Laranjeiro, R. et al. (2019) International Worm Meeting "Swim exercise improves neuronal health in multiple C. elegans neurodegenerative models."

Harinath, G., Driscoll, M., Laranjeiro, R.

[International Worm Meeting, 2019]

Human exercise provides widespread health benefits, including protection against cardiovascular diseases, stroke, diabetes, cancer, and age-associated decline in muscle and immune function. However, the molecular mechanisms by which exercise protects multiple tissues in the body, in particular those not directly affected by physical activity, are largely unknown. This gap in knowledge is partially due to the lack of short-lived genetic models in which fundamental questions on exercise trans-tissue signaling can be evaluated during the entire aging process. To address this issue, we established a long-term exercise protocol for C. elegans based on multiple daily swim sessions during the first four days of adulthood. Swim exercise both increased mid-life survival in C. elegans and improved the age-dependent decline in pharyngeal and intestinal functions. Moreover, long-term swim exercise improved the cognitive ability of wild type C. elegans in a positive associative learning assay. Specifically, exercised animals increased their ability to associate food with a particular chemical odor (butanone) by an average of 35% when compared to control counterparts. We also sought to determine whether swim exercise in C. elegans could counter challenges of pathological conditions, namely in neurodegenerative disease models. Pan-neuronal expression of a human pro-aggregant Tau fragment leads to impaired motility and morphological defects in GABAergic motor neurons. Both phenotypes in this tauopathy model were significantly improved by swim exercise. Furthermore, we found that chemotactic ability toward benzaldehyde is increased after exercise in a C. elegans model of Alzheimer's disease in which human amyloid-? peptide is pan-neuronally expressed. Finally, using a C. elegans Huntington's disease model in which the human Huntingtin protein with expanded polyglutamine is expressed in the six touch receptor neurons, we determined that exercised animals retained better touch sensitivity during lifespan. These results demonstrate that long-term swim exercise improves neuronal healthspan, at both morphological and functional levels, for different neuronal cell types in multiple C. elegans neurodegenerative models.

Laranjeiro, R. et al. (2017) International Worm Meeting "Does C. elegans benefit from physical exercise? Effects of acute and long-term swim training."

Hewitt, J. E., Meyer, J. N., Laranjeiro, R., Royal, M. A., Hartman, J. H., Vanapalli, S., Rahman, M., Harinath, G., Driscoll, M., Braeckman, B. P.

[International Worm Meeting, 2017]

Physical exercise is the most efficient and accessible intervention that can promote healthy aging in humans. In fact, exercise has been reported to prevent, or mitigate consequences of, a wide range of conditions such as diabetes, cancer, sarcopenia, cardiovascular disease, and neurodegenerative diseases. However, the molecular mechanisms by which exercise can confer systemic health benefits remain poorly understood. We used microcalorimetry to show that C. elegans swimming has a greater energy cost than crawling. Animals that swim continuously for 90 min specifically consume muscle fat supplies and exhibit post-swim locomotory fatigue, with both muscle fat depletion and fatigue indicators recovering within one hour of exercise cessation. qPCR transcript analyses also suggest an increase in fat metabolism during the swim followed by downregulation of specific carbohydrate metabolism transcripts in the hours post-exercise. During a 90 min swim, muscle mitochondria matrix environments become more oxidized as visualized by a localized mito-roGFP reporter. qPCR data support specific transcriptional changes in oxidative stress defense genes during and immediately after a swim. Consistent with potential antioxidant defense induction, we find that a single swim session suffices to confer protection against juglone-induced oxidative stress inflicted 4 hours post-exercise. Exercise adaptation occurs after long-term training. Therefore, we tested different swimming regimens in C. elegans and found that multiple swims per day over several days lead to upregulation of muscle structural genes. Once again, these results are consistent with exercise adaptation described in mammals. Importantly, and taking advantage of the unique characteristics of C. elegans, we show that our training regimen not only leads to changes in body wall muscles but also in neurons. Specifically, mitochondria in touch neurons of exercised worms exhibit a lower oxidation level and an increased turnover rate, both indicators of mitochondrial health. Moreover, long-term swim training in C. elegans delays the functional decline of touch neurons in a polyglutamine (polyQ) aggregation model. These results suggest that physical exercise can promote physiological changes in multiple tissues of C. elegans and open the door to the genetic dissection of exercise systemic health benefits.

Kumar, Sandeep et al. (2021) International Worm Meeting "Enhanced functional restoration through axon regeneration by swimming exercise"

Ghosh-Roy, Anindya, Behera, Sibaram, Dey, Shirshendu, Kumar, Sandeep, Basu, Atrayee

[International Worm Meeting, 2021]

Axonal regeneration is a promising approach to overcome impaired functionality due to axonal injury. In mammals, central nervous system has poor regenerative capacity due to both extrinsic and intrinsic factors. The regenerative capacity also declines significantly with ageing. Therefore, functional axon regeneration in adulthood is challenging and needs more understanding. The pharmacological manipulations are not very successful for functional restoration whereas rehabilitation and physical activity shows improvement. As physical exercise has complex systemic effects, understanding the downstream effectors of physical exercise that is relevant for axon regeneration might be useful. Studying this using simple model organism has several advantages. Using posterior gentle touch circuit neuron (PLM) of Caenorhabditis elegans, we are studying effect of swimming exercise on functional restoration after laser assisted axotomy. We found that a single swimming exercise session of 90 minutes, which is an established paradigm of exercise in worm (Laranjeiro et al., 2017; Laranjeiro et al., 2019) improves functional recovery irrespective of age. However multiple swimming session is required for older worms (A5 stage). Anatomical correlation showed that swimming session improves regrowth initiation, regrowth length and functional connections. We found that the energy sensor kinase AMPK/AAK-2 plays an essential role mediating swimming benefits. Characterizing tissue specific requirement, we found that it has both cell autonomous (PLM neuron) and non-autonomous (muscle) requirement. Pharmacological activation of AMPK/AAK-2 showed enhanced functional restoration similar to swimming. We are studying the downstream molecules and their specific roles in various tissues for swimming mediated functional enhancement which will be helpful for better implementation of this approach. References Laranjeiro R, Harinath G, Burke D, Braeckman BP, Driscoll M (2017) Single swim sessions in C. elegans induce key features of mammalian exercise. BMC Biology 15. Laranjeiro R, Harinath G, Hewitt JE, Hartman JH, Royal MA, Meyer JN, Vanapalli SA, Driscoll M (2019) Swim exercise in Caenorhabditis elegans extends neuromuscular and gut healthspan, enhances learning ability, and protects against neurodegeneration. Proc Natl Acad Sci U S A 116:23829-23839.

Hartman, J.H. et al. (2017) International Worm Meeting "Exercise in Caenorhabditis elegans promotes mitochondrial maintenance and protects against chemical-induced mitotoxicity."

Gordon, K.L., Driscoll, M., Sherwood, D.R., Meyer, J.N., Laranjeiro, R., Hartman, J.H.

[International Worm Meeting, 2017]

Health, disease, and aging are determined by genetic factors, environment, and lifestyle. In humans, environmental contributions to long-term health arise from ambient factors such as environmental chemical exposures and sun exposure, while lifestyle impacts health through mental health and stress, diet, drug usage, and exercise. In laboratory animals, the impact of environment and lifestyle are minimized through carefully controlled experimental conditions, and can therefore be modulated to study the effects of these factors. The effect of exercise on general health has been reported: positive impacts on cognitive function, maintenance of skeletal muscle, and protection from age-related diseases are increasingly recognized. However, molecular mechanisms underlying those protections are not well understood. Furthermore, it is unknown what impacts regular exercise training may have on other health-modifying factors such as toxic exposures from the environment. In this study, we used C. elegans to study the impact of regular exercise training on mitochondrial health and chemical toxicity. For exercise experiments, beginning at L4 stage, animals were transferred to unseeded agar plates without (control) or with liquid (causing worms to swim/thrash) for 90 minutes twice daily. This regimen was carried out for six days, and mitochondrial and toxicity outcomes were tested following exercise on adult day 6 and adult day 10. Preliminary results show that mitochondrial morphology is not significantly different between control and exercise groups at adult day 6 (p=0.64); however, on day 10, control animals have highly fragmented and disorganized mitochondria, while exercised animals exhibit significantly healthier mitochondrial morphology (p=0.0065). Furthermore, mitochondrial respiration significantly differed in spare capacity (p<0.001) on adult day 6, with exercised animals showing increased spare capacity compared to controls. Respiration experiments with day 10 adults are underway; total ATP, mitochondrial DNA copy number, and mitochondrial DNA lesions are also being investigated. Preliminary toxicity experiments showed that exercise-induced changes in mitochondrial health were accompanied by a 30-50% reduction in lethality induced by the mitochondrial toxicants arsenite and rotenone. Together, these data demonstrate that changes in physical activity result in altered mitochondrial health, which extends to protection against chemical toxicants known to damage mitochondria. Ongoing and future experiments will further explore the biochemical and metabolic changes underlying this phenomenon.

Wang, G. et al. (2019) International Worm Meeting "Molecular pathways modulating mitochondrial superoxide stress."

Raval, K., Driscoll, M., Brook, S., Wang, G., Abbott, M., Laranjeiro, R.

[International Worm Meeting, 2019]

Superoxide (O2-) is a toxic byproduct in energy metabolism that can be detoxified by superoxide dismutase (SOD). Excessive O2- can damage macromolecules, a mechanism thought to contribute to aging. Eliminating cytoplasmic SOD from mouse or fly can significantly reduce their lifespans; eliminating mitochondrial SOD (SODmito) from mouse or fly can even cause extreme embryonic lethality. In contrast, eliminating mitochondrial SODs (SOD-2 and SOD-3) from C. elegans does not reduce lifespan or induce embryonic lethality, so we infer that C. elegans is equipped with exceptional tools that counter mitochondrial O2- stress, and we here present the evidence supporting this hypothesis. We first found that mitochondrial O2- stress induces expression of the isocitrate lyase/malate synthase gene (icl-1), the only protein that catalyzes the glyoxylate shunt in C. elegans. Knocking out icl-1 in the SODmito defective worms increases embryonic lethality to ~80%. Since the glyoxylate shunt is unique to C. elegans but lacking in mouse or fly, we predict that the glyoxylate shunt is one novel tool used by C. elegans in battling mitochondrial O2- stress. We also found that eliminating SODmito induces the mitochondrial unfolded protein response (mitoUPR), which is known to induce icl-1 gene expression. Eliminating the central mitoUPR mediator ATFS-1 can cause 100% embryonic lethality in the SODmito null worms-the enhanced severity of atfs-1 mutation as compared to icl-1 deletion alone suggests that more than the glyoxylate shunt induction by the mitoUPR contributes to mitochondrial O2- stress resistance. The hypoxia inducible factor 1 (HIF-1) mediates another signaling pathway that might inhibit mitochondrial O2- production via tuning the electron transport chain. Eliminating enzymes (EGL-9 or VHL-1) that mediate HIF-1 degradation (i.e. activating HIF-1-mediated transcription) can cause nearly 100% embryonic lethality in the SODmito ICL-1 double null worms, which suggests that HIF-1-dependent responses modulated via the EGL-9 and VHL-1 signaling pathway can contribute to the embryonic lethality induced by mitochondrial O2- stress. Overall, we report that the embryonic lethality induced by unmanaged mitochondrial O2- stress can be modulated by multiple molecular pathways.

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Kohn RE, Rand JB, McManus JR, Moulder GL, Duke A, Barstead RJ, Duerr JS

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

A V Bicknell et al. (2002) European Worm Meeting "The C. elegans F47F2.1 gene encodes a cyclic AMP-dependent protein kinase (PK-A) catalytic subunit"

A V Bicknell, M J Fisher, M Tabish, R A Clegg, H H Rees

[European Worm Meeting, 2002]

PK-A mediates all known effects of cyclic AMP on cellular activity in eukaryotes. The holoenzyme is an inactive tetramer of two regulatory (R) subunits and two catalytic (C) subunits. Following binding of cyclic AMP to the R subunits, dissociation of active C-subunits occurs. In mammals, , and isoforms of C-subunit, encoded by different genes, have been identified.

Angelo RG et al. (1997) International C. elegans Meeting "DISCOVERY OF A POTENTIAL ANCHOR/SCAFFOLD PROTEIN FOR C. ELEGANS PROTEIN KINASE A (PKA)"

Angelo RG, Rubin CS

[International C. elegans Meeting, 1997]

C. elegans PKA is composed exclusively of catalytic and regulatory (R) subunits encoded by the kin-1 and kin-2 genes. Since C. elegans lacks other PKA isoforms, this enzyme must disseminate signals carried by cAMP to all cell compartments. Approximately 60% of total R (PKA) is in the particulate fraction of disrupted C. elegans, indicating that protein/protein interactions may diversify PKA signaling by anchoring the kinase at specific intracellular locations. Co-localization of PKA with upstream activators and/or downstream effectors can create target sites for cAMP action. To understand the mechanism of PKA localization in C. elegans, a cDNA expression library was screened with recombinant radiolabeled-R (0.5 nM) to obtain high-affinity binding proteins. A cDNA encoding a novel, 143 kDa A kinase anchor protein (AKAP1) was retrieved and sequenced. The corresponding gene (rap-1) is located in LGII and contains 17 exons. AKAP1 is an acidic protein (pI=4.4) that is unrelated to previously characterized proteins. C. elegans R is avidly bound by both soluble and immobilized fragments of AKAP1. Competition binding studies indicate that C. elegans R is a preferred ligand, whereas mammalian RII and RI isoforms are only weakly sequestered. Scatchard analysis yielded a Kd value of ~10 nM for the R/AKAP1 complex. Deletion mutagenesis, coupled with protein expression in E. coli and in vitro assays, demonstrated that residues 235 to 255 govern high-affinity binding of R by AKAP1. A hydrophobic surface generated by amino acids with branched aliphatic side chains may be a key determinant of R binding activity. Site directed mutagenesis will pinpoint essential residues involved in PKA binding. Studies aimed at identifying the AKAP1 binding site on R are also in progress. High-affinity anti-AKAP1 IgGs are being used to determine (a) whether AKAP1 expression is developmentally-regulated and cell- specific and (b) the relationship between R (PKA) and AKAP1 in vivo.

Hicks, Tara et al. (2021) International Worm Meeting "R-loop-induced irreparable DNA damage in C. elegans meiosis"

Hicks, Tara, Koury, Emily, Williams, Cameron, Smolikove, Sarit

[International Worm Meeting, 2021]

Most DNA-RNA hybrids are formed naturally during transcription and are composed of a nascent RNA strand hybridized to DNA as part of R-loops. The accumulation of these structures in S-phase can result in replication-transcription conflict, an outcome which can lead to the formation of double strand breaks (DSBs). While R-loops' role in mitotically dividing cells has been characterized, there are only a handful of studies describing the effect of R-loops in meiosis and these studies present a complex picture of the outcome of R-loop formation on germ cells. Here we show that DSBs formed by R-loops trigger an altered cellular response to DNA damage. RNase H is an enzyme responsible for degradation of the RNA strand in DNA-RNA hybrids and plays an essential role in preventing this outcome and its deleterious consequences. Using null mutants for the two Caenorhabditis elegans genes encoding for RNase H1 and RNase H2 (hereby rnh mutants), our studies explore the effects of replication stress-induced DNA-RNA hybrid accumulation on meiosis. As expected, rnh mutants exhibit an increase in R-loop formation. Consequently, an elevation of DSBs in germline nuclei is evidenced by the accumulation of RAD-51 foci. Despite no repair mechanism abrogation, rnh mutants fail to repair all DSBs generated, leading to a fragmentation of chromosomes in diakinesis oocytes. By combining our double mutant with a spo-11 null mutation, we show that although replicative defects are the main contributor to the phenotype, R-loops formed in meiosis are likely contributors as well. We present evidence that while rnh mutants accumulate DNA-RNA hybrids and subsequent DSBs may signal a degree of checkpoint activation in mitosis, some damaged nuclei prevail past the checkpoint, enter into meiosis, and remain unrepaired throughout. Moreover, we find no evidence of an increase in apoptosis, which indicates that DNA damage generated by R-loops remain undetected by an apoptotic checkpoint. This data altogether points to DSBs initiated by R-loops representing an irreparable type of DNA damage that evades cellular machineries designed for damage recognition.