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Extreme high temperature inhibits ACh release from C. elegans neurons

Tatyana B. Kalinnikova1, Evgenia B. Belova1, Rufina R. Kolsanova1, Rifgat R. Shagidullin1 and Marat Kh. Gainutdinov1
1Research Institute for Problems of Ecology and Mineral Wealth Use of Tatarstan Academy of Sciences, Kazan, Russian Federation
Correspondence to: Tatyana B. Kalinnikova (tbkalinnikova@gmail.com)

In our previous work we have shown that partial inhibition of ACh-esterase by neostigmine protected C. elegans behavior against disturbances caused by extreme high temperature 36°C (Kalinnikova et al., 2013). These data show that extreme high temperature can inhibit ACh release from C. elegans neurons. For further analysis of this possibility we investigated the effect of extreme high temperature on toxicity of other ACh-esterase inhibitor aldicarb for organisms of two closely-related species of free-living soil nematodes C. elegans and C. briggsae. It is known that numerous mutations leading to decrease in ACh secretion cause C. elegans resistance to aldicarb. Therefore hyperthermia must have similar effect on aldicarb resistance if extreme high temperature inhibits ACh release.

Our experiments were carried out in liquid medium (NG buffer). In these experiments either temperature 36°C or aldicarb caused C. elegans paralysis (worms’ inability to swim, induced by mechanical stimulus), depending on exposure time to high temperature or toxicant. The temperature rise from 23 to 33°C didn’t cause worms’ paralysis but accelerated time course for paralysis induced by aldicarb. In contrast, aldicarb concentrations which are toxic at 33°C (50–200 μM) were nontoxic at 36°C. Moreover, these aldicarb concentrations protected C. elegans against paralysis induced by hyperthermia. These data can be explained by inhibition of ACh release by hyperthermia, because partial inhibition of ACh-esterase can compensate ACh deficiency. The additional evidence for this explanation was obtained from experiments with worms preadapted for 2 hours to moderate high temperature 30°C. Such adaptation in accordance with our previous data (Kalinnikova et al., 2013) caused the rise in C. elegans resistance to temperature 36°C but sensitized worms to toxic aldicarb effect at 23°C. Under these conditions extreme high temperature 36°C strongly protected C. elegans against toxic aldicarb effect while aldicarb protected C. elegans against extreme high temperature.

In order to reveal the possible role of inhibition of ACh release from C. elegans neurons in the organism’s resistance to hyperthermia we have compared the dependence of behavior sensitivity to aldicarb from rise of temperature in two closely-related species – C. elegans and C. briggsae. Since behavior thermotolerance of C. briggsae is significantly higher than such of C. elegans (Kalinnikova et al., 2011) one might propose that this difference in thermotolerance can correlate with thermostability of cholinergic synapses of these species. The dependence of aldicarb toxicity for C. briggsae in temperature range 23–33°C is similar with such for C. elegans. However at 36°C there was great difference in aldicarb action on organisms of C. elegans and C. briggsae. At this temperature aldicarb caused paralysis of C. briggsae, but protected C. elegans behavior against negative action of hyperthermia. It is known that C. briggsae resistance to temperature 36°C is strongly higher than such of C. elegans (Kalinnikova et al., 2011). These data indicate that exposure to temperature 36°C caused the inhibition of ACh release in C. elegans, but not in C. briggsae organism. Therefore it is evident that in C. briggsae organism the threshold both for thermal paralysis and inhibition of ACh release is higher than in C. elegans.

Therefore it is evident that cholinergic system is one of targets for high temperature effect on nematodes’ nervous system.

References

Kalinnikova TB, Shagidullin RR, Kolsanova RR, Osipova EB, Zakharov SV, and Gainutdinov MKh. (2013). Acetylcholine deficiency in Caenorhabditis elegans induced by hyperthermia can be compensated by ACh-esterase inhibition or activation of GAR-3 mAChRs. Environment and Natural Resources Research. 3, 98–113.   PubMed

Kalinnikova TB, Timoshenko AKh, Tarasov OYu, Gainutdinov TM, and Gainutdinov MKh. (2011). Thermotolerance of soil nematodes Caenorhabditis briggsae AF16 and Caenorhabditis elegans N2 under experimental conditions. Russian Journal of Ecology. 42, 435–437.   PubMed

Identification of klf-3/F54H5.3 as a novel operon and genetic circuit controlling fat deposition and FA beta oxidation in C. elegans

Razan Bakheet1, Nafla Al-Nasser1, Ranjit Parhar1, Randy Gaugler2, Yi Wang2, Futwan Al-Mohanna1 and Sargar Hashmi2
1Cell Biology-Cardiovasclar Unit, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia,
2Laboratory of Developmental Biology, Rutgers Center for Vector Biology, Rutgers University, New Brunswick NJ
Correspondence to: Sarwar Hashmi (sarwar.hashmi@rutgers.edu)

Fatty acid biosynthesis and oxidative degradation play a key role in lipid metabolism and their homeostatic balance controls energy storage. Its deregulation and dysfunction is linked to a wide range of disease risk factors or clinical manifestations from excessive deposit (obesity) to extreme lipodystrophy (cachexia). Cancer-cachexia is a complex metabolic condition in which disease-associated metabolic changes lead to a severe loss of skeletal muscle. Alterations in mitochondrial content, morphology, and function lead to several muscle-wasting conditions. Mitochondrial dysfunction has been reported in mouse models of cancer cachexia (Constantinou et al., 2011; Fermoselle et al., 2013). We have shown that one of the three Krüppel-like factors in C. elegans, KLF-3, a highly conserved homologue, possessed an important function in lipid metabolism probably via its transcriptional regulation (Zhang et al., 2009; Zhang et al., 2011; Zhang et al., 2013). klf-3 regulates beta-oxidation of long-chain fatty acids (Zhang et al., 2011) and impacts on the biosynthesis and mobilization of triglycerides (Zhang et al., 2013). We investigated if klf-3 influenced mitochondrial activity to modulate FA beta oxidation. We analyzed mitochondrial proliferation in synchronized populations of both wild type and klf-3 mutants using a modified Mito Tracker staining protocol (Sherwood et al., 2005). We found that mitochondrial content in the wild-type worm was notably high (Figure 1A), whereas klf-3 elimination reduced the mitochondrial content in the mutant worm (Figure 1B). Our data suggests that KLF-3-associated lipid accumulation in the mutant affects mitochondrial contents, leading to its dysfunction and reduced catabolism.

Recently we explored whether there is connection between fat metabolism dysfunction, reduced mitochondrial content, and muscle-wasting conditions. We characterized a joined deletion (rh160) of klf-3 and its downstream F54H5.3 gene in C. elegans strain NJ701. Through DNA sequencing, we determined the exact breakpoints of this deletion, which spans several kb of genomic sequence. RNA interference of F54H5.3 in wild-type N2 worms revealed a partial embryonic lethality and defective reproduction. However, F54H5.3 RNAi on the background of klf-3(ok1975), a smaller deletion that affects only klf-3, alleviated the fatty phenotype of the ok1975 mutant but recapitulated the severe muscle-wasting phenotypes associated with klf-3(rh160). Together, our data suggest that klf-3 and F54H5.3 interact in the same epistatic genetic circuit and potentially form an operon-like structure to coordinate their joint regulation in lipid metabolism. These observations bear novel implications for both obesity and type 2 diabetes and cachexia in cancer patients. Although our data is preliminary it does suggest a link between mitochondrial dysfunction, fat metabolism and cancer-cachexia. Our study may provide a simpler animal model to deepen our understanding of their underlying mechanisms.

Figures



klf-3 deficiency reduces mitochondrial proliferation. The changes in mitochondrial proliferation in both wild-type and klf-3 mutants were observed after staining with MitoTracker Red in M9 buffer for two hours in dark. The worms were washed twice with M9 buffer and then transferred to NGM plate and allowed to recover for 30 min. Then worms were transferred onto 2% agarose pad, observed and imaged under LSM 510 Zeiss confocal microscope. (A) wild-type worm showing large number of mitochondria, and (B) klf-3 worm with reduced mitochondria. Three independent experiments were performed and results were consistent between experiments.

References

Constantinou C, Fontes de Oliveira CC, Mintzopoulos D, Busquets S, He J, Kesarwani M, Mindrinos M, Rahme LG, Argilés JM, and Tzika AA. (2011). Nuclear magnetic resonance in conjunction with functional genomics suggests mitochondrial dysfunction in a murine model of cancer cachexia. Int. J. Mol. Med. 27, 15-24.  PubMed

Fermoselle C, García-Arumí E, Puig-Vilanova E, Andreu AL, Urtreger AJ, de Kier Joffé ED, Tejedor A, Puente-Maestu L, and Barreiro E. (2013). Mitochondrial dysfunction and therapeutic approaches in respiratory and limb muscles of cancer cachectic mice. Exp. Physiol. 98, 1349-1365.   PubMed

Sherwood DR, Butler JA, Kramer JM, and Sternberg PW. (2005). FOS-1 promotes basement–membrane removal during anchor-cell invasion in C. elegans. Cell 121, 951-962.   PubMed

Zhang J, Yang C, Brey C, Rodriguez M, Oksov Y, Gaugler R, Dickstein E, Huang CH, and Hashmi S. (2009). Mutation in Caenorhabditis elegans Krüppel-like factor, KLF-3 results in fat accumulation and alters fatty acid composition. Exp. Cell Res. 315,  PubMed

Zhang J, Bakheet R, Parhar RS, Huang CH, Hussain MM, Pan X, Siddiqui SS, and Hashmi S. (2011). Regulation of fat storage and reproduction by Krüppel-like transcription factor KLF-3 and fat associated genes in Caenorhabditis elegans. J. Mol. Biol. 411, 537-553.  PubMed

Zhang J, Yang C, Brey C, Rodriguez M, Oksov Y, Gaugler R, Dickstein E, Huang CH, and Hashmi S. (2009). Mutation in Caenorhabditis elegans Krüppel-like factor, KLF-3 results in fat accumulation and alters fatty acid composition. Exp. Cell Res. 315, 2568-2580.  PubMed

Worm humor: Identification of a novel mutation that enhances robust experimental reproducibility in C. elegans

Caylee I. Salt
1Department of Biology, McGill University, Montreal, QC, Canada
Correspondence to: Caylee I. Salt (callista.yee@mail.mcgill.ca)

While slaving away hard at the bench, we have serendipitously discovered a new mutation that has increased repeatability and significance of any assay we have performed. This mutation significantly reduces the level of experimental error so intensely that we can actually report standard deviation instead of standard error of the mean. P-values are minuscule and our graphs have more stars than ever (Figure 1). In addition, putting our strains in this background has allowed us to perform extremely beautiful Western blots, so beautiful that we do not need to use Photoshop to ‘robustify’ our figures (Figure 2).

As typical naming of genes follows the phenotype they induce, we believe we have identified a mutation affecting a new class of genes, hereby called robust, or rbst.  Mutations affecting genes belonging to this class affect experimental performance and data reproducibility.  Tim Schedl has yet to reply to our request to be formally assigned this new class of genes, but we are confident that Jonathan Hodgkin would approve. We have mapped this mutation, win1, to either the left arm of LG III or LG IV. The graduate student currently performing the mapping studies is ironically failing to achieve a robust confirmation.

In conclusion, we report identification of a new allele, rbst-1(win1) III?; IV? which makes all results significantly more robust and will be sure to get your papers accepted without revisions. Stay tuned for more updates on our robust progress. No, we are not sharing our magical strain just yet, but if you would like to form a robust collaboration with us to pursue further robust experiments, please feel free to contact us. We look forward to hearing your robust ideas. Thanks to Steve Hodgkinson for critical reading and verbal abuse regarding this data. Thanks to BC-V for technical advice and moral support. Thanks to Chib, raela and dunnp for robust shenanigans.

Figures



rbst-1(win1) enhances mutant phenotypes and makes data seem much more believable.


rbst-1(win1) eliminates need for ‘image processing and enhancement’ by Photoshop.

Worm humor: Nematode news in brief

Decrepitus W. Picker
1Department of Molecular Biology, University of Wyoming, Laramie WY
Correspondence to: Decrepitus W. Picker (davidfay@uwyo.edu)

Slam-Dunk Grant Fails to Write Itself (Minneapolis, MN). In spite of widespread projections, R01 proposal GM088686-A1 has, “for reasons that defy all logic”, failed to write itself, according to sources close to the proposal. Moreover, the competitive renewal, which is reputed to strike the perfect balance between mildly risky high-payoff studies and guaranteed publishable units, has yet to commit a single sentence to paper. “How this set of perfectly meshed yet entirely non-interdependent aims could fail to auto-compose onto a hard drive is nearly inconceivable”, stated one of grant’s lead investigators. That the six-point bulleted Significance section, which segues seamlessly into the Innovation component, has so completely fallen short of crafting itself, was also unanticipated. “Admittedly, we thought we might have to help out with entering some Endnote references or maybe take up some slack on the budget section”, stated a co-PI close to the project, “but the response to previous criticisms should have literally fallen out of the sky and onto the page given that every experiment since the initial submission has completely put reviewer’s concerns to rest.” Regardless of the revised proposal’s failure to meet expectations, scientists associated with the grant remain optimistic that the “eight high-impact papers” generated during the previous funding period are more than likely to “submit themselves” to top journals within the next few months.

Genes within long-standing operon calling it quits (X chromosome). At a joint press conference held on the campus of Caltech, genes within the C. elegans lin-15 operon recently announced their decision to split due to “creative differences”. Despite co-existing for at least 50-100 million years, the individual genes known as lin-15a and lin-15b indicated their mutual desire to move on in a prepared statement. “We’ve done our thing together and we don’t think we have anything to prove”, affirmed the genes, which have been sharing regulatory elements prior to the separation of C. elegans and C. briggsae. According to sources close to the locus, having nearly identical names has been a long-standing source of irritation to the tandem genes, which share virtually no identity at the peptide level. “It’s already bad enough to be part of the “lin” group”, said lin-15a, “but having the same number followed by a letter is just total bullshit”. “It’s not like our discoverer was George Foreman or something”, lin-15b added. Despite their intention to separate “a minimum of 5-10 map units apart or even to different chromosomes”, the functionally interconnected genes plan to continue their long-standing collaboration on processes such as vulval induction while freeing their expression domains to pursue other developmental projects. Although details on the custody of 5′ regulatory elements and the 3′ UTR have not yet been disclosed, geneticists familiar with the process are expecting a protracted battle.

New lab announcement: Kevin Collins

Kevin Collins
1Department of Biology University of Miami, Coral Gables FL
Correspondence to: Kevin Collins (kcollins@bio.miami.edu)

I started my independent research group from August 2014 in the Department of Biology at the University of Miami in Coral Gables, Florida. Our fundamental goal is to understand how neurons communicate in circuits to establish an appropriate level of activity that produces a robust, stable behavior. Our approach is to analyze in detail a model neural circuit that controls egg-laying behavior in C. elegans. We are taking advantage of the optical clarity and powerful genetics in this experimental system to literally watch the activity of every cell in the circuit in behaving animals using fluorescent Ca2+ reporters, and also to manipulate their activity using optogenetic tools. Using mutations and transgenes to discover and alter molecular signaling events between cells, we are determining how the complex pattern of activity in a circuit creates a coherent, regulated behavior. We expect these studies will reveal general principles of neurotransmitter signaling and neural circuit function with applications to understanding the human nervous system and its dysfunction in disease.

Website:  http://www.as.miami.edu/biology/people/faculty/kevin-collins/

Ph.D. student and Postdoc positions are currently available.  To apply, please send a CV and description of research interests to kcollins@bio.miami.edu

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