Angelica Ortiz et al. (2008) C.elegans Neuronal Development Meeting "Identifying Pathways Modulated by Clozapine in Caenorhabditis elegans Behavior"

Sarah Demarco, Miriam Lundy, Edgar Buttner, Bruce Cohen, Rakesh Karmacharya, Angelica Ortiz

[C.elegans Neuronal Development Meeting, 2008]

The atypical antipsychotic clozapine effectively treats refractory schizophrenia. It also presents serious side-effects, such as agranulocytosis and metabolic syndrome. However, the molecular basis of this drug''s unique therapeutic profile remains unknown. Previously, we characterized clozapine-induced behavioral phenotypes in C. elegans and reported clozapine-specific effects in comparison to the typical antipsychotic haloperidol. The results showed that clozapine enhanced egg laying without the involvement of dopaminergic systems. We also observed that exposure to clozapine decreased the rate of pharyngeal pumping and the rate of locomotion in well-fed worms. We went on to test olanzapine, an atypical antipsychotic that has a structure similar to clozapine but that lacks clozapine''s unique efficacy in treating schizophrenia. Interestingly, we found that olanzapine failed to recapitulate clozapine''s effect on egg laying. By testing a panel of C. elegans mutants with defects in biogenic amine neurotransmitter systems, we aimed to identify novel pathways underlying clozapine-induced behaviors. We found that tdc-1, a gene required for tyramine synthesis, and ser-4, which encodes a serotonin-octopamine receptor, mediate the clozapine-induced increase in egg laying. Trace amine signaling also appears to be necessary for clozapine''s inhibition of pharyngeal pumping. Our observations suggest that the clozapine-induced decrease in pharyngeal pumping requires the presence of two distinct tyramine receptors: tyra-2, a G protein-coupled tyramine receptor, and sho-1, a tyramine-gated ion channel. We also found that tbh-1, a gene encoding tyramine beta-hydroxylase for octopamine synthesis, is needed for clozapine to decrease pharyngeal pumping rates. Clozapine''s inhibition of locomotion rate requires both the trace amine system and the biosynthesis of serotonin and dopamine. Interestingly, we found that worms lacking sho-1 did not demonstrate clozapine-induced reduction in locomotion rate. Similarly, mutations in bas-1, a gene encoding an aromatic amino acid decaboxylase necessary for serotonin and dopamine synthesis, did not show a change in locomotion rate after clozapine exposure. These results demonstrate that clozapine may be acting through a variety of pathway to produce its wide range of effects. Our observations may shed light on the molecular mechanisms underlying clozapine''s effects in human patients.

Shindou, Tomomi et al. (2015) International Worm Meeting "Electrophysiological properties of ASE neurons."

Wickens, Jeffery, MARUYAMA, Ichiro, Shindou, Tomomi, Shindou, Mayumi, Murayama, Takashi

[International Worm Meeting, 2015]

A pair of ASE chemosensory neurons, ASEL and ASER, are major salt sensors, and play critical roles in chemotaxis to NaCl. Calcium imaging has previously revealed that ASEL and ASER are activated by an increase and decrease in NaCl concentrations, respectively (Suzuki et al., 2008; Ortiz et al., 2009). These asymmetric responses by ASEL and ASER to changes of NaCl concentrations are crucial to efficient chemotaxis of C. elegans toward higher concentrations of NaCl. While Goodman et al. (1998) reported in situ whole-cell patch-clump recording of ASER, electrophysiological characterisation of ASE neurons is still required to understand how the neurons respond to the NaCl concentration changes.Toward the goal, we have investigated electrophysiological properties of ASE neurons in wild-type C. elegans by in vivo whole-cell patch-clamp recordings, and have found that both of ASE neurons showed resting membrane potentials of approximately -60 mV and membrane resistances of about 2 Gomega. In both of ASE neurons, voltage responses to current injections showed solitary action potentials. Depolarization of wild-type ASEL was observed when a puff of 150 mM NaCl was applied to the animal's nose in bath solution containing 50 mM NaCl. On the other hand, a puff of NaCl-free buffer induced ASER depolarization. These results are consistent with those of calcium imaging. To understand roles of the action potentials in ASE, we are currently trying to analyse electrophysiological properties of ASE neurons in various mutants.References1. Suzuki et al., Nature 454: 114-118 (2008)2. Ortiz et al., Current Biology 19: 996-1004 (2009)3. Goodman et al., Neuron 20: 763-772 (1998).

Carmona-Gutierrez D et al. (2019) Nat Commun "The flavonoid 4,4'-dimethoxychalcone promotes autophagy-dependent longevity across species."

Madeo F, Eisenberg T, Trausinger G, Sadoshima J, Beuschel CB, Sinner F, Kroemer G, Carmona-Gutierrez D, Aprahamian F, Mertel S, Dengjel J, Sommer C, Raml R, Sedej S, Ruckenstuhl C, Ventura N, Kepp O, Bossut N, Sica V, Sigrist SJ, Kainz K, Pietrocola F, Pieber TR, Enot DP, Pendl T, Wolinski H, Nah J, Dammbrueck C, Magnes C, Schiavi A, Abdellatif M, Schroeder S, Castoldi F, Tadic J, Chen G, Hofer SJ, Hu Z, Durand S, Zimmermann A, Bauer MA, Maglioni S

[Nat Commun, 2019]

Ageing constitutes the most important risk factor for all major chronic ailments, including malignant, cardiovascular and neurodegenerative diseases. However, behavioural and pharmacological interventions with feasible potential to promote health upon ageing remain rare. Here we report the identification of the flavonoid 4,4'-dimethoxychalcone (DMC) as a natural compound with anti-ageing properties. External DMC administration extends the lifespan of yeast, worms and flies, decelerates senescence of human cell cultures, and protects mice from prolonged myocardial ischaemia. Concomitantly, DMC induces autophagy, which is essential for its cytoprotective effects from yeast to mice. This pro-autophagic response induces a conserved systemic change in metabolism, operates independently of TORC1 signalling and depends on specific GATA transcription factors. Notably, we identify DMC in the plant Angelica keiskei koidzumi, to which longevity- and health-promoting effects are ascribed in Asian traditional medicine. In summary, we have identified and mechanistically characterised the conserved longevity-promoting effects of a natural anti-ageing drug.

Epstein HF et al. (1988) J Cell Biol "Purified thick filaments from the nematode Caenorhabditis elegans: evidence for multiple proteins associated with core structures."

Epstein HF, Berliner GC, Ortiz I, Casey DL

[J Cell Biol, 1988]

The thick filaments of the nematode, Caenorhabditis elegans, arising predominantly from the body-wall muscles, contain two myosin isoforms and paramyosin as their major proteins. The two myosins are located in distinct regions of the surfaces, while paramyosin is located within the backbones of the filaments. Tubular structures constitute the cores of the polar regions, and electron-dense material is present in the cores of the central regions (Epstein, H.F., D.M. Miller, I. Ortiz, and G.C. Berliner. 1985. J. Cell Biol. 100:904-915). Biochemical, genetic, and immunological experiments indicate that the two myosins and paramyosin are not necessary core components (Epstein, H.F., I. Ortiz, and L.A. Traeger Mackinnon. 1986. J. Cell Biol. 103:985-993). The existence of the core structures suggests, therefore, that additional proteins may be associated with thick filaments in C. elegans. To biochemically detect minor associated proteins, a new procedure for the isolation of thick filaments of high purity and structural preservation has been developed. The final step, glycerol gradient centrifugation, yielded fractions that are contaminated by, at most, 1-2% with actin, tropomyosin, or ribosome-associated proteins on the basis of Coomassie Blue staining and electron microscopy. Silver staining and radioautography of gel electrophoretograms of unlabeled and 35S-labeled proteins, respectively, revealed at least 10 additional bands that cosedimented with thick filaments in glycerol gradients. Core structures prepared from wild-type thick filaments contained at least six of these thick filament-associated protein bands. The six proteins also cosedimented with thick filaments purified by gradient centrifugation from CB190 mutants lacking myosin heavy chain B and from CB1214 mutants lacking paramyosin. For these reasons, we propose that the six associated proteins are potential candidates for putative components of core structures in the thick filaments of body-wall muscles of C. elegans.

Wang Q et al. (2016) Oxid Med Cell Longev "Bioactive Peptides from Angelica sinensis Protein Hydrolyzate Delay Senescence in Caenorhabditis elegans through Antioxidant Activities."

Li H, Zou Y, Wang Q, Mao X, Li S, Qin C, Huang Y, Ma CW, Liang M, Jia W, Huang Z

[Oxid Med Cell Longev, 2016]

Since excessive reactive oxygen species (ROS) is known to be associated with aging and age-related diseases, strategies modulating ROS level and antioxidant defense systems may contribute to the delay of senescence. Here we show that the protein hydrolyzate from Angelica sinensis was capable of increasing oxidative survival of the model animal Caenorhabditis elegans intoxicated by paraquat. The hydrolyzate was then fractionated by ultrafiltration, and the antioxidant fraction (<3kDa) was purified by gel filtration to obtain the antioxidant A. sinensis peptides (AsiPeps), which were mostly composed of peptides with <20 amino acid residues. Further studies demonstrate that AsiPeps were able to reduce the endogenous ROS level, increase the activities of the antioxidant enzymes superoxide dismutase and catalase, and decrease the content of the lipid peroxidation product malondialdehyde in nematodes treated with paraquat or undergoing senescence. AsiPeps were also shown to reduce age pigments accumulation and extend lifespan but did not affect the food-intake behavior of the nematodes. Taken together, our results demonstrate that A. sinensis peptides (AsiPeps) are able to delay aging process in C. elegans through antioxidant activities independent of dietary restriction.

Epstein HF et al. (1985) J Cell Biol "Myosin and paramyosin are organized about a newly identified core structure."

Berliner GC, Miller DM, Epstein HF, Ortiz I

[J Cell Biol, 1985]

Myosin isoforms A and B are differentially localized to the central and polar regions, respectively, of thick filaments in body wall muscle cells of Caenorhabditis elegans (Miller, D.M. III, I. Ortiz, G.C. Berliner, and H.F. Epstein, 1983, Cell, 34: 477-490). Biochemical and electron microscope studies of KCl-dissociated filaments show that the myosin isoforms occupy a surface domain, paramyosin constitutes an intermediate domain, and a newly identified core structure exists. The diameters of the thick filaments vary significantly from 33.4 nm centrally to 14.0 nm near the ends. The latter value is comparable to the 15.2 nm diameter of the core structures. The internal density of the filament core appears solid medially and hollow at the poles. The differentiation of thick filament structure into supramolecular domains possessing specific substructures of characteristic stabilities suggests a sequential mode for thick filament assembly. In this model, the two myosin isoforms have distinct roles in assembly. The behavior of the myosins, including nucleation of assembly and determination of filament length, depend upon paramyosin and the core structure as well as their intrinsic molecular properties.

Gibson, Sophia B et al. (2021) MicroPubl Biol

Harper, Clare S, Gibson, Sophia B, Lackner, Laura L, Andersen, Erik C

[MicroPubl Biol, 2021]

Parasitic nematode infections continue to have an enormous impact on human and livestock health worldwide (Hotez et al., 2014; Kaplan & Vidyashankar, 2012). A limited arsenal of anthelmintic drugs are available to combat these infections. One of the most widely used classes is benzimidazoles (BZ), and resistance against this class is widespread (Kaplan & Vidyashankar, 2012). Previous studies to understand parasitic nematode resistance using the free-living model organism Caenorhabditis elegans showed that variation in the C. elegans beta-tubulin gene ben-1, an ortholog of beta-tubulins in parasitic nematodes, confers resistance to BZ drugs (Dilks et al., 2020; Driscoll et al., 1989; Hahnel et al., 2018). The most common missense mutation resistance alleles are F167Y, E198A, and F200Y (Avramenko et al., 2019; Mohammedsalih et al., 2020). Although computational models have predicted that these amino acids are involved in the binding of BZ compounds to beta-tubulins, the binding remains to be investigated empirically at the structural level because nematode-specific beta-tubulin structures have not been created (Aguayo-Ortiz et al., 2013; Hahnel et al., 2018). To better understand the mechanisms of resistance, we sought to obtain those crystallographic structures.

Smith, Heidi K. et al. (2009) International Worm Meeting "Functional Analysis of Asymmetrically-Expressed GCY Proteins in C. elegans."

Hobert, Oliver, Ortiz, Christopher O., Smith, Heidi K.

[International Worm Meeting, 2009]

Chemosensation allows animals to evaluate their environment, detect food, other animals, and dangerous toxins while responding with appropriate behaviors essential to the animal''s survival. A robust chemosensory system can be generated even from a seemingly simple nervous system, such as that of C. elegans, which can detect and respond to a vast number of chemical cues. One important, but poorly understood, strategy used by C. elegans is to "lateralize" the function of some of its sensory neurons, such as the ASE neurons, thus increasing the discriminatory power of a system comprised of relatively few elements. We have found that the ASE neurons respond to several salt cues and these responses are asymmetric in terms of whether the left or the right ASE neuron responds to a specific salt cue (Ortiz et al., submitted). Previous work in our lab has furthermore identified a number of guanylyl cyclase genes as having a role in the chemotaxis asymmetry of ASE (Ortiz et al., submitted). Mutant analysis has revealed that individual gcy genes are specifically required for sensing particular ions. Such specificity could be conferred through either the protein''s receptor family ligand-binding region (RFLBR) in its extracellular domain or by the protein''s guanylyl cyclase (GC) domain in its intracellular region. We seek to identify the molecular mechanisms by which these asymmetrically expressed receptor type guanylyl cyclases confer the specificity that underlies this lateralization. In order to test these predictions, intra- or extra-cellular domains of individual GCY proteins were swapped, chimeric proteins were introduced into mutant background animals in a cell-specific manner, and then rescue of chemotaxis defects were tested. The individual GCY protein domains that confer the cellular specificity of ASE neurons, which enables them to mediate responses only to particular cues are identified by evaluating assay output. Results from such experiments allow for the characterization of the domain(s) essential for specificity. By identifying these molecular mechanisms, key predictions of the role that these proteins play in ASE neurons, putatively functioning either as chemoreceptors or, alternatively, as signal transducers, can begin to be tested. We will pursue further strategies for elucidating these molecular mechanisms as part of an overall effort in exploring the relationship between individual genes, their patterns of expression in specific cellular contexts, and the chemosensory behaviors exhibited by C. elegans in response to salt cues found in its environment.

Hoppe PE et al. (1996) J Cell Biol "Hydrophobicity variations along the surface of the coiled-coil rod may mediate striated muscle myosin assembly in Caenorhabditis elegans."

Hoppe PE, Waterston RH

[J Cell Biol, 1996]

Caenorhabditis elegans body wall muscle contains two isoforms of myosin heavy chain, MHC A and MHC B, that differ in their ability to initiate thick filament assembly. Whereas mutant animals that lack the major isoform, MHC B, have fewer thick filaments, mutant animals that lack the minor isoform, MHC A, contain no normal thick filaments. MHC A, but not MHC B, is present at the center of the bipolar thick filament where initiation of assembly is thought to occur (Miller, D.M.,I. Ortiz, G.C. Berliner, and H.F. Epstein. 1983. Cell. 34:477-490). We mapped the sequences that confer A-specific function by constructing chimeric myosins and testing them in vivo. We have identified two distinct regions of the MHC A rod that are sufficient in chimeric myosins for filament initiation function. Within these regions, MHC A displays a more hydrophobic rod surface, making it more similar to paramyosin, which forms the thick filament core. We propose that these regions play an important role in filament initiation, perhaps mediating close contacts between MHC A and paramyosin in an antiparallel arrangement at the filament center. Furthermore, our analysis revealed that all striated muscle myosins show a characteristic variation in surface hydrophobicity along the length of the rod that may play an important role in driving assembly and determining the stagger at which dimers associate.

Fu RH et al. (2014) PLoS One "n-butylidenephthalide protects against dopaminergic neuron degeneration and -synuclein accumulation in Caenorhabditis elegans models of Parkinson's disease."

Fu RH, Hung HS, Huang JE, Wang YC, Chen CS, Liu SP, Chang WL, Harn HJ, Shyu WC, Chen YM, Tsai SY, Li RJ, Lin SZ

[PLoS One, 2014]

BACKGROUND: Parkinson's disease (PD) is the second most common degenerative disorder of the central nervous system that impairs motor skills and cognitive function. To date, the disease has no effective therapies. The identification of new drugs that provide benefit in arresting the decline seen in PD patients is the focus of much recent study. However, the lengthy time frame for the progression of neurodegeneration in PD increases both the time and cost of examining potential therapeutic compounds in mammalian models. An alternative is to first evaluate the efficacy of compounds in Caenorhabditis elegans models, which reduces examination time from months to days. n-Butylidenephthalide is the naturally-occurring component derived from the chloroform extract of Angelica sinensis. It has been shown to have anti-tumor and anti-inflammatory properties, but no reports have yet described the effects of n-butylidenephthalide on PD. The aim of this study was to assess the potential for n-butylidenephthalide to improve PD in C. elegans models. METHODOLOGY/PRINCIPAL FINDINGS: In the current study, we employed a pharmacological strain that expresses green fluorescent protein specifically in dopaminergic neurons (BZ555) and a transgenic strain that expresses human -synuclein in muscle cells (OW13) to investigate the antiparkinsonian activities of n-butylidenephthalide. Our results demonstrate that in PD animal models, n-butylidenephthalide significantly attenuates dopaminergic neuron degeneration induced by 6-hydroxydopamine; reduces -synuclein accumulation; recovers lipid content, food-sensing behavior, and dopamine levels; and prolongs life-span of 6-hydroxydopamine treatment, thus revealing its potential as a possible antiparkinsonian drug. n-Butylidenephthalide may exert its effects by blocking egl-1 expression to inhibit apoptosis pathways and by raising rpn-6 expression to enhance the activity of proteasomes. CONCLUSIONS/SIGNIFICANCE: n-Butylidenephthalide may be one of the effective neuroprotective agents for PD.