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Below are the latest abstracts from the posters presented at conferences:
J. C. Hincks1,2, E. House1 , B. E. Taylor3 , M. Harris3 , K. Hueffer4
1) Department of Biology and Wildlife, University of Alaska Fairbanks, Fairbanks, AK; 2) Department of Chemistry, University of Alaska Fairbanks, Fairbanks, AK; 3) Department of Biology, California State University Long Beach, Long Beach, AK; 4) Department of Veterinary Medicine, University of Alaska Fairbanks, Fairbanks, AK.
The rabies virus surface glycoprotein contains an amino acid sequence similar to neurotoxin peptides known to bind to and inhibit the function of nicotinic acetylcholine receptors (nAChR). In preliminary experiments, we have shown that a peptide fraction of the neurotoxin-like-domain of the rabies virus inhibits signaling of central nervous system nAChRs. In the nematode, Caenorhabditis elegans, defined motor neuron MC acts as a neurogenic pacemaker for pharyngeal pumping, and nAChRs are critical to excitation of the pharynx. We propose to use pharyngeal pumping in C. elegans as a screening assay for bioactivity of variants of rabies virus glycoprotein peptides. We first conducted a study to confirm various elements of our method such as treatment administration, treatment incubation time, and whether time spent in the microfluidic chamber influenced pharyngeal pumping activity. In the present study, we formed 3 treatment groups from these 29 amino acid long variants along with a PBS control. C. elegans were incubated at a concentration of 1 mM for each group for 60 minutes each. Following treatments, we assessed pharyngeal pumping frequency during 2-minute observation periods using a non-invasive electropharyngeogram (NemaMetrix Katalyst 100). Pharyngeal pumping was absent or greatly attenuated in animals incubated in active peptide compared to controls. Results indicate that internal and external exposure to a peptide proposed to inhibit nAChR attenuate pharyngeal pumping in C. elegans in a manner expected by nAChR antagonism. Results support the bioactivity of this virus peptide fraction and the utility of C. elegans electropharyngeogram as a screen for bioactivity of variants of this peptide.
Abanoub Akhnoukh1 , Akhnoukh Maria1 , Christopher Barrientos1 , Pierre Fahmy1 , Marina Kaldes1 , Brianna Ortiz1 , Vanessa Valdez1 , Joshua Hincks2, Barbara Taylor1 , Michael Harris1
1) Biology, California State University Long Beach, Long Beach, CA; 2) Biology and Wildlife, University of Alaska Fairbanks, Fairbanks, AK.
In C. elegans, feeding is achieved through a well-characterized behavior, pharyngeal pumping. Pharyngeal pumping is stimulated by the presence of bacterial food (E. coli) and/or the endogenous neuromodulator of feeding behavior, serotonin (5HT). Age-related changes in tissue morphology and function and a decline in C. elegans health are correlated with a reduction in the rate of pharyngeal pumping. As such, pharyngeal pumping frequencies have been used to exemplify health and age-related decline in this model organism. Traditionally pharyngeal pumping has been assessed by eye and reported as a pump rate, computed from the number of pumps observed divided by total recording time. Although rhythmic, pharyngeal pumping is both variable and periodic, traditionally pharyngeal pumping rate assessment does not consider underlying variability of pumping behavior. The recently developed NemaMetrix ScreenChip System automates the process of accurately resolving pharyngeal pumping by recording an electrical signature, the electropharyngeogram (EPG). As EPG recordings allow for precise detection of individual pump events, we aimed to assess patterns of variability in EPG measurements to test the hypothesis that underlying variability could be a relevant and insightful index of functional decline in this organism. In the context of directed undergraduate research, we use the ScreenChip System to resolve individual pump cycle durations and assess variability over conditions including age, 5HT stimulation, and experimental disruption. Findings suggest relatively consistent patterns of variability and changes in variability coincident with reductions in pharyngeal pumping frequencies. We suggest it may be possible to resolve more subtle aspects of functional decline from assessments of changing variability than are possible from pharyngeal pumping frequencies alone. Work reported was supported by the National Heart Lung and Blood Institute and National Institute of General Medical Sciences of the National Institutes of Health under awards number 1R15HL126105 and 1SC2GM112570. The work is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health or any other funding body.
Heather Currey1 , Brian C. Kraemer1,2, Nicole F. Liachko1,2
1) Research and Development, Veterans Affairs Puget Sound Health Care System, Seattle, WA; 2) University of Washington, Seattle, WA.
Frontotemporal dementia (FTD) is the second most common pre-senile dementia, affecting more than 50,000 people in the United States alone. This devastating neurodegenerative disease is characterized by the dysfunction and death of neurons in the frontal and temporal lobes of the brain, accompanied by profound changes in behavior and cognition leading to death. Pathologically, this disease is divided into two major groups. Approximately 40% of patients accumulate aggregates of the protein tau in disease affected neurons, while another 50% of patients accumulate aggregates of the protein TDP-43. These proteins appear functionally unrelated, as Tau is a microtubule binding protein promoting assembly and stability of microtubules while TDP-43 is involved in many aspects of RNA metabolism from transcription to translation. Therefore, these two pathological variants of FTD may represent two different diseases that impact the same brain region but have different etiologies and cellular consequences and may require different therapeutic strategies for treatment. To study the cellular, molecular, and genetic underpinnings of tau and TDP-43 mediated neurotoxicity in a tractable model system, we have developed C. elegans models expressing these proteins pan-neuronally. Both tau and TDP-43 transgenic animals display early, progressive motor dysfunction, decreased lifespan, and age-dependent degeneration of specific types of neurons. To explore whether neurotoxic tau and TDP-43 affect C. elegans pharyngeal pumping, we utilized a non-invasive microfluidics-based device to record electrophysiological signals from pharyngeal muscles and neurons (electropharyngeogram, EPG). We found pump rate and pump duration have an inverse relationship within tau worms. Worms expressing human tau show a significant increase in pump frequency as compared to N2 worms. We also observed a decrease in pump duration with the same worms. Our initial data indicate that this is true in TDP-43 model worms as well. This phenotype may indicate increased neuronal hyperexcitability prior to neuronal injury and death. Future work will include EPG analysis of potential suppressors of hyperexcitability, as well as EPG analysis of loss of function genetic mutants in C. elegans homologs of tau and TDP-43.
B. Nebenfuehr, A. Golden National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD.
Humans harbor approximately 7,000 rare diseases, ~80% of which are monogenic. A rare disease is defined as affecting less than one in 1,500 people. Combined, these rare diseases affect nearly 1 in 10 Americans (25 to 30 million people), and treatments only exist for around 5% of these diseases. Thanks to the advent of whole genome sequencing, the gene(s) responsible for many rare diseases are known, opening the door for more comprehensive studies. Around 60% of the more than 20,000 protein-coding genes in Caenorhabditis elegans are estimated to have human counterparts. We can therefore study worm phenologs of human disease, or the distinct phenotype in worms that, while different from the human phenotype, stems from mutations in a homologous gene. The CRISPR-Cas9 system also allows us to mutate individual nucleotides analogous to those implicit in the human diseases to mimic the patient alleles. Presented here are examples of three ongoing projects designed to uncover cellular interactors and potential drug targets for improving and expanding the treatments available to patients suffering from a rare disease. Long-QT syndrome (LQT) is an autosomal dominant class of arrhythmia characterized by an extended time of repolarization of the heart after each beat (>440ms instead of the normal 350-440ms). This can lead to sudden cardiac arrest, particularly during exercise or other excitement. One of the genes most closely associated with LQT is the alpha-subunit of the potassium channel KvLQT1 or KCNQ1. The C. elegans ortholog of KCNQ1 is kqt-3 (87.9% homology) and deletion alleles of this gene display arrhythmic pumping in the pharynx (NemaMetrix, Eugene, OR). We are examining causative patient alleles in kqt-3 in worms to study their affects on pharyngeal pumping and will attempt suppressor, enhancer, and drug screens to try to reverse or enhance, these phenotypes. Rare mutations in the Seipin gene BSCL2 are associated with the autosomal recessive congenital generalized lipodystrophy, or Berardinelli-Seip Syndrome (BSCL). This disease manifests primarily as a nearly complete loss of adipose tissue and severe insulin resistance. The yet-unnamed worm ortholog of seipin sits at the R01B10.6 locus and has 97.1% homology to the human isoform 1 of seipin. Preliminary evidence suggests no phenotype for C. elegans seipin mutants, but a full gene knockout has not yet been generated. The iron-sulfur cluster scaffold protein NFU1 is associated with the autosomal recessive multiple mitochondrial dysfunctions syndrome, which is characterized by encephalopathy, hypotonia, and psychomotor delay, as well as a lactic acidosis and a failure to thrive. Mutants in the C. elegans ortholog lpd-8 (90.5% homology) are sterile. We are generating a new balancer for this region so that we can carry out suppressor screens. Updates on the progress of these three projects will be presented.
Where They Are
Scientists around the world are using the ScreenChip System and C. elegans to conduct research in aging and age-related diseases, cardiac diseases, neurodegenerative diseases, drug screen and development, environmental toxicology and signaling pathways & networks.
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