Hana Murakami et al. (2007) International Worm Meeting "Behavioral aging and lifespan modulated by serotonin receptors."

Hana Murakami, Karalee Bessinger, Shin Murakami, Gregory Luerman, Jason Hellmann

[International Worm Meeting, 2007]

The neurotransmitter serotonin (5HT) has been implicated to affect variation of longevity in natural Drosophila populations and age-related diseases in mammals. Based on the observations, it has been predicted that serotonin signal, perhaps at levels of serotonin biosynthesis, may control lifespan. Despite the prediction, we found that mutations in the serotonin biosynthesis genes had little or modest effects on life span. Interestingly, a deletion mutation of the ser-1 serotonin receptor gene increased longevity by up to 46%, while a deletion mutation of another serotonin receptor gene, ser-4, shortened early to mid lifespan [1]. The serotonin receptor genes modulate early phase of behavioral aging (locomotion, learning behavior, sensory behavior, etc.) [2; Murakami et al., unpublished] and in stress responses [3] in a different manner. The results provide several important suggestions: (a) Sarcopenia (i.e., muscle frailty) is not the only cause of behavioral aging. (b) Behavioral aging has early and late phases. Early phase occurs prior to the timing when sarcopenia is observed. Serotonin signal modulates early phase. Alterations in neurotransmitters may cause early phase of aging (or post-developmental changes in behaviors) [4]. (c) Late phase is associated with increased oxidative stress in neurons. We are currently defining the roles of oxidative stress in aging of behaviors. References: [1] Murakami H, Murakami S. (2007) Serotonin receptors antagonistically modulate C. elegans longevity. Aging Cell. In Press. [2] Murakami H, Bessinger K, Hellmann J, Murakami S. (2007) Manipulation of serotonin signal suppresses early phase of behavioral aging in Caenorhabditis elegans. Neurobiology of Aging. In Press. [3] Liang B, Moussaif M, Kuan CJ, Gargus JJ, Sze JY. (2006) Serotonin targets the DAF-16/FOXO signaling pathway to modulate stress responses. Cell Metab. 4:429-40. [4] Murakami S. (2007) C. elegans as a model system to study aging of learning and memory. Molecular Neurobiology. In Press.

Murakami S et al. (1996) West Coast Worm Meeting "LIFE-EXTENSION PATHWAY IN C.ELEGANS"

Johnson TE, Dames SA, Murakami S, Tedesco PM, Duhon SA

[West Coast Worm Meeting, 1996]

Life extension is conferred either by mutants extending adult life (Age), including age-1, daf-2, daf-23, daf-28, spe-26, and an allele of clk-1. Another mode of life extension is conferred by mutants delaying developmental stage (Clk), including clk-1, clk-2, clk-3, gro-1(Wong et al., 1995; Lakowski and Hekimi, 1996). Three groups have identified a life-extension pathway formed by some Daf mutations (Dorman et al., 1995; Larsen et al., 1995; Murakami and Johnson, 1996). We found that the pathway is not limited to Daf mutations but includes other types of the Age mutants (Murakami and Johnson, 1996). Interestingly, the pathway confers UV resistance, suggesting that UV resistance is a good indicator of Age. It is also consistent with the hypothesis that stress resistance is a rate limiting factor determining longevity. We are also testing whether the Age-pathway can confer other types of stress such as H2O2 and heat.

Papp, Andy et al. (2013) International Worm Meeting "Remote Control and Observation for more meaningful Behavioral Experiments."

Papp, Andy, Biondo, John

[International Worm Meeting, 2013]

C. elegans is an excellent organism for the study of the molecular genetics of a variety of neurologically-based phenomena such as mechanosensation, chemosensation, thermosensation, photosensation, behavior, learning, and memory because it manifests all of these via its simple, well characterized 302-cell nervous system (Murakami 2007). The sensitivity and interactions of these different systems necessitate carefully controlled experiments so that a single variable can be examined and meaningful conclusions can be drawn. For example, one would not want sensitive chemotaxis-based learning experiments to be interfered with due to thermotaxis, phototaxis, or mechanical stimulation caused by an inadequately controlled environment. To create a more stable environment for these experiments, we have developed a small incubator with an integrated web-cam microscope that we call the "Telebator". It can be remotely controlled, and its contents observed, via a local or Internet computer network connection (US Pat. 7618808). This system allows behavior to be observed and recorded in an environment that is isothermal with constant lighting and no changes in mechanical stimulation. The Telebator's remote-control and monitoring features also make it ideal for various other applications, including: heat shocks, temperature shifts, knowing/controlling when a plate grown for DNA extraction is just about to starve, knowing/controlling when worms of a specific developmental stage are plentiful, knowing if/when a fluorescently marked transgene is being expressed, etc. In the event of a temperature or other alarm, the system is able to notify users by e-mail, SMS text message, and YahooIM. The incubator contents and temperature log data can be observed and the incubator temperature and other parameters can be controlled via any web browser, including a "smart phone". Murakami (2007) Caenorhabditis elegans as a model system to study aging of learning and memory. Mol Neurobiol. 35(1):85-94.

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.

Edwards T J et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Axon Regeneration is Influenced by Two Heparan Sulfate Proteoglycans"

Edwards T J, Hammarlund M

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Regeneration after axonal injury is regulated by extracellular cues, including proteoglycans (Properzi and Fawcett 2004). Heparan sulfate proteoglycans (HSPGs) change expression after neuronal injury, but their role in axon regeneration is not well characterized (Murakami et al. 2006). We find that two HSPGs influence regenerative success in C.elegans: the cell surface protein syndecan (sdn-1) and the basement membrane component perlecan (unc-52). sdn-1 mutants exhibit decreased regeneration of GABAergic motor neurons, with regenerating neurites characterized by dysmorphic growth cones and impaired migration. Thus, syndecan appears to affect regenerating growth cone stability and function. On the other hand, unc-52 mutants show an overall increase in the percentage of regenerating neurites. Surprisingly, none of the other HSPG core proteins or modifying enzymes affects axon regeneration in GABA neurons. Current experiments are underway to elucidate the site of syndecan function, and to identify the regeneration pathways that mediate HSPG function.

Kim, H-S. et al. (2009) International Worm Meeting "The C.elegans spectraplakin mig-31 regulates nuclear migration in distal tip cells."

Nishiwaki, K., Kim, H-S., Ohkura, K., Murakami, R., Sakamoto, H., Tamai, K., Labouesse, M.

[International Worm Meeting, 2009]

In C.elegans, U-shaped hermaphrodite gonads are formed by directed migration of gonadal distal tip cells (DTCs). Cytoskeletal regulation has been known as one of the important processes to regulate DTC migration, but its molecular mechanism remains elusive. The mig-31 gene encodes VAB-10 spectraplakins, cytoskeletal linker proteins, and mig-31(tk27) mutants exhibit abnormal gonad formation caused by defective DTC migration. Complementation tests and western blot analysis revealed that abnormal gonad formation of mig-31 (tk27) mutant was due to the lack of DTC-specific VAB-10B isoform(s), named MIG-31. Immunohystochemical experiments revealed that MIG-31 localize to the plasma membrane of DTCs. When wild type DTCs turn dorsally, the rotation of migratory axis from anteroposterior to dorsoventral is preceded by nuclear migration within DTCs. Interestingly, however, both mig-31(tk27) mutation and RNAi-knockdown of mig-31 inhibited this nuclear migration. Although DTCs appeared to be attracted dorsally by the UNC-6 mediated guidance system even in the mig-31 knockdown animals, their nuclei stayed at their anterior or posterior ends rather than were relocated to the dorsal side as in the wild type, suggesting that MIG-31 may be required for a directional switching of DTCs. Furthermore, we found that both actin and microtubule cytoskeleton formed a transient spherical structure within the wild type DTCs during dorsal turn, presumably as a result of a cytoskeletal remodeling. In mig-31(tk27) mutant, this structure was also detectable, but both cytoskeletons seemed to be disorganized, suggesting that the interactions between MIG-31 and cytoskeletons in DTCs are affected. Taken together, we propose that MIG-31 may play a role in linking cytoskeletons to the plasma membrane of DTCs, which is required for cytoskeleton-based nuclear migration process within DTCs.

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.

Hana Murakami et al. (2005) International Worm Meeting "Aging of associative learning paradigms in C. elegans."

Shin Murakami, Karalee Murray, Jason Hellmann, Hana Murakami

[International Worm Meeting, 2005]

Associative learning in C. elegans includes chemosensory learning and thermotaxis learning. In the associative learning paradigms, worms learn to associate paired stimuli and alter the behavioral output. Conditioned worms with or without food (unconditional stimulus, US) show either preference or avoidance to the conditional stimulus (CS). Recently, slowing responses to food, called basal and enhanced slowing responses, are suggested to be a type of associative learning (Hobert, 2003). We have investigated aging effects on the three learning paradigms. The learning behaviors showed different kinetics of learning declines. The long-lived mutants, age-1 and daf-2, dramatically improves this type of learning behavior. In the mutants, there was a three-fold extension of the young period that allows a high level of thermotaxis learning. In contrast, the mutants had little or modest effects on aging of basal and enhanced slowing responses. Aging of slowing responses is likely caused by age-related changes in serotonin signal (see the other abstract; Murakami et al). Aging of chemosensory learning appears to show different kinetics during aging. In the poster we will summarize aging of the three learning paradigms.