Annette Piechulek

Heinrich Heine University; Dusseldorf, Germany

Scharf, Andrea et al. (2015) International Worm Meeting "Amyloid formation controls worm behavior."

Piechulek, Annette, Scharf, Andrea, von Mikecz, Anna

[International Worm Meeting, 2015]

The accumulation of amyloid-like structures and protein aggregation are hallmarks of aging and neurodegenerative diseases, but the role of impaired protein homeostasis in the aging process and in pathogenesis is still controversial. To analyze the role of protein fibrillation we use nanoparticles to induce proteostatic stress in Caenorhabditis elegans. Filter-trap assays show that stressed young worms accumulate SDS-insoluble, ubiquitinated proteins that are normally a feature of old adult C. elegans. The premature biochemical phenotype concurs with concentrated amyloid in the nucleoli of intestinal cells that can be detected after 24h nanoparticle exposition. Single cell imaging and light sheet microscopy reveal that nanoparticles enter cells and nuclei of the worm and accumulate in organs such as pharynx, intestine, spermathecae and vulva. The nanoparticle exposition results in a premature decline of the respective organ function such as egg laying and pharyngeal pumping (Scharf et al., 2013). In addition, nanoparticle-stressed worms show locomotion defects including locomotory senescence and reduced ability to reach food compared to untreated worms. We follow the idea that the observed changes in worm behavior are the results of nanoparticle-induced protein fibrillation. The affected behavior is driven by well described neural circuits which allows for correlation between protein fibrillation and neural function in organismal aging. The presented data will address interactions between amyloid formation, altered neural behavior phenotypes and neurotoxicity of protein aggregation in premature aging of C. elegans.

Levin ED et al. (2009) Neurotoxicology "Genetic aspects of behavioral neurotoxicology."

Kirshner A, Eddins D, French R, Helmcke K, Page GP, Linney E, Lnenicka G, Berger K, Welsh-Bohmer KA, Corl AB, Levin ED, Hirsch HV, Aschner M, Bartlett S, Possidente B, Hayden KM, Chen L, Possidente D, Ruden D, Heberlein U

[Neurotoxicology, 2009]

Considerable progress has been made over the past couple of decades concerning the molecular bases of neurobehavioral function and dysfunction. The field of neurobehavioral genetics is becoming mature. Genetic factors contributing to neurologic diseases such as Alzheimer's disease have been found and evidence for genetic factors contributing to other diseases such as schizophrenia and autism are likely. This genetic approach can also benefit the field of behavioral neurotoxicology. It is clear that there is substantial heterogeneity of response with behavioral impairments resulting from neurotoxicants. Many factors contribute to differential sensitivity, but it is likely that genetic variability plays a prominent role. Important discoveries concerning genetics and behavioral neurotoxicity are being made on a broad front from work with invertebrate and piscine mutant models to classic mouse knockout models and human epidemiologic studies of polymorphisms. Discovering genetic factors of susceptibility to neurobehavioral toxicity not only helps identify those at special risk, it also advances our understanding of the mechanisms by which toxicants impair neurobehavioral function in the larger population. This symposium organized by Edward Levin and Annette Kirshner, brought together researchers from the laboratories of Michael Aschner, Douglas Ruden, Ulrike Heberlein, Edward Levin and Kathleen Welsh-Bohmer conducting studies with Caenorhabditis elegans, Drosophila, fish, rodents and humans studies to determine the role of genetic factors in susceptibility to behavioral impairment from neurotoxic exposure.

Limke, Annette et al. (2021) International Worm Meeting "Food additives target the gut-neural axis: Impaired peptide trafficking and amyloid protein aggregation lead to premature aging phenotypes"

Berwanger, Lutz, Scharf, Andrea, Limke, Annette, von Mikecz, Anna

[International Worm Meeting, 2021]

Nanoparticles like nano silica (E551) are commonly used as food additives although bio-interactions with the gut are not well understood. Nutrients, like oligopeptides, together with nano silica are taken up via the same route via the pharynx to the intestinal tract. At the apical domain of intestinal cells, oligopeptides are transferred by the OPT-2/PEP-2 transporter to the cytoplasm and degraded downstream by hydrolases to provide amino acids for protein biosynthesis. For analyses of the peptide transport, the fluorophore-conjugated dipeptide B-Ala-Lys (AMCA) was used which normally distributes diffusely throughout the cytoplasm. Exposure with nano silica lead to an altered morphology of the intestine and disturbed peptide trafficking. Dipeptides accumulated in spherical subdomains that grow over time to a diameter of ≥6 microm. The peptide subdomains did not colocalize with known organelles such as gut granules and formed independently of the insulin/IGF-1 receptor (DAF-2) signaling pathway. Mutation of the daf-2 tyrosine kinase domain showed distinct patterns of peptide subdomains along the apical membrane of intestinal cells which indicated specific trafficking defects (Piechulek et al., 2019). We suggest that nano silica-induced peptide subdomains represent new compartments of dipeptide storage leading to the inhibition of hydrolysis and peptide metabolism which in turn results in a decline of translation. Aberrant segregation of dipeptides by phase separation leads to a petite phenotype resembling OPT-2/PEP-2 transporter deficient mutants. In addition, nano silica induce global amyloid protein aggregation that segregates components of the protein homeostasis to an insoluble aggregome. Candidate proteins include components of the translational machinery and ribosomal RNA processing (Scharf et al., 2016). Taken together, the results suggest that impairment of translation occurred by a lack of amino acid supply and amyloid segregation of proteins critically involved in protein synthesis. As translation was shown to be involved in the generation of both, premature aging and neurodegenerative diseases, further investigation of its role in the organ-cross talk between intestine and the neural system in xenobiotic-exposed C. elegans is required.

Patterson GI et al. (1996) East Coast Worm Meeting "Identification of new genes in dauer/TGF-beta signalling"

Ruvkun GB, Patterson GI

[East Coast Worm Meeting, 1996]

We are interested in understanding the mechanism by which food and pheromone signals are transduced to signal dauer arrest or non-dauer development. Don Riddle's group has identified a TGF-beta homologous pathway that is involved in this signal transduction process. daf-7 encodes a peptide ligand, and daf-1 and daf-4 encode transmembrane serine/threonine kinases that may be receptors for the daf-7 protein. Recent work by Takao Inoue, Annette Estevez and our laboratory (see abstract by A. Koweek and G. Patterson) has identified a family of genes that are likely to be involved in the signal transduction from these receptors; daf-8, daf-14, and daf-3 are all homologues of the drosophila gene Mad, which transduces signals from serine/threonine kinase receptors. The mechanism by which these receptors and Mad homologues transduce signal is unknown; we are identifying new genes that act in this pathway. In previous screens for suppressors of daf-7, the genes daf-3, daf-5 and daf-12 were identified by D. Riddle. We have identified four new genes that suppress daf-7. Two of the genes were identified by mutations that strongly suppress daf-7; the other two show weak suppression. We are trying to clone a strong suppressor called scd-1 (Suppressor of Constitutive Dauer formation--pronounced "scud"). We isolated two alleles as suppressors of daf-7; Takao Inoue provided two alleles he isolated as suppressors or daf-14 and daf-8. We mapped the gene between lin-2 and unc-9 on X, and we are doing finer mapping as well as injecting candidate cosmids for rescue. We have also begun a screen for suppressors of daf-3. We are mutagenizing a daf-7 daf-3 strain (daf-7 is dauer constitutive, daf-3 is dauer defective, and the double mutant is dauer defective). We will screen for dauers at 25 degrees C. daf-2, daf-19 and daf-23 are the known dauer constitutive mutants epistatic to daf-3. We hope to get new mutations that, while not dauer constitutive on their own, suppress the daf-3 mutation and allow the daf-7 mutation to force dauer formation. We will screen with both a null daf-3 allele and a partial loss-of function daf-3 allele.