Tomoki Kuwahara et al. (2010) East Asia Worm Meeting "The analysis of the neurotoxicity of phosphorylation-mutant alpha-synuclein in C. elegans"

Tomoki Kuwahara, Takeshi Iwatsubo, Shohei MItani, Reina Tonegawa

[East Asia Worm Meeting, 2010]

Mutations or multiplications in alpha-synuclein gene cause familial forms of Parkinson disease (PD), and the deposition of alpha-synuclein as Lewy bodies occurs as a hallmark lesion of PD brain, implicating alpha-synuclein in the pathogenesis of PD. To elucidate the molecular basis of alpha-synuclein neurotoxicity, we have generated transgenic C. elegans (Tg worms) that pan-neuronally overexpress human alpha-synuclein. Tg worms expressing wild-type or pathogenic-mutant alpha-synuclein showed mild defect in locomotory function, and immunohistochemical analysis showed that a portion of alpha-synuclein was phosphorylated at Ser129, a hallmark modification selectively detected in PD brain. Next, we generated Tg worms overexpressing non-phosphorylated form (S129A) of alpha-syncuein. Interestingly, two independent lines of S129A-alpha-synuclein Tg worms showed severe motor dysfunction and growth retardation with similar expression level to that overexpressing wild-type alpha-synuclein. Biochemical analysis revealed that S129A-alpha-synuclein exhibited higher propensity to bind membranes compared to wild-type alpha-synuclein. Further, gene expression profiling using DNA microarray showed that a set of DAF-16 target genes were dramatically upregulated in S129A-alpha-synuclein Tg worms. We also conducted a forward genetic screen for mutation that ameliorates S129A-alpha-synuclein toxicity, and several gene loci were identified. Taken together, our Tg worms would be considered useful for further genetic analysis of alpha-synuclein neurotoxicity.

Tomoki Kuwahara et al. (2004) East Asia Worm Meeting "Dopamine neuron dysfunction in transgenic C.elegans expressing human alpha-synuclein: a model for familial Parkinson,s disease"

Takeshi Iwatsubo, Shohei MItani, Keiko Gengyo-Ando, Akihiko Koyama, Tomoki Kuwahara

[East Asia Worm Meeting, 2004]

Parkinson , s disease (PD) is a neurodegenerative disorder characterized pathologically by loss of dopamine (DA) neurons and formation of Lewy bodies containing alpha-synuclein. Missense mutations or triplication of alpha-synuclein gene have been identified in pedigrees of familial PD (FPD), implicating alpha-synuclein in the pathogenesis of PD. Here we established transgenic C.elegans (TG worms) overexpressing human alpha-synuclein (wild-type, A30P or A53T FPD mutant) in DA neurons using a DA neuron-specific promotor dat-1 , encoding a DA transporter. Cell bodies and neurites of DA neurons were positive for alpha-synuclein, sometimes in a dot-like pattern. Phosphorylation of alpha-synuclein, characteristic of insoluble alpha-synuclein aggregates in human PD, was detected in a subset of DA neurons in aged worms. We generated TG worms co-expressing EYFP in monoaminergic neurons by cat-1 promoter and alpha-synuclein in DA neurons, and found that EYFP fluorescence in neurites of DA neurons was decreased without loss of cell bodies exclusively in TG worms expressing FPD mutant alpha-synuclein. We then examined the function of DA neurons by measuring decrement in body-bending frequency upon exposure to food, which has been attributed to the function of DA neurons. TG worms expressing A53T or A30P FPD mutant alpha-synuclein showed failure in changes of bending frequency upon exposure to food, whereas TG worms expressing either wild-type alpha-synuclein, beta-synuclein, or EGFP showed normal response. FPD mutation-dependent failure in bending response was recovered by administration of DA, and DA content was significantly decreased in TG worms expressing FPD mutant alpha-synuclein. These results indicate that overexpression of FPD-mutant alpha-synuclein causes dysfunction of DA neurons possibly through accumulation of misfolded alpha-synuclein. Our alpha-synuclein TG worms would be a useful experimental system for further genetic and pathological analysis of degeneration of DA neurons caused by accumulation of alpha-synuclein.

Morris, Caitlin et al. (2015) International Worm Meeting "Identification and characterization of genes controlling gut granule size."

Barrett, Alec, Hermann, Greg, Morris, Caitlin, Delahaye, Jared

[International Worm Meeting, 2015]

C. elegans intestinal cells are characterized by the presence of gut granules, lysosome-related organelles (LROs) that contain autofluorescent and birefringent material. Gut granule formation requires the activity of evolutionarily conserved genes that function in protein trafficking to LROs. While the pathways leading to LROs are beginning to be identified, the mechanisms that control LRO morphology are still poorly understood. To identify genes that function in this process, we have been screening the Million Mutation collection for strains that have altered adult gut granule size. Here we present our studies of two newly identified genes glo-8 and glo-9. glo-8 encodes one of four members of the BEACH domain containing protein family in C. elegans. The BEACH domain is highly conserved within eukaryotes and while its function is unknown, it is present within proteins that impact organelle membrane fission/fusion. Prior studies of the C. elegans BEACH domain protein SEL-2, indicate a protein trafficking role within vulval precursors and intestinal cells (de Souza et al. 2007 Development 124:691-702). While glo-8(-) significantly decreases gut granule size and increases their numbers, mutations in the other C. elegans BEACH domain encoding genes do not cause similar effects. Rather than having a gut granule restricted function, we find that glo-8 is broadly expressed and that it impacts lysosome size in intestinal cells and coelomocytes. A detailed analysis of glo-8(-) indicates that protein trafficking to gut granules and lysosomes is not significantly disrupted. However, glo-8(-) mutants display decreased numbers of RAB-5 marked early endosomes. Studies of GLO-8 are likely to provide general insight into BEACH protein function, as defects in its homologues, LYST and NBEAL2, lead to human diseases (Chediak-Higashi and Gray Platelet Syndromes, respectively) with altered morphology, formation, and function of LROs. glo-9 encodes a regulatory protein that likely impacts Arf GTPase activity. glo-9(-) mutants display enlarged gut granule that mislocalize the gut granule proteins LMP-1 and CDF-2::GFP. These phenotype resemble rab-7(-) mutants, suggesting that GLO-9 functions on late endosomes/lysosomes to regulate the trafficking of a subset of proteins to gut granules, which possibly impacts their morphology.

Hirofumi Kunitomo et al. (2001) International C. elegans Meeting "A new method of analyzing tissue-specific transcripts in Caenorhabditis elegans"

Yuichi Iino, Hirofumi Kunitomo

[International C. elegans Meeting, 2001]

The nematode C. elegans is a model organism that is ideal for the study of development, neuronal function, and other biological phenomena. The genome of this organism has been mostly sequenced, and existence of more than 19, 000 genes has been predicted. Genome-wide gene expression analysis provides a number of valuable clues to understanding biological processes. Transcripts that are rarely expressed or expressed in limited number of cells are often difficult to detect when mRNA is prepared from whole worms. We are developing a new method to purify the transcripts from an organ of interest or from small number of cells in the nematode. The poly(A) binding protein, a protein that specifically binds poly(A) RNA, was used to co-purify mRNA from worm cells. Nematode strains that express tagged poly(A) binding protein (Tg-PABP) under the control of tissue-specific promoters were generated. The poly(A) RNA transcribed in cells that harbor the Tg-PABP were then isolated by immunoprecipitation using an antibody against the tag. So far, we have found that the combination of crosslinking mRNA to Tg-PABP, immunoprecipitation with high concentrations of NaCl, and the addition of poly(A) oligonucleotides has increased the specificity of purification. We have started a comparative gene expression analysis of motor and sensory neurons using this technique.

Folick, Andrew et al. (2013) International Worm Meeting "Lysosomal lipolysis promotes longevity through a lipid-responsive nuclear hormone receptor signaling pathway."

Yu, Yong, Wang, Meng, Doebbler, Holly, Folick, Andrew

[International Worm Meeting, 2013]

Lipids are known for roles in long-term storage of energy and cellular architecture, but they also act as signaling molecules involved in gene expression and signaling transduction. Although fat storage and metabolism has been associated with metabolic health and aging, the role that lipid signaling plays in the regulation of the aging process has not been elucidated. Previous work identified lipl-4, a homolog of human lysosomal acid lipase, as a novel regulator of longevity in C. elegans, and showed that constitutive expression of this lipase in intestinal fat storage tissue decreases fat storage and increases both mean and maximum lifespan. We hypothesized that lipolysis may generate key lipid messengers to modulate lifespan-regulating signaling processes. Through metabolomic profiling, we found lipl-4 over-expression (lipl-4 Tg) increases abundance of oleoylethanolamide, a N-acyl ethanolamide known to function as an endogenous agonist of PPARa in mammals. Both the lipl-4 Tg and feeding of oleoylethanolamide are sufficient to induce the expression of the fatty acid binding protein lbp-8. This induction is dependent on the nuclear hormone receptors NHR-49 and NHR-80 and the mediator subunit MDT-15. Interestingly, lbp-8 is not only required for the lifespan extension, but also sufficient to promote longevity. Accordingly, nhr-49, nhr-80 and mdt-15 are also required for the lifespan extension phenotype of the lipl-4 Tg. These results suggest that lipl-4-mediated lipolysis extends worms lifespan by activating an oleoylethanolamide-NHR-49/NHR-80 transcriptional signaling pathway. The lipl-4 over-expression also induces expression of the acyl-CoA synthase acs-2, and acs-2 is required for the lifespan extension phenotype suggesting that this signaling pathway may promote longevity in part by increasing mitochondria fatty-acid oxidation. In summary, we have identified a new pathway by which lipid metabolism regulates lifespan, and identified a potential longevity-promoting lipid metabolite.

Latimer, Caitlin et al. (2021) International Worm Meeting "TDP-43 promotes pathological tau phosphorylation and neurotoxicity in C. elegans"

Keene, C. Dirk, Liachko, Nicole, Kraemer, Brian, Latimer, Caitlin

[International Worm Meeting, 2021]

While the neuropathology of Alzheimer's disease (AD) is defined by the presence of amyloid beta and tau aggregates, up to 50% of patients also exhibit aggregates of the protein TDP-43. AD patients with TDP-43 pathology have worse cognitive impairment with more rapid decline and some data suggest TDP-43 may synergize with tau pathology. C. elegans has been widely used to study tau and TDP-43 proteotoxicity separately and such efforts have led to the discovery of sut-2, a gene whose loss-of-function protects against tau neurotoxicity in C. elegans. However, little is known about how tau and TDP-43 interact when co-expressed in the same organism or if there are modifiers that may disrupt that relationship, necessitating models of combined pathology. We have previously shown that co-expression of tau and TDP-43 in the same organism has synergistically deleterious effects. To further explore this relationship we developed a new low-expression C. elegans model of comorbid tau and TDP-43 and found that even at low levels TDP-43 exacerbates tau pathology, resulting in increased pathological protein accumulation as well as enhanced uncoordinated locomotion and progressive neurodegeneration. We highlight the specificity of this synergy by demonstrating that a strain expressing both wild-type human amyloid and TDP-43 is phenotypically no different from strains that express either protein alone. Finally, in order to further probe the mechanisms underlying the synergy between tau and TDP-43 we crossed tau+TDP Tg with a sut-2 loss-of-function strain, which largely rescued the enhanced phenotype. The ability of this potent modifier of tau to also have an effect on a model of tau and TDP-43 co-expression suggests that it is enhanced tau toxicity, and not TDP-43, that contributes to the synergistic tau+TDP Tg phenotypes. Characterizing the neurotoxic synergies between tau and TDP-43 in vivo will be critical for understanding and treating mixed pathology AD. Employing a novel model of tau and TDP-43 proteotoxicity, these studies provide initial insights into the mechanisms underlying tau and TDP-43 proteotoxic synergy, and establish the utility and tractability of the tau+TDP Tg model for studying comorbid pathology. Ultimately this model will be useful for identifying additional genetic modifiers of mixed tau and TDP-43 neurotoxicity and aid in a deeper dissection of the biology underlying these phenotypes.

Hashmi, Sarwar et al. (2011) International Worm Meeting "Partners in Fatty Acid Beta Oxidation: Role of KLF-3 in Fat Burning and Reproductive Behavior of C. elegans."

Hussain, Mahmood, Hashmi, Sarwar, Dhansingh, I, Chen, Xiao-Liang, Zhang, Jun, Siddiqui, Shahid

[International Worm Meeting, 2011]

Fat metabolism disorders may stem from an abnormal fatty acid oxidation, which limits energy production during periods of increased energy requirement, such as muscular exertion; gastrointestinal disease; exposure to cold and fasting. Thus, obesity leads to diabetes, cardio-vascular illness, hypertension, metabolic syndrome, polycystic ovary syndrome, Syndrome X and sleep apnea. During energy utilization, fatty acids are broken down through beta-oxidation to yield acetyl-CoA. We have discovered the exciting link of a member of Kruppel-like factors (KLF) in lipid utilization by analyzing klf-3 mutants of Caenorhabditis elegans and found that many metabolic genes are potential targets under the control of klf-3 in triglycerides (TG) derived FA beta-oxidation. Three lines of data suggest the role of KLF-3 in FA beta-oxidation (1), KLF-3 protein is primarily expressed in the intestine; (2), Large lipid droplets storing TG are seen in klf-3 mutant, 3): KLF-3 binds to the promoters of fat-7 (Stearoyl-CoA desaturases, SCD), acs-1, acs-2(Acyl CoA synthase), and F08A8.1 (Acyl CoA oxidase) a set of genes essential for FA biosynthesis and beta-oxidation in C. elegans. The klf-3 gene shows strong interaction with acs-1, acs-2, F08A8.1 and F08A8.2. Mutation in klf-3 (ok1975) causes impaired reproduction, suggesting that excessive fat deposition and reproductive defects may be intimately linked. To reconcile with these seemingly unrelated phenotypes of lipid utilization and reproductive behavior, we are examining klf-3 gene function at biochemical, and developmental level, including genetic mosaic analysis of klf-3 function in C. elegans. These results may elucidate the role of KLF transcription factors in fatty acid oxidation and reproduction.

Liachko, Nicole et al. (2015) International Worm Meeting "Using stress assays to define aberrant neuronal signaling in a C. elegans model of TDP-43 proteinopathy."

Kraemer, Brian, Liachko, Nicole, Kushleika, John, Bird, Thomas

[International Worm Meeting, 2015]

Accumulation of the protein TDP-43 in neuronal aggregates is the major pathological feature of two devastating neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD-TDP). TDP-43 is an essential protein and is involved in many aspects of RNA metabolism from transcription to translation, although its most critical contribution may be regulation of the majority of cellular mRNA splicing events. Disruption of the normal activity of TDP-43, either by mutation of TDP-43 or alterations in TDP-43 regulatory pathways, promotes neuronal dysfuction and degeneration in a variety of model systems including C. elegans, Drosophila, zebrafish, mammalian cell culture, and mice. To study the cellular, molecular, and genetic underpinnings of TDP-43 mediated neurotoxicity in a tractable model system, we have developed C. elegans models of TDP-43 proteinopathy expressing either wild type or disease-causing mutant TDP-43 pan-neuronally (TDP-43 tg). These transgenic animals display early, progressive motor dysfunction, decreased lifespan, and age-dependent degeneration of specific types of neurons, including GABA-ergic and dopaminergic neurons (1). However, not all TDP-43 expressing neurons undergo apparent neurodegeneration, indicating differences in sensitivities of specific populations of neurons to the presence of TDP-43. Surveying the range of responses to TDP-43 may provide a set of shared characteristics for populations of neurons susceptible or resistant to aberrant TDP-43. To investigate neuronal function in TDP-43 tg animals, we are utilizing a panel of behavioral, stress response, and stress survival assays. Results from these assays have identified individual sensory neurons with functional impairments in the absence of early neuronal cell body degeneration. These TDP-43 sensitive neurons will allow dissection of the effects of neurotoxic TDP-43 on neuronal function, and provide insight into the upstream processes leading to TDP-43 dependent neurodegeneration. 1. N. F. Liachko, C. R. Guthrie, B. C. Kraemer, Phosphorylation Promotes Neurotoxicity in a Caenorhabditis elegans Model of TDP-43 Proteinopathy. J Neurosci 30, 16208-16219 (2010).

Natalie F de Souza et al. (2005) International Worm Meeting "Analysis of sel-2, an enhancer of lin-12 activity in vulval precursor cells"

Hanna FARES, Natalie F de Souza, Iva Greenwald, Laura Vallier

[International Worm Meeting, 2005]

LIN-12 mediates intercellular signaling in several developmental decisions in C. elegans, including lateral specification during vulval precursor cell (VPC) patterning. Elucidating the function of sel genes (suppressor or enhancer of lin-12) has furthered our understanding of LIN-12/Notch signaling at the molecular level. Null and loss-of-function alleles of sel-2 enhance lin-12 activity in the VPCs: lin-12(n302) hermaphrodites lack an anchor cell and have no vulval induction, but sel-2(0)lin-12(n302) hermaphrodites are Multivulva (Muv), with all VPCs adopting the secondary fate (the fate associated with LIN-12 activation). sel-2 encodes a protein that contains a BEACH/WD40 domain at its carboxy terminus. Other BEACH/WD40-containing proteins have been implicated in intracellular traffic in several systems, but the cellular mechanisms remain elusive. A sel-2 promoter fusion to GFP suggests that it is expressed in the VPCs. A protein fusion of GFP to the C-terminus of SEL-2 indicates that it is cytosolic in these cells. In wild type hermaphrodites, LIN-12::GFP is visualized in the apical domain of the VPCs, but in a sel-2 mutant, LIN-12 can be visualized throughout the cells, including at the basolateral membrane. By manipulating the subcellular localization of wild-type and constitutively activated transmembrane forms of LIN-12, in sel-2(+) and sel-2(0) backgrounds, we have concluded that increased lin-12 activity in sel-2 hermaphrodites is not primarily due to basolateral mislocalization of LIN-12. Since LIN-12::GFP protein levels appear raised in the VPCs of sel-2 hermaphrodites as compared to wild type, it is possible that sel-2 acts directly as a negative regulator of LIN-12 protein levels in these cells. Experiments to determine the nature of this regulation are underway. Interestingly, sel-2(0) worms are hypersensitive to aldicarb and, less robustly, to levamisole, indicating that sel-2 also acts as a negative regulator of cholinergic neurotransmission. Since mouse knockouts of the sel-2 mammalian ortholog have severely defective neurotransmission at the neuromuscular junction, sel-2 appears to have a similar function in worms.

Currey, Heather et al. (2021) International Worm Meeting "RNAi screening of a phosphatase library identifies new modifiers of TDP-43 in a C. elegans model of ALS and FTLD-TDP"

Liachko, Nicole, Hincks, Joshua, Currey, Heather

[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.