D'Souza, Serena A et al. (2011) International Worm Meeting "The LAMMER kinase MADD-3 Functions Cell-Autonomously to Direct Muscle Arm Extension."

Barbier, Louis, D'Souza, Serena A, Puckrin, Rachel, Roy, Peter

[International Worm Meeting, 2011]

The body wall muscles of nematodes project plasma membrane extensions, called muscle arms, to the motor axons to establish neuromuscular junctions. We have previously shown that an UNC-40 pathway, consisting of the UNC-40 receptor, the MADD-2 adaptor protein, and the UNC-73 Rho-GEF, functions cell-autonomously to direct muscle arm extension to the motor axon targets. Through screens for additional muscle arm development defective (Madd) mutants, we discovered three new genes that we call madd-3 X, madd-4 I, and madd-5 V. Here we present our characterization of madd-3, which encodes a LAMMER kinase. The LAMMER kinases are a well-conserved family of dual specificity kinases found ubiquitously throughout eukaryotes. The most extensively-studied family member is Drosophila Doa, which encodes multiple isoforms of the kinase. The 55 kDa Doa isoform is nuclear localized and plays an essential role in sex determination via the phosphorylation of SR proteins, which in turn regulates the sex-specific splicing of doublesex pre-mRNA. SR protein substrates are a conserved target of LAMMER kinases. The 105 kDa isoform of Drosophila Doa is localized exclusively to the cytoplasm, suggesting that it may have a role beyond regulating mRNA splicing. Like Doa, madd-3 encodes multiple isoforms. The madd-3 allele that was isolated in our screen (tr186) creates an early non-sense codon within the madd-3a-specific exon, indicating that MADD-3A is necessary for muscle arm extension. Muscle specific-expression of MADD-3A can rescue the muscle arm extension defects of madd-3 mutants, demonstrating that MADD-3A functions cell-autonomously. We therefore focused our efforts on characterizing MADD-3A in detail. Functional reporters show that MADD-3A's expression is restricted to non-pharyngeal muscles. Similar to the localization of the 105 kDa Doa isoform, we found that a functional MADD-3A::YFP fusion protein is localized to the cytoplasm of muscle cells and cannot be readily detected in nuclei. MADD-3A contains an N-terminal region with no defined domains and a C-terminal kinase domain. A structure-function analysis indicates that the kinase domain is necessary for MADD-3A function, and that the N-terminal region keeps MADD-3A from accumulating in the nucleus, but is unnecessary for function. Genetic analyses indicate that MADD-3A functions in an UNC-40 pathway. RT-PCR results, however, indicate that MADD-3A does not regulate the splicing of several components of the UNC-40 pathway, including unc-40, madd-2, and unc-73. Ongoing experiments to understand MADD-3As role in muscle arm extension may uncover new roles for the cytoplasmic isoforms of this conserved family of kinases.

D'Souza SA et al. (2016) PLoS Genet "The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans."

D'Souza SA, Bagg R, Roy PJ, Barbier L, Rajendran L, Moshiri H, van Pel DM

[PLoS Genet, 2016]

The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle's plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue.

Edgley M et al. (2002) Nucleic Acids Res "Improved detection of small deletions in complex pools of DNA."

Moerman D, Gilchrist E, Shen B, McKay S, Barstead R, D'Souza A, Moulder G, Edgley M

[Nucleic Acids Res, 2002]

About 40% of the genes in the nematode Caenorhabditis elegans have homologs in humans. Based on the history of this model system, it is clear that the application of genetic methods to the study of this set of genes would provide important clues to their function in humans. To facilitate such genetic studies, we are engaged in a project to derive deletion alleles in every gene in this set. Our standard methods make use of nested PCR to hunt for animals in mutagenized populations that carry deletions at a given locus. The deletion bearing animals exist initially in mixed populations where the majority of the animals are wild type at the target. Therefore, the production of the PCR fragment representing the deletion allele competes with the production of the wild type fragment. The size of the deletion fragment relative to wild type determines whether it can compete to a level where it can be detected above the background. Using our standard conditions, we have found that when the deletion is <600 bp, the deletion fragment does not compete effectively with the production of the wild type fragment in PCR. Therefore, although our standard methods work well to detect mutants with deletions >600 bp, they do not work well to detect mutants with smaller deletions. Here we report a new strategy to detect small deletion alleles in complex DNA pools. Our new strategy is a modification of our standard PCR based screens. In the first round of the nested PCR, we include a third PCR primer between the two external primers. The presence of this third primer leads to the production of three fragments from wild type DNA. We configure the system so that two of these three fragments cannot serve as a template in the second round of the nested PCR. The addition of this third primer, therefore, handicaps the amplification from wild type template. On the other hand, the amplification of mutant fragments where the binding site for the third primer is deleted is unabated. Overall, we see at least a 500-fold increase in the sensitivity for small deletion fragments using our new method. Using this new method, we report the recovery of new deletion alleles within 12 C.elegans genes.

Rose, Jacqueline et al. (2015) International Worm Meeting "Exposure to Nicotine or the Putative GABA agonist Toluene Differentially Modulates Expression of GABA Signaling Elements and Alters Mobility in C. elegans. ."

Stankowicz, Nicole, Leonti, Amanda, Stafford, Parker, Rose, Jacqueline

[International Worm Meeting, 2015]

In mammalian studies of nicotine addiction, nicotinic acetylcholine receptor activation reportedly acts to disinhibit reward signaling by suppressing GABA release (D'Souza and Markou, 2011). As well, it has been recently reported that a subset of neurons in the rodent hippocampus co-release GABA and acetylcholine (Saunders et al, 2015). Thus, it is of interest to describe how nicotinic receptor activation and GABA signaling may interact in neurons. C. elegans is an ideal model as both GABA and acetylcholine receptors are co-expressed at the NMJ. Mobility resulting from NMJ signaling has been well-described as relying on alternating activation of cholinergic and GABA receptors to produce contraction and simultaneous relaxation of opposing body wall muscles, respectively. Thus, the C. elegans NMJ is a site where direct interaction between excitatory cholinergic and inhibitory GABAergic signaling likely occurs. Studies from other labs confirm that cholinergic NMJ signaling can be modulated, seen as altered surface expression of acr-16 nicotinic Ach receptors (Jensen et al., 2012). Previous studies from this lab have quantified changes in nicotinic Ach and GABA receptor transcripts in adult worms following exposure to nicotine during development (Rose et al., 2013). More recently, we have found in adult worms that application of nicotine (30 microM) for 24 hours significantly increases UNC-49::GFP GABA receptor expression (p<0.05) while SNB-1::GFP synaptobrevin expression in motor neurons appears relatively unchanged. Conversely, exposure to the inhalant drug toluene (a GABA agonist) results in increased SNB-1::GFP expression in motor neurons (p<0.05) with seemingly no change in UNC-49::GFP expression. This increase in presynaptic GABA elements was further supported using qRT-PCR where a significant increase in unc-47 (vesicular GABA transporter) transcripts was found (p<0.01). With regards to mobility, these differences in expression appear to correlate with decreased swim motion (measured as body bends) following application of either nicotine or toluene (p<0.05 compared to controls). Interestingly, application of nicotine following toluene exposure seems to rescue the toluene-induced decrease in mobility. This suggests that the effects of one drug may offset the effects of the other potentially by returning to balance the drug altered pre- and post-synaptic GABA signaling elements. Taken together, these results demonstrate a potential role for GABA in both nicotine and toluene addiction. Funding support from WWU Research and Sponsored Programs Project Development Award..

Mark L Edgley et al. (2001) International C. elegans Meeting "A "Poison Primer" Technique for Enhancing Detection of Small Deletions by PCR"

Bob Barstead, Gary Moulder, Don Moerman, Erin Gilchrist, Bin Shen, Mark L Edgley, Anil D'Souza, Sheldon McKay

[International C. elegans Meeting, 2001]

Targeted PCR protocols to detect rare deletions in DNA from complex populations of mutagenized worms typically depend on a large size differential between the wild-type product and the deletion product. These protocols are used with libraries in which deletion representation is about 1 in 20,000. Recent work has shown that a significant proportion of deletions induced by trimethyl psoralen (TMP) treatment followed by UV irradiation are small, on the order of 50 to 600 base pairs (Gilchrist et al., in preparation). Detecting these deletions via PCR presents a problem because deletion amplicons are not favored over wild-type products if the amplicons are similar in size. Rare events will go undetected. To circumvent this problem we have developed a "poison primer" technique for nested PCR (D'Souza et al., unpublished data). This method works because the poison allows the formation of deletion products but titers out full-sized products. One or two extra primers, lying internal to both the regular external and internal pairs, are included in the external round as competitors to one or both external primers. These "poisons" are designed from coding sequence and are placed roughly in the center of the amplicon. The products they make with one or the other of the external primers are much shorter than the normal external product, and are thus produced in larger amounts. However, products from any deletion that removes sequence corresponding to the poison primers are made normally, with the result that deletion products are enriched relative to wild type. Since only wild-type and deletion products from this round can be amplified by the internal primers, formation of deletion products is greatly enhanced. Test reactions using sequenced deletions in the dim-1 gene and single poisons indicated that this method potentially improves sensitivity to one deletion chromosome in 5,000 wild-type chromosomes. We have isolated new deletions in more than eight genes from mutant libraries using poison primers, at complexities of 1/240 and 1/1200. Sequence data from the first three (deletions of 341, 295, and 78 base pairs) confirms that in all cases at least the 3' end of the poison primer is deleted, and control reactions on library DNA without poisons demonstrates that the poison primer was essential for detection. We are now using this system routinely for library screening, in addition to standard screens for larger deletions, to maximize the yield from each library. The specfic targeting feature of this technique should allow screens for small designer deletions, for example deletions of alternatively spliced exons or regulatory sequences.

Quartey MO et al. (2021) Sci Rep "The A(1-38) peptide is a negative regulator of the A(1-42) peptide implicated in Alzheimer disease progression."

Pennington PR, Heistad RM, Nyarko JNK, Barnes JR, Bolanos MAC, Parsons MP, Knudsen KJ, De Carvalho CE, Leary SC, Mousseau DD, Buttigieg J, Maley JM, Quartey MO

[Sci Rep, 2021]

The pool of -Amyloid (A) length variants detected in preclinical and clinical Alzheimer disease (AD) samples suggests a diversity of roles for A peptides. We examined how a naturally occurring variant, e.g. A(1-38), interacts with the AD-related variant, A(1-42), and the predominant physiological variant, A(1-40). Atomic force microscopy, Thioflavin T fluorescence, circular dichroism, dynamic light scattering, and surface plasmon resonance reveal that A(1-38) interacts differently with A(1-40) and A(1-42) and, in general, A(1-38) interferes with the conversion of A(1-42) to a -sheet-rich aggregate. Functionally, A(1-38) reverses the negative impact of A(1-42) on long-term potentiation in acute hippocampal slices and on membrane conductance in primary neurons, and mitigates an A(1-42) phenotype in Caenorhabditis elegans. A(1-38) also reverses any loss of MTT conversion induced by A(1-40) and A(1-42) in HT-22 hippocampal neurons and APOE 4-positive human fibroblasts, although the combination of A(1-38) and A(1-42) inhibits MTT conversion in APOE 4-negative fibroblasts. A greater ratio of soluble A(1-42)/A(1-38) [and A(1-42)/A(1-40)] in autopsied brain extracts correlates with an earlier age-at-death in males (but not females) with a diagnosis of AD. These results suggest that A(1-38) is capable of physically counteracting, potentially in a sex-dependent manner, the neuropathological effects of the AD-relevant A(1-42).

Danielle Thierry-Mieg et al. (2003) Worm Breeder's Gazette "www.wormgenes.org , a new gene centered database"

Vahan Simonyan, Michel Potdevin, Mark Sienkiewicz, Yuji KOHARA, Jean Thierry-Mieg, Danielle Thierry-Mieg, Tadasu SHIN-I

[Worm Breeder's Gazette, 2003]

Wormgenes is a new resource for C.elegans offering a detailed summary about each gene and a powerful query system.

Tangrodchanapong T et al. (2020) Front Pharmacol "Frondoside A Attenuates Amyloid- Proteotoxicity in Transgenic Caenorhabditis elegans by Suppressing Its Formation."

Tangrodchanapong T, Sobhon P, Meemon K

[Front Pharmacol, 2020]

Oligomeric assembly of Amyloid- (A) is the main toxic species that contribute to early cognitive impairment in Alzheimer's patients. Therefore, drugs that reduce the formation of A oligomers could halt the disease progression. In this study, by using transgenic <i>Caenorhabditis elegans</i> model of Alzheimer's disease, we investigated the effects of frondoside A, a well-known sea cucumber <i>Cucumaria frondosa</i> saponin with anti-cancer activity, on A aggregation and proteotoxicity. The results showed that frondoside A at a low concentration of 1 M significantly delayed the worm paralysis caused by A aggregation as compared with control group. In addition, the number of A plaque deposits in transgenic worm tissues was significantly decreased. Frondoside A was more effective in these activities than ginsenoside-Rg3, a comparable ginseng saponin. Immunoblot analysis revealed that the level of small oligomers as well as various high molecular weights of A species in the transgenic <i>C. elegans</i> were significantly reduced upon treatment with frondoside A, whereas the level of A monomers was not altered. This suggested that frondoside A may primarily reduce the level of small oligomeric forms, the most toxic species of A. Frondoside A also protected the worms from oxidative stress and rescued chemotaxis dysfunction in a transgenic strain whose neurons express A. Taken together, these data suggested that low dose of frondoside A could protect against A-induced toxicity by primarily suppressing the formation of A oligomers. Thus, the molecular mechanism of how frondoside A exerts its anti-A aggregation should be studied and elucidated in the future.

Hodgkin JA (1998) International Journal of Developmental Biology "Seven types of pleiotropy."

Hodgkin JA

[International Journal of Developmental Biology, 1998]

Pleiotropy , a situation in which a single gene influences multiple phenotypic tra its, can arise in a variety of ways. This paper discusses possible underlying mechanisms and proposes a classification of the various phenomena involved.

Tuck S (2011) Curr Biol "Extracellular vesicles: budding regulated by a phosphatidylethanolamine translocase."

Tuck S

[Curr Biol, 2011]

Recent work on a Caenorhabditis elegans transmembrane ATPase reveals a central role for the aminophospholipid phosphatidylethanolamine in the production of a class of extracellular vesicles.