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[
MicroPubl Biol,
2020]
Aggregates of the protein tau are the hallmark of several neurodegenerative diseases including Alzheimers disease, frontotemporal lobar degeneration (FTLD-tau), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Picks disease, and chronic traumatic encephalopathy (CTE) (VandeVrede, Boxer et al. 2020). Mutations in the gene coding for tau, MAPT, can cause FTLD-tau, directly linking tau dysfunction with disease (Dickson, Kouri et al. 2011). Another protein, TDP-43, comprises aggregates which are the primary hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP), and mutations in the gene coding for TDP-43, TARDBP, can cause disease (Kawakami, Arai et al. 2019). To model tau or TDP-43 proteinopathies, transgenic C. elegans have been generated that express the full-length human protein pan-neuronally. These worms exhibit significant uncoordinated movement on plates and impaired thrashing in liquid (Kraemer, Zhang et al. 2003; Liachko, Guthrie et al. 2010). However, tau- and TDP-43- expressing worms are not paralyzed; they still move their heads and have some motility on the plate (coiling, crawling with tail-drag, head swinging) which are not captured in standard crawling or thrashing assays. To assay differences in total activity, we used a WMicroTracker ARENA System (Phylumtech, AR and InVivo Biosystems, USA). The ARENA captures population level activity data by relying on optical interferometry, which uses a large array of infrared LED microbeams to detect both the movement and position of worms on a culture plate. Disruption of an LED microbeam by worm movement is recorded by repeat scans of the 6-well culture plate, and allows for real-time processing. The software identifies changes in the location of disrupted beams between scans and assigns an activity score based on differences identified between each consecutive scan (Simonetta SH). Both tau- and TDP-43- expressing worms had significantly less activity per minute than N2 (Figure 1). Further, we found the ARENA- assessed activity data recapitulated the relative severity of phenotypes among the strains as measured by motility assays. For example, both CK10 (tau V337M) and CK144 (tau WT) have significantly uncoordinated movement when crawling or thrashing in liquid, with CK144 having worse motility than CK10, due to its much higher burden of total tau protein expressed (Kraemer, Zhang et al. 2003). Likewise, CK410 (TDP-43 WT) worms have slightly impaired motility compared with N2 when crawling on a plate, CK423 (TDP-43 M337V) are severely uncoordinated, and CK426 (TDP-43 A315T) have the most severe uncoordinated phenotype. The relative toxicities of these strains stem from the effects of the mutations, as TDP-43 protein expression is relatively even among these transgenic strains (Liachko, Guthrie et al. 2010). Interestingly, the ARENA captures activity of these severely uncoordinated worms that move poorly in motility assays such as crawling on an NGM plate or thrashing in liquid (Kraemer, Zhang et al. 2003; Liachko, Guthrie et al. 2010). Therefore, ARENA assessment of aggregate activity may be a more accurate metric for capturing non-locomotor movement of C. elegans that are severely uncoordinated.
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[
International Worm Meeting,
2017]
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 hyperexcitibility 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.
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[
International Worm Meeting,
2021]
Accumulation of hyperphosphorylated TDP-43 protein in neuronal aggregates is the major pathological feature of two devastating neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD-TDP). To study the cellular, molecular, and genetic underpinnings of TDP-43 mediated neurotoxicity in a tractable model system, we have developed a C. elegans model of TDP-43 proteinopathy expressing human disease-causing mutant TDP-43 pan-neuronally (TDP-43 tg). These transgenic animals display early, progressive uncoordinated movement (Unc), decreased lifespan, and age-dependent neurodegeneration. Using this model, we have found that phosphorylation of TDP-43 increases mutant TDP-43 toxicity. To identify phosphatases controlling TDP-43 phosphorylation, we have screened an RNA interference (RNAi) library targeting most known or predicted C. elegans phosphatases. 167 candidate genes were individually tested for modification of TDP-43 dependent behavioral phenotypes and for changes in the phosphorylation status of TDP-43 by immunoblot. From this primary screen we have identified 15 phosphatase genes that modify TDP-43 phenotypes. Additional work on these genes will provide mechanistic insight into the environmental and cellular triggers of TDP-43 phosphorylation, and provide potential novel avenues for therapeutic interventions into TDP-43 proteinopathies such as ALS and FTLD-TDP.
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[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2010]
We leveraged next-generation sequencing technology to obtain a genome-wide and unbiased understanding of C. elegans population structure. Through collaboration and generous donations, we obtained a set of 202 wild isolates from throughout the world. In order to reduce the 100 Mb genome to a manageable size amenable to multiplexing, we employed Restriction-Assisted DNA marker sequencing (1) where the genome of each strain was cut using EcoRI and sequenced in both directions from each restriction site. This method allowed us to sequence the same eight megabases from each strain in two runs of an Illumina Genome Analyzer. We sequenced to an average of 12.6X coverage of each region, and SNPs were identified using SAMtools after mapping to the C. elegans genome using bwa. We will present the results of our analysis of population structure, linkage disequilibrium, and indications of genome-wide selection using the roughly 20,000 identified SNPs with minor allele frequencies greater than 5%. So far, we found the average pair-wise differences between strains is roughly 1/900 base pairs, as compared to the reference N2 genome. However, there is a wide range in the pair-wise differences with some strains being much more divergent from the reference N2 than the Hawaiian strain CB4856. These data will allow us to pursue genome-wide association studies and new recombinant inbred line crosses with maximally diverse wild isolates. (1) Baird NA, Etter PD, Atwood TS, Currey MC, Shiver AL, et al. 2008 Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers. PLoS ONE 3(10):
e3376.
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[
International Worm Meeting,
2005]
As exposures to the space environment become longer, information regarding the effects on biological aging will become important. We have not yet fully clarified aging processes in space environments. The aging process and lifespan are affected by various kinds of environmental factors including oxygen concentration (1), temperature and radiation. The aging phenomena that we usually see occur under certain conditions on the ground. Space environments differ from ground environments especially with regard to the radiation spectrum and gravity. We participated in the International C. elegans Experiment(ICE)First that examined the effects of a 10-day space flight on the nematode C. elegans. C. elegans has frequently been used for study of aging because of its short lifespan. Recently, Morley et al. reported that Huntington's-like polyglutamine (polyQ)-repeat proteins expressed in the muscle of C. elegans form aggregates as the animals age, and that this aggregation is delayed in long-lived mutants(2). We measured the polyQ aggregates in these nematodes after space flight as an aging marker. Herndon et al. showed that the sarcomere orientation in the body-wall muscle becomes disorderly as the animals age(3). We also observed the sarcomere orientation in the body-wall muscle of these nematodes after space flight as another aging marker. Acknowledgement: We thank Dr. R. L. Morimoto (Northwestern University) for providing us polyQ-YFP C. elegans strains. We also thank CGC for other strains. ICE-First was mainly conducted by the French Space Agency (CNES), with support of the European Space Agency and the Space Research Organization of the Netherlands. We are grateful to Dr. Michel Viso (CNES), Dr. K. Kuriyama (JAXA) and Dr. A. Higashitani (Tohoku University) for their support and suggestion for our experiment. References: 1) Honda S., Ishii N, Suzuki K, Matsuo M. J Gerontol. 48:B57-61. 1993 2) James F. Morley, Heather R. Brignull, Jill J. Weyers, and Richard I. Morimoto. Proc. Natl. Acad. Sci. USA, 99: 10417-10422. 2002 3) Herndon LA, Schmeissner PJ, Dudaronek JM, Brown PA, Listner KM, Sakano Y, Paupard MC, Hall DH, Driscoll M. Nature. 419:808-814. 2002