Steven J Husson et al. (2009) European Worm Neurobiology Meeting "Optogenetics tools to dissect a nociceptive neuronal network and neuropeptide signalling pathways in C. elegans"

Alexander Gottschalk, Jana Liewald, Steven J Husson, Liliane Schoofs, Martin Brauner, Wagner Steuer Costa

[European Worm Neurobiology Meeting, 2009]

Light-gated cation channels such as the blue light-activated depolarizing Channelrhodopsin-2 (ChR2), allow optical activation of individual neurons of live and behaving C. elegans at the millisecond time-scale in a non-invasive manner (Nagel et al., 2005, Zhang et al., 2007, Liewald et al., 2008). This optogenetics approach paves the way for further functional dissection of peptide signalling pathways or individual neuronal networks in a detail that is not possible in higher organisms. The huge advantage is that we can specifically stimulate the sensory input neurons, while other potentially contributing neurons are kept silent. The sensory PVD neurons that envelop the nematode with highly branched dendritic arbors are involved in harsh touch nociception. Expression and activation of ChR2 in PVD results in a forward escape movement and sometimes a reversal. These results are in line with the fact that the PVD neurons make synaptic contacts with the locomotory command interneurons PVC and AVA that regulate forward and backward movement, respectively. Using electrophysiology, we will assess the physiology of the PVD cells, as well as the downstream interneurons in response to photoactivation, while the involvement of different ion channels, receptors or neurotransmitters will be assessed by RNAi. We are also investigating the flp-15 and nlp-38 neuropeptidergic signalling pathways by optogenetics. Neuropeptide release can be triggered by photo-activating the respective neurons in an acute fashion while effects on behaviour can be observed at the same time. This way, we can correlate neuropeptide action with acute behavioural changes or effects, about which very limited knowledge is currently available in any system. References Liewald JF, Brauner M, Stephens GJ, Bouhours M, Schultheis C, Zhen M, and Gottschalk A (2008) Optogenetic analysis of synaptic function. Nat Methods, 5, 895-902. Nagel G, Brauner M, Liewald JF, Adeishvili N, Bamberg E, and Gottschalk A (2005) Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr Biol, 15, 2279-2284. Zhang F, Wang LP, Brauner M, Liewald JF, Kay K, Watzke N, Wood PG, Bamberg E, Nagel G, Gottschalk A, and Deisseroth K (2007) Multimodal fast optical interrogation of neural circuitry. Nature, 446, 633-639.

Husson, Steven J et al. (2009) International Worm Meeting "Optogenetics tools to dissect small neuronal networks and neuropeptide signalling systems."

Liewald, Jana, Husson, Steven J, Schultheis, Christian, Schoofs, Liliane, Gottschalk, Alexander, Brauner, Martin, Erbguth, Karen, Schedletzky, Thorsten

[International Worm Meeting, 2009]

Light-gated ion channels or pumps such as the blue light-activated depolarizing Channelrhodopsin (ChR2) and the yellow light-driven hyperpolarizing Halorhodopsin (HR) allow optical activation or inhibition in muscles and neurons of live and behaving C. elegans (Zhang et al., 2007). Furthermore, inward currents evoked by either ChR2 or HR, as well as muscle currents in response to activating ChR2 in motor neurons, can be directly measured by electrophysiology, while photo-evoked body contraction or elongation of the animal could be monitored at the behavioural level (Nagel et al., 2005; Liewald et al., 2008). These state-of-the-art technologies pave the way for further functional dissection of individual neuronal networks in a detail that is not possible in higher organisms. Doing so, we are investigating some defined neuropeptidergic signalling pathways. Neuropeptide release can be triggered by photo-activating the respective neurons in an acute fashion while effects on behaviour can be observed at the same time. This way, we can correlate neuropeptide action with acute behavioural changes or effects, about which very limited knowledge is currently available in any system. While higher organisms display millions of contributing neurons, only a handful of neurons take part in individual neuronal networks in C. elegans. This opens the possibility to study the contribution of each neuron to the function of a small network, for example involved in nociception. The huge advantage of our optogenetics tools is that we can specifically stimulate the sensory input neurons, while other potentially contributing neurons are kept silent. The involvement of different ion channels, receptors or neurotransmitters can be assessed by using different genetic backgrounds, while the physiological properties of each individual neuron will be monitored by electrophysiology. Liewald JF, Brauner M, Stephens GJ, Bouhours M, Schultheis C, Zhen M, and Gottschalk A (2008) Optogenetic analysis of synaptic function. Nat Methods, 5, 895-902. Nagel G, Brauner M, Liewald JF, Adeishvili N, Bamberg E, and Gottschalk A (2005) Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr Biol, 15, 2279-2284. Zhang F, Wang LP, Brauner M, Liewald JF, Kay K, Watzke N, Wood PG, Bamberg E, Nagel G, Gottschalk A, and Deisseroth K (2007) Multimodal fast optical interrogation of neural circuitry. Nature, 446, 633-639.

Yoko Honda et al. (2005) International Worm Meeting "Study of space-flight effect on the aging of C. elegans in the International C. elegans Experiment(ICE)-First"

Shuji Honda, Noriaki Ishioka, Yoko Honda

[International Worm Meeting, 2005]

As exposures to the space environment become longer, information regarding the effects on biological aging will become important. We have not yet fully clarified aging processes in space environments. The aging process and lifespan are affected by various kinds of environmental factors including oxygen concentration (1), temperature and radiation. The aging phenomena that we usually see occur under certain conditions on the ground. Space environments differ from ground environments especially with regard to the radiation spectrum and gravity. We participated in the International C. elegans Experiment(ICE)First that examined the effects of a 10-day space flight on the nematode C. elegans. C. elegans has frequently been used for study of aging because of its short lifespan. Recently, Morley et al. reported that Huntington's-like polyglutamine (polyQ)-repeat proteins expressed in the muscle of C. elegans form aggregates as the animals age, and that this aggregation is delayed in long-lived mutants(2). We measured the polyQ aggregates in these nematodes after space flight as an aging marker. Herndon et al. showed that the sarcomere orientation in the body-wall muscle becomes disorderly as the animals age(3). We also observed the sarcomere orientation in the body-wall muscle of these nematodes after space flight as another aging marker. Acknowledgement: We thank Dr. R. L. Morimoto (Northwestern University) for providing us polyQ-YFP C. elegans strains. We also thank CGC for other strains. ICE-First was mainly conducted by the French Space Agency (CNES), with support of the European Space Agency and the Space Research Organization of the Netherlands. We are grateful to Dr. Michel Viso (CNES), Dr. K. Kuriyama (JAXA) and Dr. A. Higashitani (Tohoku University) for their support and suggestion for our experiment. References: 1) Honda S., Ishii N, Suzuki K, Matsuo M. J Gerontol. 48:B57-61. 1993 2) James F. Morley, Heather R. Brignull, Jill J. Weyers, and Richard I. Morimoto. Proc. Natl. Acad. Sci. USA, 99: 10417-10422. 2002 3) Herndon LA, Schmeissner PJ, Dudaronek JM, Brown PA, Listner KM, Sakano Y, Paupard MC, Hall DH, Driscoll M. Nature. 419:808-814. 2002

Susana M Garcia et al. (2005) International Worm Meeting "Novel Molecular Modulators of Protein Homeostasis in C. Elegans"

Susana M Garcia, Margarida Amaral, Richard Morimoto

[International Worm Meeting, 2005]

Protein folding in the cells highly crowded macromolecular environment is a complex process. Many factors can affect the folded state of proteins, these include alterations of the cellular environment and the protein folding machinery. Disruptions in the folding process result in accumulation of misfolded proteins and the formation of aggregates, which have been linked to a number of human diseases such as polyglutamine (polyQ) expansion disorders. Our lab has previously expressed polyQ repeats as YFP (yellow fluorescent protein) fusion proteins in body wall muscle cells in C. elegans (Satyal et al. 2000; Morley et al 2002). These animals showed a polyQ length dependent aggregation phenotype. At intermediate polyQ length, animals show age-dependent transition from soluble to aggregated polyQ, optimal for screening. Previously, our C. elegans strains expressing different polyQ lengths were used in a genome-wide RNAi screen and 186 modulators of polyQ aggregation were identified (Nollen et al 2004). Recently, we performed a forward genetic screen, using EMS mutagenesis, to identify additional modifiers of protein homeostasis, including weaker alleles. Two recessive mutations found in our screen, rm7 and rm8, cause an enhancement of the aggregation phenotype in our polyQ C. elegans strains. These were mapped to chromosomes IV and X respectively. rm7 is a deletion in the C. elegans gene unc-30. UNC-30 is a neuronal transcription factor that regulates GABA expression in a subset of motor neurons. The effect of rm7 and other mutants of this pathway in modulating polyQ aggregation establishes a link between neuronal signaling and protein homeostasis in muscle cell.

Ao-lin Hsu et al. (2003) International Worm Meeting "Regulation of Aging and Age-related Disease by DAF-16 and HSF-1"

Ao-Lin Hsu, Cynthia Kenyon, Coleen Murphy

[International Worm Meeting, 2003]

The C. elegans heat-shock transcription factor HSF-1, which regulates the heat-shock response, plays a key role in aging. Reducing hsf-1 activity accelerates tissue aging and shortens lifespan (1), and we find that overexpressing hsf-1 extends lifespan. Thus HSF-1 appears to function in normal animals to slow the rate of aging and extend lifespan. Interestingly, HSF-1, like the transcription factor DAF-16, is required for daf-2-insulin/IGF-1 receptor mutations to extend lifespan. Conversely, DAF-16 is required for overexpression of HSF-1 to extend lifespan. Why are both DAF-16 and HSF-1 required for lifespan extension? We find that although both HSF-1 and DAF-16 can regulate expression of some of their target genes independently of one another, both proteins are required to activate expression of a specific subset of genes. This subset includes the small heat shock proteins (sHSPs), whose activites, in turn, promote longevity. A fundamental question in the biology of aging is what couples the normal aging process to age-related disease. When polyglutamine repeat proteins are expressed in C. elegans, they form Huntingtons like aggregates. Interestingly, the accumulation of these aggregates increases with age, and is delayed by DAF-2 insulin/IGF-1 pathway mutations that extend lifespan (2). We find that the small heat-shock proteins delay the onset of polyglutamine-expansion protein aggregation. Thus, possibly by preventing the aggregation of damaged or misfolded proteins, the small heat shock proteins may directly link the normal aging process to this age-related disease.(1) Garigan et al., Genetics vol. 161, 1101-12, 2002; (2) Morley et al., PNAS vol. 99, 10417-22, 2002.

Yoko Honda et al. (2006) East Asia C. elegans Meeting "Study of space-flight effect on the aging of C. elegans in the International C. elegans Experiment (ICE)-First."

Akira Higashibata, Noriaki Ishioka, Masashi Tanaka, Shuji Honda, Yoko Honda

[East Asia C. elegans Meeting, 2006]

As exposures to the space environment become longer, information regarding the effects on biological aging will become important. We have not yet fully clarified aging processes in space environments. The aging process and lifespan are affected by various kinds of environmental factors including oxygen concentration<SUP>1)</SUP>, temperature and radiation. The aging phenomena that we usually see occur under certain conditions on the ground. Space environments differ from ground environments especially with regard to the radiation spectrum and gravity. We participated in the International Caenorhabditis elegans Experiment (ICE)-First that examined the effects of a 10-day space flight on the nematode C. elegans. C. elegans has frequently been used for study of aging because of its short lifespan and powerful genetic system. Recently, Morley et al. reported that Huntington's-like polyglutamine (polyQ)-repeat proteins expressed in the muscle of C. elegans form aggregates as the animals age, and that this aggregation is delayed in long-lived mutants<SUP>2)</SUP>. We measured the polyQ aggregates in these nematodes after space flight as an aging marker. We also examined the effects of space flight on the gene expression by DNA microarray analysis and real time PCR. The expression of several genes specifically for nervous systems was suppressed in space-flown animals, suggesting inactivation of nervous functions in space. The relationship between aging and changes in the gene expression in space will be discussed. 1) Honda S et al. Oxygen-dependent perturbation of life span and aging rate in the nematode. J Gerontol. 48:B57-61, 1993. 2) James F et al. The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA, 99: 10417-10422, 2002.

Ellen Nollen et al. (2003) International Worm Meeting "Genome-wide RNAi screen for genes involved in polyglutamine aggregate formation"

Ronald H Plasterk, Richard I Morimoto, Gijs van Haaften, Susana Garcia, Ellen Nollen

[International Worm Meeting, 2003]

Accumulation of polyglutamine containing proteins into intracellular aggregates is associated with various CAG trinucleotide expansion disorders, including neurodegenerative diseases such as Huntingtons disease and spinocerebellar ataxias. The aggregation properties of polyglutamine proteins are directly related to the length of the polyglutamine stretch. With polyglutamine stretches above a threshold of approximately 30 glutamine residues, the aggregation rate increases with increasing numbers of glutamine residues. The length-dependent kinetics of aggregation recapitulates the length-dependent increase in cellular toxicity and age of onset of disease. Expression of polyglutamine stretches of 0, 24, 33, 35, 40, 44, and 82 glutamine residues as YFP-fusion proteins under control of the muscle specific unc-54 promoter in C. elegans reconstitutes the length and age dependence of aggregation. 1 Whereas worms expressing YFP-fusions with polyglutamine stretches up to 24 (Q24) show a diffuse YFP staining in all muscle cells, Q82 animals show a punctate staining in most of the cells. Interestingly, all Q lengths show variability in aggregation within individual animals, depending on the cell and the age of the worm, which is influenced by the genetic background of the worms. For example, aggregation of Q82-YFP is greatly delayed in the aging mutant age-11. This heterogeneity of aggregation suggests that genes exist that influence the formation of polyglutamine aggregates. We are using a genome-wide RNAi screen to identify genes involved in polyglutamine aggregation. In a candidate gene approach with RNAi against genes encoding molecular chaperones and molecules involved in proteins degradation, we have already identified genes that may play a role in aggregate formation. 1Morley JF, Brignull HR, Weyers JJ, Morimoto RI. Proc Natl Acad Sci U S A 2002 99(16):10417-22

Anusha Narayan et al. (2007) International Worm Meeting "Transfer at C. elegans synapses."

Shawn Lockery, Paul Sternberg, Tod Thiele, Gilles Laurent, Anusha Narayan

[International Worm Meeting, 2007]

Neural circuits in C. elegans have been studied using light and electron microscopic techniques, focal laser ablations and, more recently, calcium imaging techniques. For a clearer functional understanding of these circuits, however, some knowledge of the rules of synaptic information transfer is required. How is the dynamic range of the post-synaptic neuron set? What are the mechanisms for synaptic integration and gain control? Questions such as these can best be answered by monitoring or controlling connected pre- and post-synaptic neurons simultaneously. We chose to focus on the synapses between the AFD/ASER and AIY neurons, since the functional relevance of these neurons has been established and there is anatomical evidence for synapses between them. Channelrhodopsin-2 (chR2) is a light activated cation channel with fast kinetics (order of milliseconds1). We express chR2 under a neuron-specific promoter2 in the presynaptic neuron, and use whole-cell patch-clamp recording techniques to monitor membrane voltage or currents in the postsynaptic neuron. We are first calibrating the response to light of chR2-expressing neurons. Currently, we are calibrating this light response in worms expressing chR2 in ASER. We have observed depolarizations of 10-30 mV in response to light (450-490 nm) in current clamp, and inward currents of 5-10 pA in voltage-clamp. We have also seen evidence of spontaneous synaptic activity, in the form of discrete synaptic events (potentials or currents) with different reversal potentials (some depolarizing, others Cl-dependent). We are beginning to characterize the ASER-AIY synapse, and will then move on to the AFD-AIY synapse. References 1. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K. Millisecond-timescale, genetically targeted optical control of neural activity, Nat. Neurosci. 8 (2005), pp. 1263-1268. 2. Nagel G, Brauner M, Liewald JF, Adeishvili N, Bamberg E, Gottschalk A. Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr. Biol. 15 (2005), pp. 2279-2284.

Ashkavand, Z. et al. (2019) International Worm Meeting "Altered ER-mitochondrial calcium homeostasis promotes proteostasis collapse in presenilin mutants."

Norman, K., Sarasija, S., Ryan, K., Laboy, J., Ashkavand, Z.

[International Worm Meeting, 2019]

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease. A hallmark of AD is the disruption of proteostasis that leads to the accumulation of protein aggregates such as amyloid-beta (Abeta) in the brain of patients. Familial AD is largely caused by mutations in the presenilin genes; however their role in AD is not understood. We have found that mutations in the C. elegans presenilin gene (sel-12 mutants) cause elevated ER to mitochondria Ca2+ signaling, which leads to an increase in mitochondrial Ca2+ content that results in increased oxidative stress. This, in turn, promotes neurodegeneration. To understand the cellular mechanisms driving neurodegeneration, we have utilized two transgenic models expressing metastable proteins to evaluate protein homeostasis in sel-12 mutants. These include animals expressing 1) polyQ peptide fused to YFP (Q35::YFP)1or 2) human Abeta2. Expression of Q35::YFP shows diffuse uniform fluorescence in young adult wild type animals; however as the wild type animals age Q35::YFP begins to aggregate. We found that expression of Q35::YFP in sel-12 mutants shows precocious aggregation of Q35::YFP, which can be suppressed be reducing ER to mitochondria Ca2+ signaling or application of a mitochondrial specific antioxidant. Similarly, we found that Abeta overexpression in sel-12 mutant animals causes an enhanced reduction in swimming behavior that can be rescued by reducing ER to mitochondria Ca2+ signaling. Since these metastable proteins are overexpressed and may not fully address endogenous protein homeostasis, we used heat stress and examined the ability of sel-12 animals to recover from this insult. Strikingly, sel-12 mutants are highly susceptible to heat stress and, unlike wild type animals, cannot recover from acute high temperature exposure. Furthermore, we found if we reduced ER to mitochondrial Ca2+signaling in sel-12 mutants, we could significantly improve the survival of sel-12 mutants after acute heat stress. These data indicate that defective ER to mitochondria Ca2+ signaling promotes proteostasis collapse in sel-12 mutants. 1. Morley et al., 2002. PNAS 99: 10417-10422. 2. McColl et al., 2012. Mol. Neurodeger 7: 57.

Yoko Honda et al. (2007) International Worm Meeting "Study of space-flight effect on the aging of C. elegans in the International C. elegans Experiment (ICE)-First."

Yoko Honda, Masashi Tanaka, Noriaki Ishioka, Akira Higashibata, Shuji Honda

[International Worm Meeting, 2007]

. Yoko Honda1, Akira Higashibata2, Noriaki Ishioka2, Masashi Tanaka1, Shuji Honda1. 1) Genomics for Longevity & Health, Tokyo Metropolitan Institute of Gerontology(TMIG), Tokyo, Japan; 2) Japan Aerospace Exploration Agency(JAXA), Tsukuba, Japan. As exposures to the space environment become longer, information regarding the effects on biological aging will become important. We have not yet fully clarified aging processes in space environments. The aging process and lifespan are affected by various kinds of environmental factors including oxygen concentration (1), temperature and radiation. The aging phenomena that we usually see occur under certain conditions on the ground. Space environments differ from ground environments especially with regard to the radiation spectrum and gravity. We participated in the International Caenorhabditis elegans Experiment (ICE)-First that examined the effects of a 10-day space flight on the nematode C. elegans. C. elegans has frequently been used for study of aging because of its short lifespan and powerful genetic system. Recently, Morley et al. reported that Huntington''s-like polyglutamine (polyQ)-repeat proteins expressed in the muscle of C. elegans form aggregates as the animals age, and that this aggregation is delayed in long-lived mutants (2). We measured the polyQ aggregates in these nematodes after space flight as an aging marker. We also examined the effects of space flight on the gene expression by DNA microarray analysis and real time PCR. The expression of more than ten genes specifically for nervous systems was suppressed in space-flown animals, suggesting inactivation of nervous functions in space. Next, effects of inactivation of these genes on lifespan were tested by RNAi in the laboratory. 1) Honda S et al.Oxygen-dependent perturbation of life span and aging rate in the nematode. J Gerontol. 48:B57-61. 1993 2) James F et al. The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA, 99: 10417-10422. 2002.