Sandrine Fraboulet et al. (2009) European Worm Neurobiology Meeting "Chemical rescue of paralysis in a transgenic model of Caenorhabditis elegans overexpressing human Beta-amyloid"

David I C Scopes, Andrew K Jones, David B Sattelle, Aileen Moloney, Christopher D Link, Steven D Buckingham, Mark J Treherne, Hozefa Amijee, Sandrine Fraboulet

[European Worm Neurobiology Meeting, 2009]

Authors acknowledge support from MRC. Numerous studies support a role for aggregated Ab as a mediator of the toxicity that underlies Alzheimer.s disease (AD) and other diseases such as Inclusion Body myositis (IBM), an acquired muscle disease affecting people over 50 years old. In this pathology, muscle weakness and degeneration is accompanied by chronic muscle inflammation. Remarkably, despite the inflammation component of the disease, IBM patients are only poorly responsive to anti-inflammatory drugs suggesting that inflammation per se may not be the primary cause of the pathology. We have used a C. elegans transgenic line over-expressing human amyloid 1-42 peptide in the muscles as a model with which to test the efficiency of new compounds on in vivo amyloid toxicity. This C. elegans line becomes paralyzed shortly after the induction of amyloid expression in the muscles, which makes it an easily-recordable phenotype useful for exploring candidate treatments to alleviate the paralysis. We report on the actions of two novel chemicals, which inhibit amyloid aggregation and partially rescue the amyloid-induced phenotype. References: Wu Y, W.Z., Butko P, Christen Y, Lambert MP, Klein WL, Link CD, Luo Y. (2006) J. Neurosci., 26, 13102-13. Link CD, T.A., Kapulkin V, Duke K, Kim S, Fei Q, Wood DE, Sahagan BG. (2003) Neurobiol Aging., 24, 397-413. Jones, A.K., Buckingham, S.D. and Sattelle, D.B. (2005). Nat Rev Drug Discov, 4, 321-30.

G Kao et al. (1999) International C. elegans Meeting "Characterization of Laminin beta and gamma Genes."

G Kao, C Huang, WG Wadsworth

[International C. elegans Meeting, 1999]

We have characterized laminin b (lam-1) and laminin g (lam-2) genes. The sequencing project has uncovered genes for one isoform each of the two subunits and two isoforms of laminin a , epi-1 and lam-3. (See abstract by Cheng et al.). This indicates that two types of trimers likely are form in basement membranes : a A bg and a B bg . A hypomorphic mutation (rh219) exists in lam-1, whose phenotypes we have described in previous meetings (IWM 1997). Both lam-1(RNAi) and lam-2(RNAi) animals arrest development during morphogenesis. Significantly, this phenotype is identical to the arrest seen in double knockout of epi-1 and lam-3, and is more severe than either knockout alone. We examined the expression patterns of lam-1 and lam-2 using promoter:GFP fusions. Both genes are expressed in body wall muscles, pharynx, vulval muscles, the rectal muscle group and the distal tip cells. The expression patterns of b and g subunit genes overlap with and are more widespread than either epi-1 or lam-3 alone. Taken together these results suggest that lam-1 and lam-2 are found in all basement membranes and indicate that trimers a A bg and a B bg are formed.

Link CD et al. (2003) Neurobiol Aging "Gene expression analysis in a transgenic Caenorhabditis elegans Alzheimer's disease model."

Duke K, Taft A, Wood DE, Kim S, Link CD, Kapulkin V, Fei Q, Sahagan BG

[Neurobiol Aging, 2003]

We have engineered transgenic Caenorhabditis elegans animals to inducibly express the human beta amyloid peptide (Abeta). Gene expression changes resulting from Abeta induction have been monitored by cDNA hybridization to glass slide microarrays containing probes for almost all known or predicted C. elegans genes. Using statistical criteria, we have identified 67 up-regulated and 240 down-regulated genes. Subsets of these regulated genes have been tested and confirmed by quantitative RT-PCR. To investigate whether genes identified in this model system also show gene expression changes in Alzheimer's disease (AD) brain, we have also used quantitative RT-PCR to examine in post-mortem AD brain tissue transcript levels of alphaB-crystallin (CRYAB) and tumor necrosis factor-induced protein 1 (TNFAIP1), human homologs of genes found to be robustly induced in the transgenic C. elegans model. Both CRYAB and TNFAIP1 show increased transcript levels in AD brains, supporting the validity of this approach.

AM van der Linden et al. (1999) International C. elegans Meeting "Mutations that suppress the lethargic and egg-laying defective phenotype of Gb overexpression"

HC Korswagen, AM van der Linden, RH Plasterk, F Simmer

[International C. elegans Meeting, 1999]

G proteins act as signal-transducing molecules that connect serpentine transmembrane receptors to various intracellular effectors. In C. elegans several G proteins have been identified. There are 20 G a subunits, two G b subunits (GPB-1 and GPB-2) and at least two G g subunits (Jansen, pers. comm.). Previously, Zwaal et al. (1) studied GPB-1 in detail. GPB-1 is over 80% identical to mammalian G b subunits and is required throughout development. Animals homozygous for the gpb-1 deletion arrest in the first stage of larval development. This lethal phenotype is morphologically similar to gsa-1 and acy-2 null mutant animals, suggesting the absence of an essential Gs-coupled signaling pathway in early development (2). Progeny of mosaic animals that lost maternal expression of GPB-1 in the germline are embryonic lethal due to the inappropriate orientation of cleavage planes in early cell division. Overexpression of GPB-1 reduces locomotion and egg-laying and resembles the phenotype of goa-1(gf) mutants. The second G beta subunit (GPB-2) is not required for viability, but it is shown to modulate muscle activity (Simmer, pers. comm). Whereas GPB-1 and GPB-2 show similar expression patterns and are both involved in locomotion and egg-laying, they seem to have opposite functions in regulating muscle and neuron activity. Presently, we are investigating the role of these proteins during embryonic development. The effects of GPB-1 on behavior may represent either a direct signal function of the G bg -subunit on downstream targets, or G bg -levels may indirectly influence G alpha activity, or both. To identify downstream components of the G bg -signaling pathway, we screened for mutations that suppress the lethargic and egg-laying defective phenotype of GPB-1 overexpression. We isolated 13 independent mutations that suppress this phenotype, named gbs ( G b eta s uppressor). Two mutations are hyperactive (loopy) and egg-laying constitutive in the absence of GPB-1 overexpression, and are phenotypically similar to goa-1(lf) mutants. We mapped gbs-1 to chromosome I. We are currently mapping and characterising these suppressors. 1) Zwaal et al. (1996) Cell 86; 2) Korswagen et al. (1998) EMBO J. 17

M Gotta et al. (1999) International C. elegans Meeting "Control of cell division axis choice in C.elegans"

M Gotta, J Ahringer

[International C. elegans Meeting, 1999]

The orientation of the cleavage plane is a critical aspect of development. The cleavage plane is established by the position and orientation of the mitotic spindle, which is set by the position of the centrosomes. In C. elegans embryos, cell division planes follow a fixed pattern, allowing abnormalities to be easily analysed. In previous work, we found that gpb-1 , which encodes the beta subunit of a heterotrimeric GTP-binding protein, has a crucial role in spindle orientation. In embryos lacking GPB-1 activity, mitotic spindles in early cell divisions are formed properly but they are randomly oriented instead of following a fixed pattern. Lack of GPB-1 does not appear to affect the polarity of the zygote. Interestingly, GPB-1 localises to the asters during cell division. To further characterise the G protein involved in spindle orientation, we sought the G g partner. There are two G g genes in the genome, gpc-1 and gpc-2 . RNA interference to gpc-2 , but not to gpc-1 , causes embryo lethality. Staining of early embryos with anti-tubulin antibodies shows that cleavage planes in early cell divisions are abnormal. However, the first cleavage is asymmetric and P granules are correctly localised to the germline precursor cells. This suggests that GPC-2 is probably the partner of GPB-1 in controlling spindle orientation. We are currently trying to find interactors of G bg by a three hybrid screen. As a second approach, we are trying to identify other factors involved in spindle orientation by RNAi to candidate genes based on the homology with yeast polarisation during mating which is controlled by a heterotrimeric G protein. Upon activation by the receptor, the G bg dimer activates downstream factors such as Cdc42p and Ste20p. Injection of dsRNA for two of the many Ste20p homologues show no phenotype, although this may reflect a redundant function. RNAi to the C. elegans Cdc42p homologue causes embryo lethality and the early embryos are osmotically sensitive. In these embryos cleavage planes are abnormally oriented. In addition the first cleavage can be symmetric and P granules are not properly localised. We have undertaken a two hybrid screen with CDC-42 and we are currently analysing the interacting clones by RNAi.

C Huang et al. (1999) International C. elegans Meeting "Each C. elegans Laminin a Subunit Mediates Distinct Aspects of Morphogenesis"

WG Wadsworth, C Huang, PD Yurchenco

[International C. elegans Meeting, 1999]

Laminin is a heterotrimeric glycoprotein composed of a , b , and g subunits. The genome sequencing project reveals two a , one b and one g laminin subunit genes. Based on sequence analyses, major features and known binding sites are conserved. The predicted a chains, laminin a A and laminin a B, are most similar to the vertebrate a 1/ a 2 and a 5 chains respectively, while the b and g resemble the b 1/ b 2 and g 1 chains. Thus, there are two predicted laminin trimers, a A bg and a B bg . Mutations in epi-1 , which encodes laminin a B, have been isolated and characterized (abstract ECWM 98). They cause defects that are consistent with the developmental roles for basement membranes (BMs). We report the expression patterns of laminin a A and a B. By immunostaining, both first appear at gastrulation between germ layers. However, laminin a A is heavily deposited anteriorly, surrounding pharygeal precursors, whereas laminin a B is more posteriorly localized. Laminin a B becomes localized to the BMs associated with pharynx, intestine, gonad, body wall, body wall muscle, and muscles throughout development. In contrast, laminin a A accumulates in pharyngeal BM, intestinal BM and body wall muscle BM during elongation and its level in intestinal BM and body wall muscle BM gradually decreases. In larvae and adults, laminin a A is only sometimes detected weakly in pharyngeal BM and body wall muscle BM. It is primarily localized in the spermatheca. Injection of dsRNA directed to epi-1 produces phenotypes similar to those observed in epi-1 alleles and the injection of dsRNA directed to lam3 , which encodes laminin aB, causes L1 larval arrest with severe pharyngeal defects. Double dsRNA-mediated interference directed to both genes produces embryos that arrest at morphogenesis with phenotypes more severe than those caused by the RNAi of either epi-1 or lam3 alone, indicating that both genes contribute to morphogenesis. Together, our RNAi and immunostaining results indicate that laminin a B has a broad role in maintaining the structural integrity of BMs and for regulating many aspects of morphogenesis, while laminin a A is restricted to specialized membranes and is required for the morphogenesis of specific tissues.

Kim B et al. (2019) Sci Rep "Differential effects of alkyl gallates on quorum sensing in Pseudomonas aeruginosa."

Choi HY, Kwak JH, Kim B, Kim WG, Park JS

[Sci Rep, 2019]

Virulence factors and biofilms constitute attractive targets for the prevention of infections caused by multidrug-resistant bacteria. Among alkyl gallates, propyl gallate (PG) and octyl gallate (OG) are used as food preservatives. Here we found that alkyl gallates differentially affect virulence, biofilm formation, and quorum sensing (QS) in Pseudomonas aeruginosa. Ethyl gallate (EG), PG, and butyl gallate (BG) inhibited biofilm formation and virulence factors including elastase, pyocyanin, and rhamnolipid, in P. aeruginosa without affecting cell viability by antagonizing the QS receptors LasR and RhlR. PG exhibited the most potent activity. Interestingly, hexyl gallate (HG) inhibited the production of rhamnolipid and pyocyanin but did not affect elastase production or biofilm formation. Notably, OG inhibited the production of rhamnolipid and pyocyanin but stimulated elastase production and biofilm formation. Analysis of QS signaling molecule production and QS gene expression suggested that HG inhibited RhlR, while OG activated LasR but inhibited PqsR. This mechanism was confirmed using QS mutants. Additionally, PG prevented the virulence of P. aeruginosa in Caenorhabditis elegans and a mouse model. This is the first report of the differential effects of alkyl gallates on QS systems and PG has great potential as an inhibitor of the virulence and biofilm formation of P. aeruginosa.

Zwaal RR et al. (1996) Midwest Worm Meeting "EARLY CLEAVAGE ORIENTATION REQUIRES G PROTEIN ACTIVITY"

Ahringer JA, Zwaal RR, Plasterk RHA

[Midwest Worm Meeting, 1996]

Heterotrimeric GTP binding proteins form a class of widespread signal transduction molecules involved in taste, smell and vision, and in the recognition of hormones and neurotransmitters. G proteins cycle between an on and off state. In the off state, they form a heterotrimeric complex in which the a-subunit binds a molecule of GDP. Upon activation by a seven transmembrane receptor, GDP is replaced by GTP and the complex dissociates into an a-monomer and a bg-dimer. The a and the bg are able to independently modify specific second messenger pathways in the cell, together producing the proper response. The intrinsic GTPase activity of the a-subunit hydrolyzes GTP to GDP to end the on state. In C. elegans, a number of Ga subunits have been cloned, which are differentially expressed and might have specific functions in development and behavior. There appears to be only a single b-subunit, encoded by the gpb-1 gene. This b-subunit should thus interact with all different a-subunits, and is therefore essential for all G protein regulated processes in the worm. A polyclonal antibody raised against GPB-1 was used to determine its expression pattern. gpb-1 appears to be expressed in most if not all cell types, and is most pronounced in the neurons. Expression is also detected in the hermaphrodite gonad and in the early embryo from the 1-cell stage onwards, suggesting the protein is maternally contributed to function during embryonic development. Interestingly, immunostaining is detected not only in the cell membranes, but also appears to colocalize with the asters just before and during early cell division. We obtained a null-allele for gpb-1 using the transposon insertion/deletion method(1). Heterozygous animals are wild-type, but homozygous progeny die soon after hatching, possibly due to an osmoregulatory defect. Embryogenesis in these embryos proceeds normally, presumably due to the presence of maternally provided Gb. We performed mosaic analysis by studying homozygous mutant animals that are rescued by an extrachromosomal gpb-1 transgene. Effects caused by presumed loss of the transgene include defects in movement and egg-laying, as could be expected based on the phenotypes of Gao mutant animals(2). A small fraction of the rescued adults produced broods of mainly or only dead embryos. Using immunohistochemistry, we observed no or strongly reduced levels of GPB-1 in the gonads of these mothers, and no GPB-1 in the dead embryos, indicating that the absence of maternal GPB-1 perturbs embryonic development. In these embryos, the orientation of the early cleavage spindles appears randomly oriented. Consequently, cell positions are abnormal, resulting in abnormal differentiation. Other characteristics of early cleavages, such as the asymmetric distribution of P-granules and factors determining cell size and timing of cleavage, seemed to be unaffected in gpb-1 embryos. This suggests that maternally provided G protein activity is important for the proper segregation of asymmetrically distributed cytoplasmic components by mediating the orientation of the cleavage spindle. (1)Zwaal et al., PNAS (1993) 90: 7431-7435. (2)Sgalat et al., Science (1995) 267: 1648-1651; Mendel et al., ibid 1652-1655.

Park Y-S et al. (1990) Worm Breeder's Gazette "col-4 isn't."

Kramer JM, Park Y-S

[Worm Breeder's Gazette, 1990]

Of the six blister genes, we have been particularly interested in bli-6 IV because Bob Herman has shown that alleles of sqt-1, dpy-10 and rol-8 can suppress the dominant mutation bli-6(mn4). Although Dpy is often epistatic to Bli, Bob identified non- or very weak Dpy alleles of dpy-2 and dpy-10 that suppress mn4. Since sqt-1, dpy-10 and rol-8 show genetic interactions and all except rol-8 have been shown to encode collagens (Adam Levy has identified a collagen that is likely to be rol-8) there is a good possibility that they may physically interact with the bli-6 gene product. A Bristol/Bergerac polymorphism, identified by hybridizing genomic Southern blots with col-2 at low stringency, had been previously mapped to the vicinity of bli-6, and was named col-4 (Cox et al. Genetics 109:513,1985). Initial three-factor mapping with unc-5 and dpy-20 failed to separate bli-6 and col-4(Berg), so we decided to examine col-4 more closely. Further mapping using unc-44 and unc-24 showed that col-4 is approx. 0.4 mu to the right of bli-6, but concurrent molecular analyses of col-4 produced some unexpected results. The col-4 gene was cloned by identifying a col-2 crosshybridizing clone, in a size selected genomic sublibrary, that showed the expected Br/Bg polymorphism when probed to genomic Southern blots. The first surprise was that col-4 doesn't encode a collagen. The first case, that we know of, where low stringency collagen cross-hybridization has pulled out something other than a collagen. The col-4 sequence predicts a polypeptide consisting primarily of 23 tandem copies of the following 15 amino acid repeat: GAPPSGGPPGPF(D/N)PS (note: NPS is a potential glycosylation site)