Methods. 2003;30(4):313–21. pmid:12828945
  31. 31. Gonzales-Moreno C, Fernandez-Hubeid LE, Holgado A, Virgolini MB. Low-dose N-acetyl cysteine prevents paraquat-induced mortality in Caenorhabditis elegans. MicroPubl Biol. 2023;2023:10.17912/micropub.biology.000815. pmid:37065769
  32. 32. Drake J, Link CD, Butterfield DA. Oxidative stress precedes fibrillar deposition of Alzheimer’s disease amyloid beta-peptide (1-42) in a transgenic Caenorhabditis elegans model. Neurobiol Aging. 2003;24(3):415–20. pmid:12600717
  33. 33. Mendenhall A, Crane MM, Leiser S, Sutphin G, Tedesco PM, Kaeberlein M, et al. Environmental canalization of life span and gene expression in caenorhabditis elegans. J Gerontol A Biol Sci Med Sci. 2017;72(8):1033–7. pmid:28369388
  34. 34. Servello FA, Apfeld J. The heat shock transcription factor HSF-1 protects Caenorhabditis elegans from peroxide stress. Trans Med Aging. 2020;4:88–92.
  35. 35. Kramer JM, Johnson JJ. Analysis of mutations in the sqt-1 and rol-6 collagen genes of Caenorhabditis elegans. Genetics. 1993;135(4):1035–45. pmid:8307321
  36. 36. da Silveira TL, Zamberlan DC, Arantes LP, Machado ML, da Silva TC, Câmara D de F, et al. Quinolinic acid and glutamatergic neurodegeneration in Caenorhabditis elegans. Neurotoxicology. 2018;67:94–101. pmid:29702159
  37. 37. Cypser JR, Johnson TE. Multiple stressors in Caenorhabditis elegans induce stress hormesis and extended longevity. J Gerontol A Biol Sci Med Sci. 2002;57(3):B109-14. pmid:11867647
  38. 38. Ristow M, Zarse K. How increased oxidative stress promotes longevity and metabolic health: the concept of mitochondrial hormesis (mitohormesis). Exp Gerontol. 2010;45(6):410–8. pmid:20350594
  39. 39. Oh S-I, Park J-K, Park S-K. Lifespan extension and increased resistance to environmental stressors by N-acetyl-L-cysteine in Caenorhabditis elegans. Clinics (Sao Paulo). 2015;70(5):380–6. pmid:26039957
  40. 40. Gusarov I, Shamovsky I, Pani B, Gautier L, Eremina S, Katkova-Zhukotskaya O, et al. Dietary thiols accelerate aging of C. elegans. Nat Commun. 2021;12(1):4336. pmid:34267196
  41. 41. Geiss GK, Bumgarner RE, Birditt B, Dahl T, Dowidar N, Dunaway DL, et al. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol. 2008;26(3):317–25.
  42. 42. Murshid A, Eguchi T, Calderwood SK. Stress proteins in aging and life span. Int J Hyperthermia. 2013;29(5):442–7.
  43. 43. Hodge F, Bajuszova V, van Oosten-Hawle P. The intestine as a lifespan- and proteostasis-promoting signaling tissue. Front Aging. 2022;3:897741. pmid:35821863
  44. 44. Hesp K, Smant G, Kammenga JE. Caenorhabditis elegans DAF-16/FOXO transcription factor and its mammalian homologs associate with age-related disease. Exp Gerontol. 2015;72:1–7. pmid:26363351
  45. 45. Park S-K, Tedesco PM, Johnson TE. Oxidative stress and longevity in Caenorhabditis elegans as mediated by SKN-1. Aging Cell. 2009;8(3):258–69. pmid:19627265
  46. 46. Shamalnasab M, Dhaoui M, Thondamal M, Harvald EB, Færgeman NJ, Aguilaniu H, et al. HIF-1-dependent regulation of lifespan in Caenorhabditis elegans by the acyl-CoA-binding protein MAA-1. Aging (Albany NY). 2017;9(7):1745–69. pmid:28758895
  47. 47. Oliveira RP, Porter Abate J, Dilks K, Landis J, Ashraf J, Murphy CT, et al. Condition-adapted stress and longevity gene regulation by Caenorhabditis elegans SKN-1/Nrf. Aging Cell. 2009;8(5):524–41. pmid:19575768
  48. 48. Wang J, Robida-Stubbs S, Tullet JMA, Rual J-F, Vidal M, Blackwell TK. RNAi screening implicates a SKN-1-dependent transcriptional response in stress resistance and longevity deriving from translation inhibition. PLoS Genet. 2010;6(8):e1001048. pmid:20700440
  49. 49. Offenburger S-L, Ho XY, Tachie-Menson T, Coakley S, Hilliard MA, Gartner A. 6-OHDA-induced dopaminergic neurodegeneration in Caenorhabditis elegans is promoted by the engulfment pathway and inhibited by the transthyretin-related protein TTR-33. PLoS Genet. 2018;14(1):e1007125. pmid:29346382
  50. 50. Miller HA, Dean ES, Pletcher SD, Leiser SF. Cell non-autonomous regulation of health and longevity. Elife. 2020;9:e62659. pmid:33300870
  51. 51. Chen PX, Zhang L, Chen D, Tian Y. Mitochondrial stress and aging: lessons from C. elegans. Semin Cell Dev Biol. 2024;154(Pt A):69–76. pmid:36863917
  52. 52. Dutta N, Garcia G, Higuchi-Sanabria R. Hijacking cellular stress responses to promote lifespan. Front Aging. 2022;3:860404. pmid:35821861
  53. 53. Simmer F, Tijsterman M, Parrish S, Koushika SP, Nonet ML, Fire A, et al. Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi. Curr Biol. 2002;12(15):1317–9. pmid:12176360
  54. 54. Brothers HM, Gosztyla ML, Robinson SR. The physiological roles of Amyloid-β Peptide hint at new ways to treat Alzheimer’s Disease. Front Aging Neurosci. 2018;10:118. pmid:29922148