Healthspan Genetics and Pharmacology: Molecular Promotion of Healthy Aging
The problem of how to maintain strong health and functionality to counter the impact of aging and age-associated disease is a major public health challenge of our time. C. elegans, which lives ~ 21 days in the lab, is a premier model organism in which longevity and healthspan can be studied to reveal conserved molecular mechanisms operative. We have a strong interest in understanding how C. elegans ages at the tissue and cellular levels, and in defining strategies that extend the period of healthy functionality, called healthspan. By deciphering this fundamental biology in a simple model, we expect to generate the molecular ties for analysis in humans and to suggest plausible therapies in the clinic.
In brief, we showed that basic age-associated tissue degeneration is strikingly reminiscent of that that transpires in human muscle, heart and nervous system; and we find evidence of a significant stochastic component(s) that influences the quality of aging (Herndon et al., 2002). In addition to the influence of reduced insulin signaling pathway in promoting neuronal healthspan (Toth et al., 2012; Vayndorf et al., 2016), we have documented a profound impact of growth factor EGF signaling in promoting locomotory health in adults, in part by signal modulation by novel EGF binding protein (high performance in late age hpl genes) availability (Iwasa, Yu, Xue, & Driscoll, 2010). We remain interested in metabolic changes that can promote health, with work including analysis of the effects of the anti-diabetes drug metformin in C. elegans healthspan promotion (Onken & Driscoll, 2010) and the finding that the deleterious effects of glucose, and the benefits of dietary restriction depend on gluconeogenesis pathways (Onken, Kalinava, & Driscoll, 2020).
Understanding of Single Neuron Aging Over Lifetime in Physiological Context
Subtle changes in neuronal morphology are hypothesized to contribute to age-associated functional decline in human brain and may be exacerbated in neurodegenerative disease. Defining the mechanisms by which adult nervous systems maintain their structural integrity is thus of considerable importance to both normal aging and neurodegenerative disease. Our interests in neuronal healthspan led us to show that morphological features of synapses deteriorate with age and that aberrant dendrite restricting is a dramatic feature of some aging neurons (Toth et al., 2012). Two distinct abnormality types, dendrite branching and novel soma outgrowths, may be differentially controlled at the genetic level. Insulin signaling pathways that modulate aging and healthspan on other fronts also influence dendritic restructuring (Scerbak et al., 2014). Our work suggests that proteostasis crises can modulate older age morphological restructuring in single neurons in the living animal (Vayndorf et al., 2016). We also collaborated with Dr. Zu-Hang Sheng to document touch neuron mitochondrial change over adult life to show phases of increase, maintenance, and decrease in mitochondrial volume and in oxidative stress response capacity (Morsci, Hall, Driscoll, & Sheng, 2016).
Exophers–A Novel Neuronal Trash Disposal Mechanism Relevant to Mechanism of Aggregate and Organelle Transfer Between Cells
While observing aging and stress neurons in physiological context, we discovered a previously unknown capacity of young adult C. elegans neurons to extrude noxious material—neurons can release large soma-sized packets of cellular contents, which can include aggregated human neurodegenerative disease proteins, mitochondria and ER(Arnold, Melentijevic, Smart, & Driscoll, 2018; Melentijevic et al., 2017). The ability to throw out cell contents changes with age and is markedly enhanced if proteostasis is disrupted, suggesting the extrusion of “exophers” and their offensive contents may be a previously undescribed last-resort effort to restore proteostasis. Physiological stresses can markedly enhance exopher production (Arnold, Cooper, Grant, & Driscoll, 2020). We propose that the neuronal extrusion phenomenon constitutes a significant but currently uncharacterized pathway in which stressed neurons maintain their functions by ridding themselves of toxic contents. There are many unanswered questions regarding exopher biology that we are currently seeking to answer, including the identification of cell autonomous and cell nonautonomous signals and pathways that regulate exopher production, the nature of neuronal substrates for extrusion, details of an aggresome-like mechanism that distinguishes neurotoxic species and is needed for exopher-genesis, nature of cellular machines that promote extrusion and scission of the exopher from the parent neuronal soma, how and why organelles are extruded, fates of both extruding neuron and the cell that takes up the neuronal debris, and the mechanism by which neighboring glial-like cell encounters and attempts degradation of exopher contents. Study of the basic biology of exophers in aging and in compromised C. elegans neurons should provide novel insight into the aggregate spreading process relevant to human disease. Our collaborators in this project are our neighbors Dr. Barth Grant and his lab.
The Caenorhabditis Intervention Testing Program
The multi-lab CITP project seeks to identify compounds that robustly extend longevity and/or healthspan across a panel of genetically diverse nematode species/strains from the Caenorhabditis genus. There are two distinctive aspects of the CITP. First, the collaborative group includes three laboratories, that of Dr. Patrick Phillips at the University of Oregon, Dr. Gordon Lithgow at the Buck Institute for Aging Research, and our group at Rutgers, tasked with close reproduction of studies among sites (Lithgow, Driscoll, & Phillips, 2017). Our work underscores both how challenging it actually is to perform precisely the same experiment in different labs, and how critical constant communication is in this effort. Second, the project adds a genetic diversity component to outcome assessment. The idea here is that multiple interventions have been claimed to promote longevity and healthy aging in the reference N2 C. elegans strain. However, real world human populations are heterogeneous in genetic makeup, so attention to intervention impact across the variants of a given species is imperative. CITP tests interventions across a panel of Caenorhabditis strains that encompass genetic diversity on the order of from mouse to humans. The idea is that interventions that improve health or extend longevity across this genetically diverse test set should target highly conserved pathways and should take priority for efficacy testing in higher organisms. Some intervention targets are suggested by outside scientists interested in the potential of compounds they identified. Overall work has produced data on multiple interventions and identified anti-amyloid agent Thioflavin T as a robust anti-aging intervention (Lucanic et al., 2017). The link to CITP website
As the aging and neuroscience communities progress through the era of an intensive drive to address Alzheimer’s disease, CITP is focusing on assessing anti-AD strategies using novel sets of diverse species genetically engineered to have “at risk” neurons and on testing compounds specifically proposed to have anti-AD or anti-aging impacts.
A C. elegans Exercise Model With Which to Decipher Molecular Mechanisms of Systemic and Sustained Benefits of Exercise
Exercise exerts remarkably powerful effects on metabolism and health, with anti-disease and anti-aging outcomes. To anchor a new field in molecular genetics of exercise and aging, we showed that a single swim exercise confers physiological changes that increase robustness (Laranjeiro, Harinath, Burke, Braeckman, & Driscoll, 2017), and we showed that extended swim training can both confer molecular muscle adaptation similar to mammalian exercise and induce long-lasting benefit to muscle, neurons, intestine and pharynx (Hartman et al., 2018; Laranjeiro et al., 2019). C. elegans swim training is also protective in four different neurodegenerative disease models and in a simple memory paradigm, which anchors our capacity to decipher cell and tissue circuitry by which exercise benefits generate resilience across tissues and against disease-inducing genetics. We have found that some exercise benefits are maintained for long periods after training has ended (Hartman et al., 2018)—we are using the C. elegans model to determine the molecular underpinnings of sustained physiological outcomes of these “legacy”-life effects. Our ongoing work is focused on the identification of molecular factors that maintain the physiological benefits of exercise late into life after exercise training has ended.
The development of a basic exercise model in which we can reproducibly generate systemic long-term health benefits that can be quantitatively measured in multiple tissue types, and can be readily perturbed using molecular genetics approaches opens exciting new avenues in deciphering resilience outcomes of exercise and determining the factors that promote significant anti-aging anti-disease outcomes.
Worms in Space—addressing challenges of extended microgravity, one neuron at a time
Our work on age-associated muscle decline and exercise-promoted maintenance of health stimulated interaction with colleagues who consider these aspects of biology for space travel. One conversation led to another, and we were offered an incredible opportunity to send C. elegans to the International Space Station for study as part of a multinational collaborative project – the Molecular Muscle Experiment (MME) (https://www.mme-spaceworms.com/). The MME aims to study the molecular mechanisms involved in muscle mass loss during spaceflight (astronauts can lose up to 40% of their muscle after 6 months in space).
On earth, stressed neurons can exhibit striking morphological changes including resprouting and new process outgrowth. As little was known of the impact of microgravity conditions on the integrity of individual adult neurons consequent to spaceflight, especially at the singe neuron level, we sent C. elegans adults expressing specific fluorescently labeled neurons to the ISS. Our goal was to examine neuronal morphology consequent to flight experience, relevant to the maintenance of muscle function and to the challenges of long-term adult experience in extended space travel.
We were part of the MME team that sent multiple C. elegans strains to the International Space Station (ISS) aboard a SpaceX Falcon 9 rocket from Florida’s Kennedy Space Center; animals were frozen after 5 days of adult life on the ISS and returned for our analysis.
We found that animals that lived 5 days of their adult life on the ISS exhibited considerable morphological remodeling of adult neurons when compared to ground control animals (Laranjeiro et al., 2021). Our results indicate hyperbranching as a common response of adult neurons to spaceflight. We also found that, in the presence of a neuronal proteotoxic stress, spaceflight promotes a remarkable accumulation of neuronal-derived waste in the surrounding tissues (especially hypodermis), suggesting an impaired transcellular degradation of debris that is released from neurons. Our data reveal that spaceflight can significantly affect adult neuronal morphology and clearance of neuronal trash, highlighting the need to carefully assess the risks of long-duration spaceflight on the nervous system and to develop countermeasures to protect human health during space exploration.
Mechanisms of Necrotic Cell Death in Injury and Disease
Brain injury and neurodegenerative conditions are often associated with exacerbated ion channel activity, inducing necrotic cell death. We pioneered study of neuronal necrosis in C. elegans. Novel mutant forms of the channel encoded by mec-4(d) mutants specify hyper-activated ion channels that conduct excess ions, resulting in neuronal swelling and cell death (Driscoll & Chalfie, 1991). We developed this genetic necrosis model to show that elevated ion conductance is a critical component of necrosis initiation, and, unexpectedly, that Ca+2 influx is essential for neurotoxicity (originally this channel family was thought to be Na+-selective) (Bianchi et al., 2004). We conducted powerful genetic interaction screens to identify molecules critical for the progression through necrosis using the MEC-4(d) model (Zhang et al., 2008), and we developed a second model of C. elegans excitotoxicity in which we disrupted glutamate transporters to elevate endogenous glutamate levels to mimic the neurotoxic condition of glutamate excitotoxicity in humans (Mano, Straud, & Driscoll, 2007). Our genetic enhancer and suppressor screens defined a downstream necrosis pathway that requires channel hyperactivation, calcium influx (Bianchi et al., 2004; Royal et al., 2005; Zhang et al., 2008), catastrophic release of stored calcium in the ER compartment of the cell (Mano et al., 2007), and the later activation of lysosomal cathepsin enzymes that degrade cellular proteins (Syntichaki, Xu, Driscoll, & Tavernarakis, 2002); as well as nicalin-dependent mechanism of channel surface expression (Kamat, Yeola, Zhang, Bianchi, & Driscoll, 2014). Overall, our work in this area has been appreciated to have provided the earliest evidence that necrosis is not merely a run-away breakdown of the neurons, but rather can be modulated by specific regulated steps, which are logical targets for therapeutic intervention against stroke.
Molecular Mechanisms of Mechanotransduction—Touch and Feeling at the Molecular Level. The process by which mechanical signals, such as pressure or force, are interpreted to direct biological responses is not well understood. I contributed to the identification of a new family of ion channels (the C. elegans degenerin channels, some of which (MEC-4 and MEC-10) (Driscoll & Chalfie, 1991) normally function as the core mediators of touch transduction in specialized mechanosensory neurons, and proprioception in C. elegans (UNC-8) (Tavernarakis, Shreffler, Wang, & Driscoll, 1997). We contributed some of the first in vivo genetic structure/function studies that defined the MEC channel pore (Hong & Driscoll, 1994; Hong, Mano, & Driscoll, 2000), used in vivo calcium measurements to show that the MEC channel is the likely primary sensor of force in touch sensation with collaborator Dr. William Schafer (Suzuki et al., 2003), identified an unexpected Ca2+ current associated with the MEC channel (Bianchi et al., 2004), and defined factors that regulate DEG/ENaC trafficking (Royal et al., 2005). Overall, our work advanced understanding of a novel ion channel class with members that can recognize and respond to force to initiate neuronal responses. More recently we have become interested in the possibility that mechanical signaling can contribute to animal-wide proteostasis regulation.
We often encounter questions that require establishment of new experimental measures to address C. elegans aging. We established new methods that can be used to quantitate age-associated decline: rapid in vivo quantitation of fluorescent age pigment levels (the conserved accumulation of lipofuscin, fluorescent signature of DR state also identified) (Gerstbrein, Stamatas, Kollias, & Driscoll, 2005), in collaboration with Dr. Zhen Yan mitochondrial health (Laker et al., 2014) and high-level computer vision analysis of swimming vigor decline (C. elegans Swim Test CeleST) (Restif et al., 2014). We have been collaborative partners with Dr. Siva Vanapalli’s lab at Texas Tech University in the development of microfluidics Nemaflex, in which animal strength can be measured (Rahman et al., 2018); NemaLife, a crawling-based microfluidics device that enables lifespan and healthspan to be analyzed with easy media change and natural progeny elimination features (Rahman et al., 2020), and burrowing capacity assays as a vigor indicator (Lesanpezeshki et al., 2019).
Arnold, M. L., Cooper, J., Grant, B. D., & Driscoll, M. (2020). Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans. J Vis Exp(163). doi:10.3791/61368
Arnold, M. L., Melentijevic, I., Smart, A. J., & Driscoll, M. (2018). Q&A: Trash talk: disposal and remote degradation of neuronal garbage. BMC Biol, 16(1), 17. doi:10.1186/s12915-018-0487-6
Bianchi, L., Gerstbrein, B., Frokjaer-Jensen, C., Royal, D. C., Mukherjee, G., Royal, M. A., . . . Driscoll, M. (2004). The neurotoxic MEC-4(d) DEG/ENaC sodium channel conducts calcium: implications for necrosis initiation. Nat Neurosci, 7(12), 1337-1344. doi:10.1038/nn1347
Driscoll, M., & Chalfie, M. (1991). The mec-4 gene is a member of a family of Caenorhabditis elegans genes that can mutate to induce neuronal degeneration. Nature, 349(6310), 588-593. doi:10.1038/349588a0
Gerstbrein, B., Stamatas, G., Kollias, N., & Driscoll, M. (2005). In vivo spectrofluorimetry reveals endogenous biomarkers that report healthspan and dietary restriction in Caenorhabditis elegans. Aging Cell, 4(3), 127-137. doi:10.1111/j.1474-9726.2005.00153.x
Hartman, J. H., Smith, L. L., Gordon, K. L., Laranjeiro, R., Driscoll, M., Sherwood, D. R., & Meyer, J. N. (2018). Swimming Exercise and Transient Food Deprivation in Caenorhabditis elegans Promote Mitochondrial Maintenance and Protect Against Chemical-Induced Mitotoxicity. Sci Rep, 8(1), 8359. doi:10.1038/s41598-018-26552-9
Herndon, L. A., Schmeissner, P. J., Dudaronek, J. M., Brown, P. A., Listner, K. M., Sakano, Y., . . . Driscoll, M. (2002). Stochastic and genetic factors influence tissue-specific decline in ageing C. elegans. Nature, 419(6909), 808-814. doi:10.1038/nature01135
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Hong, K., Mano, I., & Driscoll, M. (2000). In vivo structure-function analyses of Caenorhabditis elegans MEC-4, a candidate mechanosensory ion channel subunit. J Neurosci, 20(7), 2575-2588. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/10729338
Iwasa, H., Yu, S., Xue, J., & Driscoll, M. (2010). Novel EGF pathway regulators modulate C. elegans healthspan and lifespan via EGF receptor, PLC-gamma, and IP3R activation. Aging Cell, 9(4), 490-505. doi:10.1111/j.1474-9726.2010.00575.x
Kamat, S., Yeola, S., Zhang, W., Bianchi, L., & Driscoll, M. (2014). NRA-2, a nicalin homolog, regulates neuronal death by controlling surface localization of toxic Caenorhabditis elegans DEG/ENaC channels. J Biol Chem, 289(17), 11916-11926. doi:10.1074/jbc.M113.533695
Laker, R. C., Xu, P., Ryall, K. A., Sujkowski, A., Kenwood, B. M., Chain, K. H., . . . Yan, Z. (2014). A novel MitoTimer reporter gene for mitochondrial content, structure, stress, and damage in vivo. J Biol Chem, 289(17), 12005-12015. doi:10.1074/jbc.M113.530527
Laranjeiro, R., Harinath, G., Burke, D., Braeckman, B. P., & Driscoll, M. (2017). Single swim sessions in C. elegans induce key features of mammalian exercise. BMC Biol, 15(1), 30. doi:10.1186/s12915-017-0368-4
Laranjeiro, R., Harinath, G., Hewitt, J. E., Hartman, J. H., Royal, M. A., Meyer, J. N., . . . 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(47), 23829-23839. doi:10.1073/pnas.1909210116
Laranjeiro, R., Harinath, G., Pollard, A. K., Gaffney, C. J., Deane, C. S., Vanapalli, S. A., . . . Driscoll, M. (2021). Spaceflight affects neuronal morphology and alters transcellular degradation of neuronal debris in adult Caenorhabditis elegans. iScience, 24(2), 102105. doi:10.1016/j.isci.2021.102105
Lesanpezeshki, L., Hewitt, J. E., Laranjeiro, R., Antebi, A., Driscoll, M., Szewczyk, N. J., . . . Vanapalli, S. A. (2019). Pluronic gel-based burrowing assay for rapid assessment of neuromuscular health in C. elegans. Sci Rep, 9(1), 15246. doi:10.1038/s41598-019-51608-9
Lithgow, G. J., Driscoll, M., & Phillips, P. (2017). A long journey to reproducible results. Nature, 548(7668), 387-388. doi:10.1038/548387a
Lucanic, M., Plummer, W. T., Chen, E., Harke, J., Foulger, A. C., Onken, B., . . . Phillips, P. C. (2017). Impact of genetic background and experimental reproducibility on identifying chemical compounds with robust longevity effects. Nat Commun, 8, 14256. doi:10.1038/ncomms14256
Mano, I., Straud, S., & Driscoll, M. (2007). Caenorhabditis elegans glutamate transporters influence synaptic function and behavior at sites distant from the synapse. J Biol Chem, 282(47), 34412-34419. doi:10.1074/jbc.M704134200
Melentijevic, I., Toth, M. L., Arnold, M. L., Guasp, R. J., Harinath, G., Nguyen, K. C., . . . Driscoll, M. (2017). C. elegans neurons jettison protein aggregates and mitochondria under neurotoxic stress. Nature, 542(7641), 367-371. doi:10.1038/nature21362
Morsci, N. S., Hall, D. H., Driscoll, M., & Sheng, Z. H. (2016). Age-Related Phasic Patterns of Mitochondrial Maintenance in Adult Caenorhabditis elegans Neurons. J Neurosci, 36(4), 1373-1385. doi:10.1523/JNEUROSCI.2799-15.2016
Onken, B., & Driscoll, M. (2010). Metformin induces a dietary restriction-like state and the oxidative stress response to extend C. elegans Healthspan via AMPK, LKB1, and SKN-1. PLoS One, 5(1), e8758. doi:10.1371/journal.pone.0008758
Onken, B., Kalinava, N., & Driscoll, M. (2020). Gluconeogenesis and PEPCK are critical components of healthy aging and dietary restriction life extension. PLoS Genet, 16(8), e1008982. doi:10.1371/journal.pgen.1008982
Rahman, M., Edwards, H., Birze, N., Gabrilska, R., Rumbaugh, K. P., Blawzdziewicz, J., . . . Vanapalli, S. A. (2020). NemaLife chip: a micropillar-based microfluidic culture device optimized for aging studies in crawling C. elegans. Sci Rep, 10(1), 16190. doi:10.1038/s41598-020-73002-6
Rahman, M., Hewitt, J. E., Van-Bussel, F., Edwards, H., Blawzdziewicz, J., Szewczyk, N. J., . . . Vanapalli, S. A. (2018). NemaFlex: a microfluidics-based technology for standardized measurement of muscular strength of C. elegans. Lab Chip, 18(15), 2187-2201. doi:10.1039/c8lc00103k
Restif, C., Ibanez-Ventoso, C., Vora, M. M., Guo, S., Metaxas, D., & Driscoll, M. (2014). CeleST: computer vision software for quantitative analysis of C. elegans swim behavior reveals novel features of locomotion. PLoS Comput Biol, 10(7), e1003702. doi:10.1371/journal.pcbi.1003702
Royal, D. C., Bianchi, L., Royal, M. A., Lizzio, M., Jr., Mukherjee, G., Nunez, Y. O., & Driscoll, M. (2005). Temperature-sensitive mutant of the Caenorhabditis elegans neurotoxic MEC-4(d) DEG/ENaC channel identifies a site required for trafficking or surface maintenance. J Biol Chem, 280(51), 41976-41986. doi:10.1074/jbc.M510732200
Scerbak, C., Vayndorf, E. M., Parker, J. A., Neri, C., Driscoll, M., & Taylor, B. E. (2014). Insulin signaling in the aging of healthy and proteotoxically stressed mechanosensory neurons. Front Genet, 5, 212. doi:10.3389/fgene.2014.00212
Suzuki, H., Kerr, R., Bianchi, L., Frokjaer-Jensen, C., Slone, D., Xue, J., . . . Schafer, W. R. (2003). In vivo imaging of C. elegans mechanosensory neurons demonstrates a specific role for the MEC-4 channel in the process of gentle touch sensation. Neuron, 39(6), 1005-1017. doi:10.1016/j.neuron.2003.08.015
Syntichaki, P., Xu, K., Driscoll, M., & Tavernarakis, N. (2002). Specific aspartyl and calpain proteases are required for neurodegeneration in C. elegans. Nature, 419(6910), 939-944. doi:10.1038/nature01108
Tavernarakis, N., Shreffler, W., Wang, S., & Driscoll, M. (1997). unc-8, a DEG/ENaC family member, encodes a subunit of a candidate mechanically gated channel that modulates C. elegans locomotion. Neuron, 18(1), 107-119. doi:10.1016/s0896-6273(01)80050-7
Toth, M. L., Melentijevic, I., Shah, L., Bhatia, A., Lu, K., Talwar, A., . . . Driscoll, M. (2012). Neurite sprouting and synapse deterioration in the aging Caenorhabditis elegans nervous system. J Neurosci, 32(26), 8778-8790. doi:10.1523/JNEUROSCI.1494-11.2012
Vayndorf, E. M., Scerbak, C., Hunter, S., Neuswanger, J. R., Toth, M., Parker, J. A., . . . Taylor, B. E. (2016). Morphological remodeling of C. elegans neurons during aging is modified by compromised protein homeostasis. NPJ Aging Mech Dis, 2. doi:10.1038/npjamd.2016.1
Zhang, W., Bianchi, L., Lee, W. H., Wang, Y., Israel, S., & Driscoll, M. (2008). Intersubunit interactions between mutant DEG/ENaCs induce synthetic neurotoxicity. Cell Death Differ, 15(11), 1794-1803. doi:10.1038/cdd.2008.114