Source: LOUISIANA STATE UNIVERSITY submitted to
DEVELOPMENT OF A CAENORHABDITIS ELEGANS (C. ELEGANS) MODEL - AN INTERMEDIATE STEP BETWEEN CELL CULTURE STUDIES AND LABORATORY RODENT STUDIES
Sponsoring Institution
State Agricultural Experiment Station
Project Status
TERMINATED
Funding Source
STATE
Reporting Frequency
Annual
Accession No.
0222387
Grant No.
(N/A)
Project No.
LAB04048
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
May 1, 2010
Project End Date
Sep 30, 2012
Grant Year
(N/A)
Project Director
Zheng, J. Z.
Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100
Performing Department
Food Science
Non Technical Summary
JUSTIFICATION: These novel studies using the C. elegans model address areas of important medical needs by contributing to the development of treatments and preventive strategies for obesity, obesity associated diseases, aging, and neuronal degenerative disease(s). Despite extensive research efforts, the cause(s) of these diseases are still illusive and there is no cure to date(1). These projects which screen bioactives for anti-obesity, anti-aging, and PD therapeutic potential have the potential to improve public health and bring increased economic benefits to the State of Louisiana and its citizens. C. elegans is an animal model that has been used for research into human diseases including aging(2-4), diabetes(5), Azheimers Disease(6); satiety(7), and obesity(8-10). C. elegans was the first multi-cellular organism with a completely sequenced genome. Its biosynthetic and metabolic pathways are highly preserved with mammalian systems, with more than 65% of the genes relating to human diseases being conserved in C. elegans(11-13). Its short lifespan, small size, large brood sizes, genetic amenity, easy maintenance, and cost efficiency in the laboratory significantly reduce the cost of research. The quick turnover of tested compounds is extremely suitable for large-scale and high through-put screening programs of bioactive compounds like our major ongoing projects(14, 15). Using the C. elegans model is an intermediate stage prior to confirmation in a rodent model and human clinical trials. A thorough CRIS search has been conducted. Although a total of 27 to 66 projects are related to nematodes in that database, no duplicated or proposals similar to ours were found. All of these C. elegans research projects have the expectation of being supported by external funding from the National Institute of Health, National Science Foundation, Department of Defense, industrial funding, or other sources. As a new faculty member of the AgCenter, one of my strategies to attract larger external grants is to obtain small grant funding that will support pilot studies as a start, since pilot data is needed to obtain the larger grants. With the support of these small grants, I was able to develop sufficient data to attract an intermediate size grant from a private donor interested in supporting Parkinson's disease research and from the Board of Regents to support the bioactive screening in food and food by-products. Generation of more preliminary data will allow funding of larger competitive grant applications.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7027010104050%
7231499103040%
7241599102010%
Knowledge Area
724 - Healthy Lifestyle; 702 - Requirements and Function of Nutrients and Other Food Components; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
1599 - Grain crops, general/other; 1499 - Vegetables, general/other; 7010 - Biological Cell Systems;

Field Of Science
1020 - Physiology; 1030 - Cellular biology; 1040 - Molecular biology;
Goals / Objectives
Objective 1. To use the Caenorhabditis elegans (C. elegans) model for screening to find the most effective and safest bioactives in foods or food by-products for the treatment of obesity. This objective is approached on three levels. 1) By demonstrating that resistant starch (RS) is a safe food component. 2) By using C. elegans to analyze the gut microbiota profile to determine the populations of gastrointestinal flora that are involved in the fermentation of RS to short chain fatty acids (SCFA) such as acetate, lactate and butyrate. 3) By determining the mechanisms and pathways by which SCFA, the gut fermentation products of RS, reduce body fat, and prevent or treat diet-induced insulin-resistance. Objective 2. To use the C. elegans model and its many genetically manipulated strains to screen the effect of anti-aging bioactives on different gene pathways such as the silent information regulator 2 (Sir2) proteins or sirtuin proteins and the DAF-2 gene, the only gene encoding the insulin/IGF-1 like receptor in C. elegans. Objective 3. To use the C. elegans model to test the novel hypothesis that dietary phytochemical lectins may enter central nervous system (CNS) from the gastrointestinal tract in an ascending manner and be or chaperone the "unknown pathogen" in Parkinson's disease. This proposal will screen a library of pathogenic and non-pathogenic dietary phytochemical lectins in the C. elegans model in order to: 1) Determine if dietary phytochemical lectins are the unknown pathogen responsible for Parkinson's Disease or act as a chaperon to viruses or toxin(s), including those that may be responsible for -SYN and LB inclusions in PD, and play a role in the etiology of the disease. 2) Investigate the potential route through which non-pathogenic lectins conjugated with nanoparticles (NPs-lectins) access the CNS for targeted and time-controlled drug release.
Project Methods
PROCEDURE: 1. Using RS to reduce body fat in rodents. Three hundred and five genes in C. elegans have been shown to be involved in reducing body fat, and 112 genes are involved in increasing fat storage as demonstrated by RNAi and Nile Red staining. C. elegans is well-suited to obesity studies, because deposits of fat for energy storage can be found along its intestinal tract and the bodies of C. elegans are transparent. Thus, lipid-staining dyes such as Nile Red or BODIPY can be visualized directly and quantitated photometrically in the intact animals. 2. Two genes have been extensively studied and recognized as different pathways for extending lifespan. One is the silent information regulator 2 (Sir2) proteins or sirtuin proteins which extends lifespan in drosophila. Doubling the copy number of Sir2 gene, sir-2.1(e1370), will induce a 50% increase of the lifespan in C. elegans, and the null mutant sir-2.1(ok434)IV has a shorter lifespan than wild type N2. The other pathway for extending lifespan in C. elegans is through the DAF-2 gene which is a homolog of the insulin and insulin-like growth factor (IGF) receptors. The insulin/IGF-1-like signaling is conserved from worms to humans, DAF-2 is the only gene that encodes the insulin/IGF-1 like receptor in the worm, and insulin-resistance is indirectly proportional to lifespan. Mutations that reduce activity of daf-2 can double or triple the lifespan by allowing inappropriate activation of daf-16 function. Nevertheless, a 2% glucose treatment eliminates these effects of daf-2(e1370)III. The increase in longevity associated with decreased DAF-2 signaling is analogous to mammalian longevity that is associated with caloric restriction. 3. C. elegans is the preferred model for dietary phytochemical lectin-transport and release studies due to its low cost and incredibly lower manpower needs. This project is designed to screen hundreds of lectins which may be transported trans-synoptically along the nervous system. Generally, the size of neurons are between 20micon - 40micron. A small mouse has a distance of 1 - 2 inches from its stomach to its brain. To be able to view the fluorescence of the tested lectins, thin sections of nervous tissues are required which necessitates tremendous hours of labor work. Since the rodent lives ~2yr, the process of transporting, seeing the transported lectins, and their turnover time is much longer. In comparison, the whole C. elegans can be viewed under microscope and no tissue sectioning is needed. The short turn over time is another plus to speed up the studies for multiple and/or repeated screening procedures. C. elegans has 8 DAergic neurons out of a total of 302 neurons. Nevertheless, their complex neuronally controlled behaviors and dysfunctions can be defined at the cellular levels(44-48). These characteristics offer specific advantages of C. elegans for PD study. Genetically manipulated C. elegans (BZ555, egIs1) express green fluorescence protein (GFP) in the eight DAergic neurons. If the TRITC-lectins (red) were transported to the DAergic neurons (green), a co-localization will be detected (orange) using fluorescent microscopy.

Progress 05/01/10 to 09/30/12

Outputs
OUTPUTS: During this period one paper was published and four abstracts were presented. PARTICIPANTS: Jolene Zheng (PI), Roy Martin, Frederick Enright, Michael Keenan, John Finley, Wenqian Wei, Chenfei Gao, LSU AgCenter. TARGET AUDIENCES: Food and bio-medical scientists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Using Caenorhabidits elegans (C. elegans) model organism: Change in knowledge occurred 1. Subgroup lectins was transported to the CNS by gastrointestinal absorption and impacted dopaminergic (DAergic) neurons that are damaged in Parkinson's disease (PD). Subgroup lectins reduced the number of GFP-DAergic neurons, the endogenous GFP-dopamine transporter fluorescent, or the size of GFP-DAergic neurons, suggesting they were toxic. These changes may be dose-dependently inhibited by specific haptenic sugars. 2. Studies of dietary fiber sources like Prowashonupana barley (Sustagrain), plant extracts, or agricultural by-products are Bioactive compounds that may benefit hyperglycemia-impaired lipid metabolism, reduce body fat, and improve healthy aging. Incorporating optimal functional food components into the daily diet and developing these drugs will result in future prevention of obesity and improvements in public health.

Publications

  • C Gao, M King, Z Fitzpatrick, W Wei, J King, F Greenway, W Johnson, J Finley, M Keenan, F Enright, R Martin, and J Zheng. Prowashonupana barley as dietary fiber sources reduced body fat in Canorhabditis elegans model. Institution of Food Technologists, Louisiana Gulf Coast Section, March 2012.
  • Zheng J, Sonnier T, Vase A, Korivi N, Ajmera P, Morrison SF, DiLorenzo DJ, Greenway FL. A less invasive surgical approach for splanchnic nerve stimulation to treat obesity. Obes Surg. 2012 Nov;22(11):1783-4.
  • J Zheng, J King, M King, S Heymsfield, J Finley, and F Greenway. Effect of sugar, aspartame, or acesulfame potassium alone or in beverages on C. elegans model organism. Experimental Biology Annual Meeting, April 2012.
  • J King, M King, M Keenan, WQ We, Z Fitzpatrick, F Greenway, J Finley, F Enright, R Martin, and J Zheng. Compound 206 reduced body fat: Identification of the mechanism of action in C. elegans model. Institution of Food Technologists, Louisiana Gulf Coast Section, March 2012.
  • Zachary L. Fitzpatrick and Jolene Zheng. Green fluorescent membrane-labeling technology, pkh67: a proxy for studying adipose regulation in Caenorhabditis elegans. Institution of Food Technologists, Louisiana Gulf Coast Section, March 2012.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Participation in three international scientific conferences disseminated the research results of resistant starch and other functional food or medications. A database of plant glycoproteins with a potential relationship to Parkinson's Disease was begun, and improvements to a specific data mining skill set for C. elegans studies were made. Patients of at a local children's hospital received education on nutrition and function foods from students involved in the project. PARTICIPANTS: J. Zheng (PI), W. Wei, C. Gao, R. Martin, F. Enright, M. Keenan, J. Finley, J. King, M. King, Z. Fitzpatrick, and C. Sabliov, LSU AgCenter; J. Ye, F. Greenway, and J. Keller, Pennington Biomedical Research Center-LSU; R. Laine, LSU. TARGET AUDIENCES: Presentations were received by fellow scientific professionals and the public audience (patients with diabetes, obese, coronary cardiovascular diseases, hypertension).   PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Using Caenorhabidits elegans (C. elegans) as a model organism, the unique properties of 16 dietary plant lectins have been tested. The lectins may be transported to the central nervous system by gastrointestinal absorption. They impacted dopaminergic (DAergic) neurons that are damaged in Parkinson's disease (PD) and altered mobility. Dolichos biflorus agglutinin, Glycine max lectin, and Phaseolus vulgaris erythroagglutinin appear to be transported trans-synaptically to the nervous system and, at variable times post- feeding, co-localized in GFP-DAergic neurons. Some lectins that were not detected to co-localize, in fact, reduced the number of GFP-DAergic neurons, suggesting they were toxic to these neurons; reduced the endogenous GFP-dopamine transporter fluorescent intensity indicating a dopamine transporter protein diminunition; or decreased the size of GFP-DAergic neurons. Others increased neuron size. These changes may be dose-dependently inhibited by specific haptenic sugars. This study may provide profound insight into neuronal degenerative diseases and result in clinical applications from an ongoing proprietary drug development program. The mechanism(s) of dietary fiber sources like Prowashonupana barley (Sustagrain), compound ON-206 and Histalea, and plant extracts were examined. These treatments reduced body fat and improved healthy aging by different mechanisms. The effect of PW barley appeared mainly due to β-glucans, mediated via daf-2 or daf-2/daf-16, and may benefit hyperglycemia-impaired lipid metabolism. The ON-206 action is through activation of CPT-1. Histalea reduced intestinal fat deposition in second generation antipsychotics (SGA)-induced obesity via a combination of an H1R agonist and an H3R antagonis. This drug development may be used to prevent SGA-induced adverse effect. Incorporating optimal functional food components into the daily diet and developing these drugs will result in future prevention of obesity and improvements in public health. With the assistance of information technology, three methods of specific data mining were created for this research, speeding up data processing which significantly reduced the labor requirement for analyzing C. elegans data. More methods of automatic data acquisition and analyses, for example, digital counting pumping rate, are being developed.

Publications

  • Zheng, J. (2011) Resistant starch is a functional food that reduces intestinal fat deposition and promotes health in Caenorhabditis elegans model. BITs 1st Annual World Congress of Endobolism. Vol.1, Page 24.


Progress 05/01/10 to 12/31/10

Outputs
OUTPUTS: We have successfully developed a platform using the Caenorhabditis elegans (C. elegans) model for screening bioactives that may benefit health in humans. Study progress of resistant starch (RS) and aging have been published in a peer reviewed journal, submitted to the LSU AgCenter magazine, and presented at the Obesity Society annual meeting. The study of dietary phytochemical lectins in Parkinson's disease has been presented at the American Gastrointerology Society annual meeting and awarded a competitive internal grant and received a private donation. PARTICIPANTS: Principal investigator: Jolene Zheng, M.D. Ph.D., Fred Enright, Ph.D., Wenqian Wei, research associate. Organization: Department of Veterinary Science. Roy Martin, Ph.D. and Michael Keenan, Ph.D., Organization: School of Human Ecology. John Finley, Ph.D., Organization: Department of Food Science. Cristina Sabliov, Ph.D., Organization: Biological Agricultural Engineer Department. Partner Organizations: Pennington Biomedical Research Center-LSU system and USDA-ARS-SRRC (New Orleans). TARGET AUDIENCES: Targeted audiences for this project include nutrition and food professionals, crop producers, pharmaceuticals, neuroscientists, and medical professionals. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Using mutant strains of sir-2.1(ok434)IV or daf-16(mgDf50)I, we have identified two genetic pathways, sir-2 or daf-16, that were involved in the RS-induced fat reduction which may promote anti-obesity. We have observed that the apparent anti-aging effect induced with the tested RS was mediated through the daf-16 pathway, which was eliminated in the mutant strain. These results indicated that RS may support anti-aging. Among nine dietary phytochemical lectins that were screened, we have found that three of them appeared in Dopaminergic (DAergic) neurons, three of them reduced the size the DAergic neurons, and five of the lectins reduced locomotion. These characteristics may imply an unfavorable effect on human health and may be involved in neurodegenerative disease such as Parkinson's disease. We also found Glycine max may be utilized as a vehicle for targeted drug delivery. These studies have been supported through five extramural grants from a granting agency, a public company, and private industries.

Publications

  • Jolene Zheng, Fred Enright, Michael Keenan, John Finley, Jun Zhou, Jianping Ye, Frank Greenway, Reshani Senevirathne, Chris Gissendanner, Rosaly Manaois, Alfredo Prudente, Joan King, and Roy Martin. 2010. Resistant Starch, Fermented Resistant Starch, and Short-Chain Fatty Acids Reduce Intestinal Fat Deposition in Caenorhabditis elegans. J Agric Food Chem. 2010 Apr 28;58(8):4744-8.
  • Jolene Zheng, Fred Enright, Frank Greenway, John Finley, Michael Keenan, Jianping Ye, and Roy Martin. 2010. Barley flour fermentable fiber (FF) reduces intestinal fat deposition and increases the pharyngeal pumping rate in C. elegans. Obesity (2):S143.
  • Jolene Zheng. 2010. Dietary lectins altered C. elegans' behavior in the environmental Unknown Pathogen in Parkinson's disease. Gastroenterology. 138 (5) Supplement 1, Page S-762.