Mitochondrial p38 Mitogen-Activated Protein Kinase: Insights into Its Regulation of and Role in LONP1-Deficient Nematodes

p38 Mitogen-Activated Protein Kinase (MAPK) cascades are central regulators of numerous physiological cellular processes, including stress response signaling. In C. elegans, mitochondrial dysfunction activates a PMK-3/p38 MAPK signaling pathway (MAPKmt), but its functional role still remains elusive. Here, we demonstrate the induction of MAPKmt in worms deficient in the lonp-1 gene, which encodes the worm ortholog of mammalian mitochondrial LonP1. This induction is subjected to negative regulation by the ATFS-1 transcription factor through the CREB-binding protein (CBP) ortholog CBP-3, indicating an interplay between both activated MAPKmt and mitochondrial Unfolded Protein Response (UPRmt) surveillance pathways. Our results also reveal a genetic interaction in lonp-1 mutants between PMK-3 kinase and the ZIP-2 transcription factor. ZIP-2 has an established role in innate immunity but can also modulate the lifespan by maintaining mitochondrial homeostasis during ageing. We show that in lonp-1 animals, ZIP-2 is activated in a PMK-3-dependent manner but does not confer increased survival to pathogenic bacteria. However, deletion of zip-2 or pmk-3 shortens the lifespan of lonp-1 mutants, suggesting a possible crosstalk under conditions of mitochondrial perturbation that influences the ageing process. Furthermore, loss of pmk-3 specifically diminished the extreme heat tolerance of lonp-1 worms, highlighting the crucial role of PMK-3 in the heat shock response upon mitochondrial LONP-1 inactivation.

Several studies in C. elegans have uncovered additional roles of PMK-1/p38 MAPK in environmental and cellular stress responses.For example, the PMK-1 pathway has a key role in regulating the expression of innate immune genes in response to various pathogens [11,12].PMK-1 is the sole effector of the TIR-1/NSY-1/SEK-1 cascade, functioning in the intestines of worms to promote host pathogen resistance [13][14][15][16].The PMK-1 signaling pathway is also activated in response to abiotic stressors such as cadmium [17,18] and cholesterol deprivation [19].Additionally, during oxidative stress, it acts in the intestine to phosphorylate SKN-1, the worm ortholog of mammalian Nuclear Factor-Erythroid 2-Related Factor (NRF) transcription factors, mainly regulating transcription of phase II detoxification genes [20][21][22].Furthermore, PMK-1 and SKN-1 activities have been linked to the heat tolerance of C. elegans, as heat stress induces nuclear translocation of PMK-1 in intestinal cells and depletion of pmk-1 reduces the survival rate of worms at high temperatures [23].
In contrast to PMK-1, there is little knowledge about the role of the PMK-3 signaling pathway in stress responses.A mitochondrial MAPK (MAPK mt ) cascade defined by DLK-1 (MAP3K), SEK-3 (MAPKK) and PMK-3 kinases was identified to be required for lifespan extension of C. elegans mutants with disrupted mitochondrial activity [24].It seems that the PMK-3 pathway defines another cellular retrograde response that is activated in complement to the mitochondrial unfolded protein response (UPR mt ), a conserved surveillance mechanism by which metazoans respond to mitochondrial stress [25].During UPR mt activation, an increased unfolded/misfolded protein load inside the mitochondria redirects the basic leucine zipper (bZIP) transcription factor ATFS-1 from the matrix to the nucleus, regulating the expression of both mitochondrial protective genes and metabolismrelated genes [25,26].In the absence of mitochondrial stress, ATFS-1 is trafficked to the mitochondrial matrix and rapidly degraded by the ATP-dependent protease LONP-1, the ortholog of mammalian LonP1 [25].
LonP1 protease belongs to the ATPase associated with diverse cellular activities (AAA + ) family with major roles in mitochondrial protein quality control [27][28][29].We have previously shown that deletion of the lonp-1 gene in C. elegans increases endogenous levels of ROS, impacts the mitochondrial function and impairs physiological processes, such as growth, behavior and lifespan [30].Loss of LONP-1 induces the UPR mt , while the accompanied activation of several cytoplasmic UPR (UPR cyt ) responses was shown to increase the resistance of mutants to heat and other forms of stress.In addition to worms, genetic or pharmacological inhibition of human LonP1 in cancer cells induces similar mitochondrial and cytoplasmic UPR mechanisms [30].Although the induction of such UPR pathways could result in lifespan extension of C. elegans, the short-lived phenotype of the lonp-1 mutants implies the existence of additional factors/mechanisms that modulate their lifespan and possibly their viability under adverse environmental conditions.In this study, we demonstrated the induction of the MAPK mt signaling pathway in lonp-1 mutants and we examined its relationship with other mitochondrial surveillance mechanisms, in addition to the effects of its disruption on physiological functions of lonp-1-deficient animals.The described relationships and effects might be evolutionarily conserved, thus shedding some light on human health and mitochondrial dysfunction-related diseases.

The MAPK mt Pathway Is Active in Lonp-1-Deficient Mutants
Mitochondrial dysfunction caused by disruption of the mitochondrial electron transport chain, such as mutations in the Rieske iron-sulfur polypeptide 1 (isp-1) of complex

The MAPK mt Pathway Is Active in Lonp-1-Deficient Mutants
Mitochondrial dysfunction caused by disruption of the mitochondrial electron transport chain, such as mutations in the Rieske iron-sulfur polypeptide 1 (isp-1) of complex III, has previously been shown to activate the MAPK mt pathway, which consists of DLK-1, SEK-3, and PMK-3 kinases and induces transcription of the tbb-6 gene [24] (Figure 1A).tbb-6 encodes for a structurally diverged member of the beta-tubulin family in C. elegans [31], with no reported specific functions or overt mutant phenotypes.We examined the expression levels of tbb-6 in synchronized young adults of wild-type (wt) and lonp-1 knockout strains and we observed elevated mRNA levels of the endogenous tbb-6 gene (Figure 1B), as well as a strong induction of a transcriptional reporter tbb-6p::gfp (Figure 1C) in the intestine and pharyngeal muscles of lonp-1 compared to wt animals.Mild exposure to isp-1(RNAi) further increased the fluorescence of the reporter in lonp-1, indicating a strong additive mitochondrial dysfunction in lonp-1;isp-1(RNAi) adults (interaction tbb-6 encodes for a structurally diverged member of the beta-tubulin family in C. elegans [31], with no reported specific functions or overt mutant phenotypes.We examined the expression levels of tbb-6 in synchronized young adults of wild-type (wt) and lonp-1 knockout strains and we observed elevated mRNA levels of the endogenous tbb-6 gene (Figure 1B), as well as a strong induction of a transcriptional reporter tbb-6 p ::gfp (Figure 1C) in the intestine and pharyngeal muscles of lonp-1 compared to wt animals.Mild exposure to isp-1(RNAi) further increased the fluorescence of the reporter in lonp-1, indicating a strong additive mitochondrial dysfunction in lonp-1;isp-1(RNAi) adults (interaction p < 0.0005 in a two-way ANOVA; Figure S1A).In addition to tbb-6, the C. elegans beta-tubulin family comprises five more members, of which tbb-1 and tbb-2 are ubiquitously expressed and partially redundant, ben-1/tbb-5 is broadly expressed in the nervous system, and mec-7/tbb-3 and tbb-4 are expressed only in touch receptor neurons [31].The expressions of all three major beta-tubulins, tbb-1, tbb-2, and ben-1, were not changed in lonp-1 mutant animals (Figure S1B), confirming a specific, yet unidentified, role of TBB-6 in mitochondrial dysfunction.However, the expression levels of the tbb-6 gene and reporter were found to severely drop upon RNAi-mediated disruption of DLK-1 and SEK-3 or deletion of PMK-3 in both wt and lonp-1 mutants (Figures 1D,E and S2).Thus, we concluded that MAPK mt signaling regulates both the basal and the lonp-1-induced expression of the tbb-6 gene.

ATFS-1 Counteracts Activation of the MAPK mt Pathway upon Lonp-1 Inactivation
Mitochondrial perturbation by LONP-1 deficiency has been demonstrated to elicit activation of several stress-responsive transcription factors, including SKN-1 and DAF-16, the worm orthologs of the mammalian NRF and Forkhead Box O (FOXO) factors, respectively [30].Since the promoter of tbb-6 contains putative binding sites for SKN-1 and DAF-16 transcription factors (WormBase version WS289), we tested their requirement for tbb-6 induction in lonp-1 mutants.RNAi targeting SKN-1 or DAF-16 did not reduce fluorescence of the tbb-6 p ::gfp reporter or tbb-6 mRNA levels (Figure S3), suggesting that these factors are not involved in the expression of tbb-6 in lonp-1 adults.Interestingly, genetic loss of atfs-1 by the gk3094 allele led to high tbb-6 mRNA levels in an otherwise wt background (Figure 2A), and RNAi-induced depletion of atfs-1 in a lonp-1 background further increased the expression of the tbb-6 gene or the tbb-6 p ::gfp reporter (Figure 2A,B).These results support the notion that ATFS-1 counteracts the activity of the MAPK mt pathway, as has been previously suggested for isp-1(qm150) mutants [24].However, when two other UPR mt -associated factors, namely DVE-1 and UBL-5 [32], were knocked down by RNAi, no difference in the signal of the tbb-6 reporter was detected (Figure S4), suggesting that ATFS-1 exerts a repressive function independently of the canonical UPR mt signaling branch.The repressive function of ATFS-1 on tbb-6 expression occurs upstream of These results support the notion that ATFS-1 counteracts the activity of the MAPK mt pathway, as has been previously suggested for isp-1(qm150) mutants [24].However, when two other UPR mt -associated factors, namely DVE-1 and UBL-5 [32], were knocked down by RNAi, no difference in the signal of the tbb-6 reporter was detected (Figure S4), suggesting that ATFS-1 exerts a repressive function independently of the canonical UPR mt signaling branch.The repressive function of ATFS-1 on tbb-6 expression occurs upstream of PMK-3, as atfs-1(RNAi) was not able to increase the tbb-6 p ::gfp fluorescence in lonp-1;pmk-3 double mutants (Figure 2B).

ATFS-1 Suppresses the Induction of cbp-3 in Lonp-1 Mutants
It has been reported that tbb-6 induction in isp-1 mutant worms depends on cbp-3/F40F12.7,a homolog of the mammalian acetyltransferase CBP/p300 [24].However, cbp-3 is probably a pseudogene of cbp-1, bearing only the kinase-inducible domain interacting domain (KIX) and missing the histone acetyltransferase (HAT) domain of CBP-1 [33].Thus, in contrast to cbp-1(RNAi), we showed that RNAi against cbp-3 does not cause developmental defects and arrest in worms, possibly due to lack of the HAT domain.When lonp-1 mutant adults were subjected to cbp-3(RNAi), the expression levels of tbb-6 p ::gfp or the endogenous tbb-6 gene were significantly decreased (Figure 3A,B  We further showed that the mRNA levels of cbp-3, and not those of cbp-1, were upregulated in lonp-1 mutants, independently of PMK-3 function (Figure 3C).Although the expression of cbp-3 has been previously shown to increase upon induction of UPR mt in an ATFS-1-dependent manner [25], we found that depletion of atfs-1 enhances, rather than decreases, the expression of cbp-3 in lonp-1 mutants (Figure 3D), similarly to tbb-6.Depletion of SKN-1 had no effect on cbp-3 transcript levels (Figure 3D), which is consistent with the lack of effects of skn-1(RNAi) in tbb-6 mRNA levels.Thus, our data suggest that ATFS-1 suppresses the induction of cbp-3 in lonp-1 mutants and imply a role of the KIX domain of CBP-3 in the activation of the MAPK mt pathway, placing it upstream of PMK-3/TBB-6 signaling.We further showed that the mRNA levels of cbp-3, and not those of cbp-1, were upregulated in lonp-1 mutants, independently of PMK-3 function (Figure 3C).Although the expression of cbp-3 has been previously shown to increase upon induction of UPR mt in an ATFS-1-dependent manner [25], we found that depletion of atfs-1 enhances, rather than decreases, the expression of cbp-3 in lonp-1 mutants (Figure 3D), similarly to tbb-6.Depletion of SKN-1 had no effect on cbp-3 transcript levels (Figure 3D), which is consistent with the lack of effects of skn-1(RNAi) in tbb-6 mRNA levels.Thus, our data suggest that ATFS-1 suppresses the induction of cbp-3 in lonp-1 mutants and imply a role of the KIX domain of CBP-3 in the activation of the MAPK mt pathway, placing it upstream of PMK-3/TBB-6 signaling.Mitochondrial dysfunction in yeast strongly activates the mitochondria-associated degradation (MAD) pathway [34], and inhibition of MAD components could block tbb-6 p ::gfp induction in isp-1 mutants [24].The MAD pathway mediates ubiquitination and degradation of misfolded or damaged mitochondrial proteins under mitochondrial stress, analogously to the endoplasmic reticulum (ER)-associated degradation pathway (ERAD).Physical interaction of CBP-3 with proteins involved in protein ubiquitination, such as F57C12.2[35,36] and ubq-1 [37], has been reported; thus, we wondered whether CBP-3 functions with the MAD pathway to enable induction of MAPK mt in lonp-1 mutants.C. elegans has two orthologs of a highly conserved AAA + ATPase, named p97/VCP/CDC48 in mammals, which direct ubiquitinated proteins from cellular membranes to the 26S proteasome for degradation [38][39][40].The worm orthologs, CDC-48.1 and CDC-48.2,share 88% identity over the entire p97 protein, and both have essential and redundant functions [41].We introduced a null mutation of cdc-48.2into lonp-1 worms, but did not find a significant change in fluorescence of the reporter tbb-6 p ::gfp or the mRNA levels of tbb-6 gene (Figure 4A).In a similar manner, depletion of cdc-48.1 through RNAi, alone or in combination with cdc-48.2(tm659),did not reduce the expression of tbb-6 in lonp-1 mutants (Figure 4B).These results indicate that the activity of the MAD pathway is not a prerequisite for MAPK mt induction, at least in lonp-1-deficient worms, and cbp-3 possibly influences PMK-3 activity through mechanisms independent of MAD signaling.

Induction of the MAPK mt Pathway in Lonp-1 Mutants Is Not MAD Pathway-Dependent
Mitochondrial dysfunction in yeast strongly activates the mitochondria-associated degradation (MAD) pathway [34], and inhibition of MAD components could block tbb-6p::gfp induction in isp-1 mutants [24].The MAD pathway mediates ubiquitination and degradation of misfolded or damaged mitochondrial proteins under mitochondrial stress, analogously to the endoplasmic reticulum (ER)-associated degradation pathway (ERAD).Physical interaction of CBP-3 with proteins involved in protein ubiquitination, such as F57C12.2[35,36] and ubq-1 [37], has been reported; thus, we wondered whether CBP-3 functions with the MAD pathway to enable induction of MAPK mt in lonp-1 mutants.C. elegans has two orthologs of a highly conserved AAA + ATPase, named p97/VCP/CDC48 in mammals, which direct ubiquitinated proteins from cellular membranes to the 26S proteasome for degradation [38][39][40].The worm orthologs, CDC-48.1 and CDC-48.2,share 88% identity over the entire p97 protein, and both have essential and redundant functions [41].We introduced a null mutation of cdc-48.2into lonp-1 worms, but did not find a significant change in fluorescence of the reporter tbb-6p::gfp or the mRNA levels of tbb-6 gene (Figure 4A).In a similar manner, depletion of cdc-48.1 through RNAi, alone or in combination with cdc-48.2(tm659),did not reduce the expression of tbb-6 in lonp-1 mutants (Figure 4B).These results indicate that the activity of the MAD pathway is not a prerequisite for MAPK mt induction, at least in lonp-1-deficient worms, and cbp-3 possibly influences PMK-3 activity through mechanisms independent of MAD signaling.

Loss of Lonp-1 Induces the ZIP-2 Innate Immune Response Effector
Several studies of C. elegans have established that disruption of essential core processes, including mitochondrial function, can induce innate immune responses in worms [42,43].In particular, UPR mt has been shown to induce a subset of ATFS-1-dependent innate immunity genes, providing resistance to pathogenic bacteria Pseudomonas aeruginosa, independently of PMK-1/p38 MAPK [44].One such gene is the infection response gene-1 Values were presented as mean ± SEM, and numeric data were analyzed with a two-way ANOVA followed by post hoc Tukey's test, showing no significant (ns) differences.
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 8 of 18 (irg-1), a target of the bZIP transcription factor ZIP-2 [42,45,46], which was reported to be upregulated by ATFS-1 upon UPR mt [25,44].The expression of irg-1 and zip-2 was examined and was found to be significantly induced in lonp-1 mutants compared to wt animals (Figure 5A).In addition to irg-1, the infection response gene-2 (irg-2), another ZIP-2 target [42,45], was upregulated upon lonp-1 deletion (Figure 5A).Using a zip-2(ok3730) loss-offunction allele, we verified that ZIP-2 was required for basal expression of both irg-1 and irg-2, whereas in a lonp-1 mutant background, ZIP-2 was necessary only for irg-2 induction (Figure 5A).Instead, irg-1 could be upregulated in lonp-1 worms by ZIP-2-independent mechanisms, as was previously reported for other conditions, e.g., suppression of translation [42].Nevertheless, mitochondrial damage caused during infection by P. aeruginosa elicits a host defense, mediated in part by the ZIP-2 transcription factor, to promote organismal survival [47,48].However, the increased activity of ATFS-1 and ZIP-2 in the lonp-1 background was not sufficient to promote host resistance to infection by the P. aeruginosa strain PA14, and lonp-1 adults were significantly susceptible to the pathogen compared to wt animals (Figure 5B).Given that ATFS-1 activity counteracts the activation of the MAPK mt Nevertheless, mitochondrial damage caused during infection by P. aeruginosa elicits a host defense, mediated in part by the ZIP-2 transcription factor, to promote organismal survival [47,48].However, the increased activity of ATFS-1 and ZIP-2 in the lonp-1 background was not sufficient to promote host resistance to infection by the P. aeruginosa strain PA14, and lonp-1 adults were significantly susceptible to the pathogen compared to wt animals (Figure 5B).Given that ATFS-1 activity counteracts the activation of the MAPK mt pathway, we tested whether genetic disruption of pmk-3 could enhance the host defense response to PA14.As shown in Figure 5B, loss of pmk-3 did not alter the hypersensitive phenotype of lonp-1 mutants to pathogenic bacteria, although it had a negative impact on the immune response of wt worms.In spite of that, ZIP-2-dependent expression of irg-1 and irg-2 genes was compromised in both pmk-3 and lonp-1;pmk-3 mutants, similarly to zip-2 deletion (Figure 5A).These results suggest a genetic interaction between PMK-3 and ZIP-2 when lonp-1 is missing, which determines the activity of ZIP-2 and affects other functions than the innate immune response.

Disruption of the MAPK mt Pathway Influences Lonp-1 Mutants' Lifespan and Stress Responses
Induction of the ZIP-2 pathway provides surveillance for several core cellular processes, and can also delay the ageing process by preserving mitochondrial homeostasis in aged worms [49].Moreover, the extended lifespan of some, but not all, long-lived mitochondrial mutants was shown to require an active MAPK mt pathway [24].Considering the above genetic interaction between ZIP-2 and PMK-3, we examined the effects of disruption of each pathway on the lifespan of lonp-1 mutants.Although worms deficient in the lonp-1 gene display a shorter lifespan than the wt [30], deletion of either zip-2 or pmk-3 severely reduced the lifespan of lonp-1 mutants, without affecting the normal lifespan of wt worms (Figure 6A and Table S3).This shortening of lifespans was not observed when pmk-1/p38 MAPK was deleted in the lonp-1 mutant background, with loss of pmk-1 to reduce only the brood size of both wt and lonp-1 strains (Figure S5A,B and Table S3).Furthermore, deletion of pmk-3, pmk-1 and zip-2 (Figure S5C) did not have any impact on the growth of wt or lonp-1 animals.
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 9 of 18 pathway, we tested whether genetic disruption of pmk-3 could enhance the host defense response to PA14.As shown in Figure 5B, loss of pmk-3 did not alter the hypersensitive phenotype of lonp-1 mutants to pathogenic bacteria, although it had a negative impact on the immune response of wt worms.In spite of that, ZIP-2-dependent expression of irg-1 and irg-2 genes was compromised in both pmk-3 and lonp-1;pmk-3 mutants, similarly to zip-2 deletion (Figure 5A).These results suggest a genetic interaction between PMK-3 and ZIP-2 when lonp-1 is missing, which determines the activity of ZIP-2 and affects other functions than the innate immune response.

Disruption of the MAPK mt Pathway Influences Lonp-1 Mutants' Lifespan and Stress Responses
Induction of the ZIP-2 pathway provides surveillance for several core cellular processes, and can also delay the ageing process by preserving mitochondrial homeostasis in aged worms [49].Moreover, the extended lifespan of some, but not all, long-lived mitochondrial mutants was shown to require an active MAPK mt pathway [24].Considering the above genetic interaction between ZIP-2 and PMK-3, we examined the effects of disruption of each pathway on the lifespan of lonp-1 mutants.Although worms deficient in the lonp-1 gene display a shorter lifespan than the wt [30], deletion of either zip-2 or pmk-3 severely reduced the lifespan of lonp-1 mutants, without affecting the normal lifespan of wt worms (Figure 6A and Table S3).This shortening of lifespans was not observed when pmk-1/p38 MAPK was deleted in the lonp-1 mutant background, with loss of pmk-1 to reduce only the brood size of both wt and lonp-1 strains (Figure S5A,B and Table S3).Furthermore, deletion of pmk-3, pmk-1 and zip-2 (Figure S5C) did not have any impact on the growth of wt or lonp-1 animals.Worms bearing deficiency in the lonp-1 gene are short-lived relative to the wt, but exhibit an increased resilience in some forms of exogenous stress, for instance, to specific oxidants or heat shock [30].Here, we showed that, similarly to pmk-1 deletion, loss of pmk-3 renders both wt and lonp-1 worms sensitive to oxidative stress induced by the organic peroxide tert-butyl hydroperoxide (tBHP) (Figures 6B and S6A).Treatment of worms with antimycin, an inhibitor of complex III of the electron transport chain, revealed a general requirement for pmk-3 to confront superoxide anion generation in mitochondria (Figure 6B), while pmk-1 was required only in lonp-1-deficient animals (Figure S6A).These results suggest that PMK-3 is important for both wt and lonp-1 adults to cope with oxidative stress.Moreover, deletion of pmk-3 severely reduced the high survival rate of lonp-1 mutants at 35 • C (Figure 6B), while loss of pmk-1 had no effect on their extreme heat tolerance, with the double lonp-1;pmk-1 mutants being similarly tolerant to single lonp-1 mutants (Figure S6A).However, in a wt background, pmk-1 deletion had a greater negative impact on thermotolerance compared to pmk-3 deletion.Further studies are needed to address the key role of PMK-3 in the heat resistance of lonp-1, but it does not seem to involve tbb-6 or zip-2 functions (Figure S6B,C).Overall, our data support a central role of PMK-3/p38 MAPK in mediating the lifespan length and stress resistance under conditions of lonp-1-induced mitochondrial perturbations.

Discussion
In humans, mutations of the LonP1 gene are associated with a broad spectrum of diseases, including a rare multisystem developmental disorder known as CODAS (Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies) syndrome, as well as neurodegeneration and cancer [27][28][29]50].The mammalian protease LonP1 is a multifunctional enzyme with essential roles during development of cardiomyocyte and skeletal muscle cells [51,52].Also, acting as both a chaperone and a protease, LonP1 plays a crucial role in protein quality control, preserving mitochondrial homeostasis in adults.An age-related decline in the expression and activity of LonP1 seems to contribute to structural and functional impairment of organs with age [29,51].While genetic disruption of the LonP1 gene in mice and Drosophila results in embryonic lethality [53][54][55], C. elegans lonp-1 knockout mutants are viable but exhibit disturbed mitochondrial and ROS homeostasis, resulting in impaired growth and a shortened lifespan [30].lonp-1-deficient worms are extremely resistant to specific oxidants and heat stress due to induction of both mitochondrial and cytosolic stress signaling pathways.These include UPR mt , where increased activity of the ATFS-1 transcription factor was found to be essential for normal development of lonp-1 mutants and determines the resistance of lonp-1 adults to external oxidative stress [30].Interestingly, lack of LONP-1 was recently reported to induce constitutive formation of mitochondrial stress granules, perhaps as an adaptive response [56].Moreover, ATFS-1 activity in longlived isp-1 mitochondrial mutants could prevent the induction of another mitochondrial stress signaling pathway named MAPK mt , in which PMK-3/p38 MAPK upregulates the expression of the tbb-6 beta-tubulin target gene in the intestines [24].MAPKs are potent regulators of microtubule dynamics in mammals, and the DLK-1/PMK-3 pathway in C. elegans can regulate microtubule dynamics and endocytosis in neurons [57,58].Although the exact role of TBB-6 in intestinal cells is still unknown, it has been suggested that it may function to enhance ADP entry into mitochondria during mitochondrial stress [24].
Here, we demonstrated the induction of the MAPK mt pathway in lonp-1 mutants, which is consistent with the presence of a disturbed mitochondrial network and an accumulation of ROS in these animals.Based on our results discussed below, a schematic representation of the genetic interactions and molecular functions of MAPK mt in worms deficient in the lonp-1 gene is shown in Figure 7. Depletion of atfs-1 further enhances the expression of the tbb-6 marker, supporting the notion that ATFS-1 suppresses MAPK mt activity, probably to fine-tune it.One regulator of ATFS-1 in PMK-3 activity may be the CBP ortholog CBP-3, which contains only the kinase-inducible domain interacting domain [33], and is required for tbb-6 induction in isp-1 mutants [24].We found that cbp-3 is upregulated in lonp-1 worms and ATFS-1 suppresses this induction upstream of PMK-3/TTB-6 signaling.The combination of such protein interaction and phosphorylation events could be important for orchestrating the cellular processes and changing the metabolic status under two opposing signals in response to mitochondrial stress.For example, under conditions of moderate mitochondrial dysfunction, C. elegans can adapt and often exhibits an altered metabolism and an increased lifespan [59].However, the ageing process can be accelerated when the activity of the different mitochondrial protein quality control systems is not in balance.In support of this, activity of the MAPK mt pathway is required for the lifespan extension of some mitochondrial mutants [24].We have shown that disruption of atfs-1 in lonp-1 adults reduced their lifespan [30] and here, deletion of pmk-3 also shortened the lifespan of lonp-1 animals.Moreover, our data as presented below uncovered a genetic interaction between PMK-3 and ZIP-2, which could influence the lifespan of lonp-1 mutants.
resentation of the genetic interactions and molecular functions of MAPK mt in worms deficient in the lonp-1 gene is shown in Figure 7. Depletion of atfs-1 further enhances the expression of the tbb-6 marker, supporting the notion that ATFS-1 suppresses MAPK mt activity, probably to fine-tune it.One regulator of ATFS-1 in PMK-3 activity may be the CBP ortholog CBP-3, which contains only the kinase-inducible domain interacting domain [33], and is required for tbb-6 induction in isp-1 mutants [24].We found that cbp-3 is upregulated in lonp-1 worms and ATFS-1 suppresses this induction upstream of PMK-3/TTB-6 signaling.The combination of such protein interaction and phosphorylation events could be important for orchestrating the cellular processes and changing the metabolic status under two opposing signals in response to mitochondrial stress.For example, under conditions of moderate mitochondrial dysfunction, C. elegans can adapt and often exhibits an altered metabolism and an increased lifespan [59].However, the ageing process can be accelerated when the activity of the different mitochondrial protein quality control systems is not in balance.In support of this, activity of the MAPK mt pathway is required for the lifespan extension of some mitochondrial mutants [24].We have shown that disruption of atfs-1 in lonp-1 adults reduced their lifespan [30] and here, deletion of pmk-3 also shortened the lifespan of lonp-1 animals.Moreover, our data as presented below uncovered a genetic interaction between PMK-3 and ZIP-2, which could influence the lifespan of lonp-1 mutants.ZIP-2 is a bZIP transcription factor with an established role in innate immunity [42,45,46].Although mitochondrial dysfunction can induce immune responses in worms, mediated in part by ATFS-1 and ZIP-2 [44,47,48], the observed activation of ZIP-2 in lonp-1 mutants was not sufficient to increase survival of mutants against pathogenic bacteria PA14.Moreover, the ZIP-2-dependent transcription of irg-1 and irg-2 target genes was abolished after depletion of pmk-3, signifying a requirement of the PMK-3 kinase for both basal and induced activity of the ZIP-2 factor, even though PMK-3 is not involved in innate immunity.Separate from its role in immunity, ZIP-2 is activated in response to age- ZIP-2 is a bZIP transcription factor with an established role in innate immunity [42,45,46].Although mitochondrial dysfunction can induce immune responses in worms, mediated in part by ATFS-1 and ZIP-2 [44,47,48], the observed activation of ZIP-2 in lonp-1 mutants was not sufficient to increase survival of mutants against pathogenic bacteria PA14.Moreover, the ZIP-2-dependent transcription of irg-1 and irg-2 target genes was abolished after depletion of pmk-3, signifying a requirement of the PMK-3 kinase for both basal and induced activity of the ZIP-2 factor, even though PMK-3 is not involved in innate immunity.Separate from its role in immunity, ZIP-2 is activated in response to age-associated mitochondrial dysfunction, independently of ATFS-1, and delays aging phenotypes by preserving mitochondrial homeostasis in aged worms [49].In addition to this, ZIP-2 is activated and mediates lifespan extension under various dietary restriction conditions [60,61].Since deletion of either zip-2 or pmk-3 significantly reduced the lifespan of lonp-1 mutants, we speculate that this novel crosstalk between MAPK mt and ZIP-2 is important in modulating the ageing process under mitochondrial dysfunction.This is strengthened by the observation that the DLK-1/PMK-3 signaling pathway declines with age, and this is overcome by mild mitochondrial stress [62].Future studies investigating the underlying molecular mechanism and transcriptional targets are expected to shed light on the relevant ageing modulators.
We have previously shown that knockout of lonp-1 induces a subset of stress-responsive genes associated with protective mechanisms, thereby safeguarding the mitochondrial and cytosolic proteome to defend the mitochondrial function [30].Thus, here, we evaluated the consequences of pmk-3 deletion on the resistance of lonp-1 mutant adults to exogenous stresses.We confirmed that the activity of PMK-3 is indispensable for both oxidative and heat stress responses.More specifically, loss of PMK-3 significantly diminished the extreme heat resistance of lonp-1 mutants, while deletion of pmk-1 did not block this response.However, thermotolerance of wt animals was reduced when pmk-1 was missing, which is consistent with a previous work, where heat was found to induce nuclear translocation of PMK-1 [23].It has been suggested that PMK-1 might contribute to the elevated expression of hsp and other chaperone genes through hyperphosphorylation of heat shock factor I (HSF-1), analogously to the stress activation of mammalian HSF1 [63].HSF-1 is the single worm homolog of the four mammalian HSFs, and its functions in thermal stress response are tightly regulated by post-translational modifications [64].Given that the extreme thermotolerance of lonp-1 mutants requires HSF-1 activity [30] and PMK-3, but not PMK-1 (this study), we propose that PMK-3 kinase mediates the function of HSF-1 or its regulators in lonp-1 mutants in response to heat stress.These relationships might be evolutionarily conserved, as we have shown that genetic or pharmacological inhibition of human LonP1 in cancer cells induces the expression of genes involved in the heat shock response (HSR) [30].Interestingly, new evidence exists for nuclear localization of mitochondrial LonP1 in mammalian cells, and this translocation is increased under heat stress, up-regulating HSF1 target genes [65].Thus, it was suggested that LonP1 modulates the heat shock response through interaction with and possible degradation of HSF1 in the nucleus [65].It is tempting to speculate that mammalian p38 MAPK could have a similar regulatory role with PMK-3 on LonP1 and HSF1 functions.Although in mammals there are four p38 isoforms that are highly similar to PMK-1 in worms, they exhibit differential expression/activation patterns and specific downstream effectors [1,66], raising the possibility that they may fulfill analogous cellular functions to the PMK-3 kinase in worms, promoting organismal fitness and stress responses.

C. elegans Strains and Culture Conditions
C. elegans strains were maintained at 20 • C on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 as a food source.Whenever it was necessary, NGM plates with UV-killed bacteria supplemented with 40 µg/mL 5-fluoro-2 -deoxyuridine (FUdR; Sigma-Aldrich, St. Louis, MO, USA) were used to prevent progeny growth.All strains used in this study are listed in Table S1.

RNA Interference
RNAi experiments were conducted at 20 • C on NGM plates seeded with E. coli HT115 (DE3) bacteria, transformed with the indicated RNAi construct (primers listed in Table S2) or the appropriate empty vector (plasmid L4440 or T444T, Addgene, Watertown, MA, USA).Plates containing 2 mM isopropyl b-D-1-thiogalactopyranoside (IPTG; Applichem GmbH, Darmstadt, Germany) were incubated at 37 • C overnight to induce RNAi.Worms were synchronized from eggs on RNAi plates at 20 • C and transferred onto new RNAi plates when they reached L4 or the young prefertile adult stage (YA) for analysis.For postdevelopmental RNAi experiments, L4 to YA worms raised on OP50 plates were transferred onto RNAi plates for the indicated time.

Microscopic Analysis
Age-synchronized transgenic worms expressing the indicated fluorescence stress reporter were grown on OP50 or RNAi plates at 20 • C, and a microscopic analysis was performed in 1-day-old adults, considering the developmental delay in lonp-1 mutants.Worms were immobilized with levamisole (Sigma-Aldrich, St Louis, MO, USA) and mounted on 2% agarose pads on glass microscope slides.Images of worms were captured via fluorescent microscopy using a Leica DMRA upright fluorescent microscope equipped with a Hamamatsu ORCA-flash 4.0 camera with a 10× or 40× objective lens.All strains were assayed in parallel and microscopy settings were kept the same in each experiment.Fluorescence was measured as the average pixel intensity with ImageJ 1.52p (Fiji) [67] in the whole captured worm image or in standardized regions of the intestine.At least 50 worms per strain and condition were measured in three biological replicates, and the mean of calculated values was plotted as the mean fluorescence intensity ± SEM.Representative fluorescence and bright field micrographs are shown for each genotype and condition.

Growth Rate and Fecundity Assays
Worms were synchronized by placing 10 gravid adults of each strain on NGM plates with adequate food (OP50) and they were allowed to lay eggs for 2-3 h before removing.Eggs were left to hatch at 20 • C, and the number of progenies in each developmental stage after 68 h was scored.The total brood size was determined at 20 • C by picking 10 L4 stage nematodes from an NGM plate and placing one nematode onto separate individual NGM plates with OP50.Individual animals were transferred onto new plates daily until the end of the spawning period of worms.The total number of F1 progeny hatched in each plate for each individual was counted.

Lifespan Analysis
Animals were synchronized by egg-laying, and 100-150 healthy L4 larvae of each strain, were transferred to three NGM plates (approximately 40 per plate) seeded with OP50 bacteria, at 20 • C. Animals were moved to fresh plates every 2-4 days and survival was scored daily by gentle prodding.Worms that failed to respond were considered dead, whereas worms raptured, burrowed into the agar, bagged (that displayed internal hatching of eggs), or animals that crawled off the plates were censored in the analysis.Statistical analysis was performed by comparing each population to the appropriate control and pvalues were determined using the log-rank (Mantel-Cox) test.Replicates were carried out as indicated in Table S3.

Stress Sensitivity Assays
In all assays, animals were synchronized by egg laying on NGM plates with OP50 bacteria at 20 • C. Heat shock assays were performed by shifting 1-day-old adults onto three fresh plates containing approximately 30 animals per plate from 20 • C to 35 • C for the indicated time points.The percentage of alive animals was scored following 16 h of recovery at 20 • C by assessing the response to gentle touch.For the tBHP-induced oxidative stress assay, 1-day-old adult worms were placed on freshly prepared tBHP plates [30] containing 10 mM tBHP (from a stock solution of 70% in water, Sigma-Aldrich, St Louis, MO, USA) devoid of bacteria.During the initial two hours, worms were periodically relocated to the center of the plate, as they tried to "escape" from the plate and avoid tBHP, and survival was counted every hour.For antimycin A (Sigma-Aldrich, St Louis, MO, USA)-induced oxidative stress, synchronized L4 larvae of each strain were placed on plates containing 40 µM FUdR for 24 h.The next day, approximately 30 worms per plate were transferred onto fresh plates without FUdR seeded with UV-killed bacteria containing 40 µM antimycin A. Survival was monitored after 24 h on three plates per strain.At least three independent experiments were performed for each stress assay.Resilience against the human opportunistic pathogen P. aeruginosa was tested using the pathogen infection assay, performed by SunyBiotech (https://www.sunybiotech.com,accessed on 1 February 2023).In brief, age-synchronized worms of the indicated genotype were grown at 20 • C on NGM plates seeded with OP50 bacteria.Approximately 80 of the synchronized young adults of each strain were transferred to fresh FUdR-containing plates seeded with the P. aeruginosa strain PA14.Viability was scored daily and p-values were determined using the log-rank (Mantel-Cox) test.

RNA Extraction and Quantitative Reverse Transcription PCR (qRT-PCR)
Total RNA was extracted from frozen worm pellets, each containing 200-300 worms, as described previously [30].In each experimental set, a minimum of three biological replicate samples were collected and analyzed independently.Relative mRNA quantities were determined using the comparative Ct method for quantification, with each sample independently normalized to the endogenous reference gene (ama-1).Gene expression data are presented as the average fold change of all biological replicates relative to the control.The primer sequences used for qRT-PCR are shown in Table S2.

Statistics
Graphs were constructed and statistical analyses were performed in GraphPad Prism version 8.0.0 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com/scientificsoftware/prism, accessed on 10 December 2021).Statistical analyses were performed by comparing sample to the appropriate control in the same condition and p-values were determined by a Student's t-test and depicted as follows: **** p < 0.0001; *** p = 0.0001-0.001;** p = 0.001-0.01;* p = 0.01-0.05;ns indicates not significant with a p-value ≥ 0.05.For analyses involving multiple strains and conditions, a two-way ANOVA with a Tukey's multiple comparisons analysis was used to assess the significance and differences between groups.

Conclusions
In C. elegans, the lonp-1 gene encodes a mitochondrial AAA + protease, which has been shown to play a crucial role in protein quality control across species.In this work, we found that worms deficient in lonp-1 have an activated mitochondrial PMK-3/p38 MAPK signaling pathway.In lonp-1 mutants, the enhanced activity of the ATFS-1 transcription factor counteracted activation of the MAPK mt pathway, pointing to the existence of interconnected and fine-tuned defense mechanisms in response to mitochondrial stress.In support of this notion, both ATFS-1 and PMK-3 activities positively regulated the lonp-1 mutants' lifespan and oxidative stress response.In addition to this, we demonstrated that PMK-3 was required for both the basic and lonp-1-induced transcriptional activity of ZIP-2, a bZIP transcription factor that has previously been shown to preserve mitochondrial homeostasis in aged worms independently of innate immunity.Since loss of ZIP-2 activity reduced the lifespan of lonp-1 animals, we suggest that this genetic interaction between PMK-3 and ZIP-2 can influence the ageing process in the face of mitochondrial damage.Finally, the extreme thermotolerance of lonp-1 mutants was found to require PMK-3 activity, uncovering a new role of MAPK mt in the heat stress response when LONP-1 is missing.

Figure 1 .
Figure 1.Activation of the mitochondrial MAPK (MAPK mt ) signaling pathway in lonp-1-deficient worms.(A) Schematic representation of the MAPK mt pathway consisting of the DLK-1/SEK-3/PMK-3 kinase cascade and the downstream target gene, tbb-6.(B) Relative mRNA quantification of endogenous tbb-6 gene expression levels in wt and lonp-1-knockout (ko) mutants.(C) Representative microscopy images and GFP fluorescence quantification of the tbb-6p::gfp reporter in wt and lonp-1(ko) worms.(D,E) Quantification of endogenous tbb-6 gene expression or the tbb-6p::gfp reporter in wt and lonp-1(ko) mutants upon disruption of the MAPK mt signaling components.Experiments were performed on 1-day-old adult worms grown on NGM plates, seeded with E. coli OP50 or with E. coli HT115 bacteria transformed with the indicated RNAi construct (and empty vector as a control) at 20 °C.Scale bar indicates 100 µm.Values are presented as means ± SEM, and asterisks denote statistical significance assessed with an unpaired Student s t-test or with a two-way ANOVA followed by a post hoc Tukey s test, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 1 .
Figure 1.Activation of the mitochondrial MAPK (MAPK mt ) signaling pathway in lonp-1-deficient worms.(A) Schematic representation of the MAPK mt pathway consisting of the DLK-1/SEK-3/PMK-3 kinase cascade and the downstream target gene, tbb-6.(B) Relative mRNA quantification of endogenous tbb-6 gene expression levels in wt and lonp-1-knockout (ko) mutants.(C) Representative microscopy images and GFP fluorescence quantification of the tbb-6 p ::gfp reporter in wt and lonp-1(ko) worms.(D,E) Quantification of endogenous tbb-6 gene expression or the tbb-6 p ::gfp reporter in wt and lonp-1(ko) mutants upon disruption of the MAPK mt signaling components.Experiments were performed on 1-day-old adult worms grown on NGM plates, seeded with E. coli OP50 or with E. coli HT115 bacteria transformed with the indicated RNAi construct (and empty vector as a control) at 20 • C. Scale bar indicates 100 µm.Values are presented as means ± SEM, and asterisks denote statistical significance assessed with an unpaired Student's t-test or with a two-way ANOVA followed by a post hoc Tukey's test, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
), supporting the role of cbp-3 in the induction of tbb-6 in a lonp-1 background.Int.J. Mol.Sci.2023, 24, x FOR PEER REVIEW 6 of 18Thus, in contrast to cbp-1(RNAi), we showed that RNAi against cbp-3 does not cause developmental defects and arrest in worms, possibly due to lack of the HAT domain.When lonp-1 mutant adults were subjected to cbp-3(RNAi), the expression levels of tbb-6p::gfp or the endogenous tbb-6 gene were significantly decreased (Figure3A,B), supporting the role of cbp-3 in the induction of tbb-6 in a lonp-1 background.

2. 4 .
Induction of the MAPK mt Pathway in Lonp-1 Mutants Is Not MAD Pathway-Dependent

Figure 4 .
Figure 4.The MAD pathway does not mediate activation of the MAPK mt pathway in lonp-1 mutants.(A) Representative microscopy images and GFP fluorescence quantification of the tbb-6p::gfp reporter in 1-day adults of wt, cdc-48.2(tm659),lonp-1(ko) and lonp-1(ko);cdc-48.2(tm659), at 20 °C.(B) Relative mRNA quantification of endogenous tbb-6 gene expression levels in 1-day adults of the aforementioned strains, grown on NGM plates seeded with E. coli OP50 or with E. coli HT115 bacteria transformed with the cdc-48.1(RNAi)construct (and empty vector as control), at 20 °C.Scale bar indicate 100 µm.Values were presented as mean ± SEM, and numeric data were analyzed with a two-way ANOVA followed by post hoc Tukey s test, showing no significant (ns) differences.

Figure 4 .
Figure 4.The MAD pathway does not mediate activation of the MAPK mt pathway in lonp-1 mutants.(A) Representative microscopy images and GFP fluorescence quantification of the tbb-6 p ::gfp reporter in 1-day adults of wt, cdc-48.2(tm659),lonp-1(ko) and lonp-1(ko);cdc-48.2(tm659), at 20 • C. (B) Relative mRNA quantification of endogenous tbb-6 gene expression levels in 1-day adults of the aforementioned strains, grown on NGM plates seeded with E. coli OP50 or with E. coli HT115 bacteria transformed with the cdc-48.1(RNAi)construct (and empty vector as control), at 20 • C. Scale bar indicate 100 µm.Values were presented as mean ± SEM, and numeric data were analyzed with a two-way ANOVA followed by post hoc Tukey's test, showing no significant (ns) differences.

Figure 7 .
Figure 7.A schematic representation of the genetic interactions and molecular functions of MAPK mt in worms deficient in the lonp-1 gene.

Figure 7 .
Figure 7.A schematic representation of the genetic interactions and molecular functions of MAPK mt in worms deficient in the lonp-1 gene.