Antony Stretton - US grants

The aim of this project is to devise immunohistochemical labels for neurons and synapses in the motornervous system of the large parasitic nematode Ascaris. We will generate monoclonal antibodies against subsets of neurons, against synapses and against enzymes known to be important in neuronal function, e.g. choline acetyltransferase, the biosynthetic enzyme for acetylcholine. Since we have electrophysiological and anatomical information about many of the neurons in the motor nervous system, we can correlate the presence of specific antigens with known physiological activities. Our long-term objective is to generate a series of labels so that the function of a neuron can be determined using histochemical techniques that label the molecules that mediate function. This will enable us to determine the functional anatomy of the entire nematode nervous system in the detail that will be required before we can test our understanding of the cellular and molecular mechanisms that the nervous system uses to process information. Gaining a basic understanding of the Ascaris nervous system is important since Ascaris is a model nematode in which to investigate the control of locomotion in nematodes. The Ascaris motor nervous system is anatomically similar to that of other nematodes, so it is reasonable to assume that this similarity will also apply at the functional level. By studying the mechanisms underlying the activity of the neuromuscular system of Ascaris, general principles applying to other nematodes will be found. This basic understanding of the nematode nervous system will make a contribution to parasite biology, and allow differences in the biology between host and parasite to be identified as targets for anthelmintic drugs. In view of the agricultural impact of nematodes on food supplies, and the problems they cause as animal and human parasites, especially in tropical and economically less-developed countries, this is an important health-related goal.

Our aim is to devise immunohistochemical labels for neurons and synapses in the motornervous system of the large parasitic nematode Ascaris. We will generate and use antibodies against synapses and against neurotransmitters and the enzymes responsible for their synthesis. Since we have electrophysiological and anatomical information about many of the neurons in the motor nervous system, we can correlate the presence of specific antigens with known physiological activities. Our objective is to generate a series of labels so that the function of a neuron can be determined using histochemical techniques that label the molecules that mediate function. This will enable us to determine the functional anatomy of the entire nematode nervous system in the detail that will be required before we can test our understanding of the cellular and molecular mechanisms that the nervous system uses to process information. Gaining a basic understanding of the Ascaris nervous system is important since Ascaris is a model nematode in which to investigate the control of locomotion in nematodes. The Ascaris motor nervous system is anatomically similar to that of other nematodes, so it is reasonable to assume that this similarity will also apply at the functional level. By studying the mechanisms underlying the activity of the neuromuscular system of Ascaris, general principles applying to other nematodes will be found. This basic understanding of the nematode nervous system will make a contribution to parasite biology, and allow differences in the biology between host and parasite to be identified as targets for anthelmintic drugs. In view of the agricultural impact of nematodes on food supplies, and the problems they cause as animal and human parasites, especially in tropical and economically less-developed countries, this is an important health-related goal.

Invertebrate nervous systems are structurally less elaborate than those of vertebrates. This simplicity along with the accessibility of their nervous system have permitted detailed electrophysiological and morphological studies resulting in the construction of functional circuit diagrams. Dr. Stretton has used Ascaris summ, a large nematode parasite of pigs, as a model system to understand the neural circuits controlling locomotion. Their relatively simple movements are coordinated by a motor nervous system that contains approximately 77 neurons distributed within the ventral nerve cord along the length of the worm. He has found that along with the classic neurotransmitters that operate within the nematode nervous system are a large number of immunoreactive neuropeptide-like substances. These data indicate that previously unknown elaborate communication system exists in these simple organisms similar to that reported in more complex animals. Dr. Stretton along with Dr. Wright, his postdoctoral fellow, will isolate, clone, and sequence the gene that encodes two bioactive neuropeptides. Since each neuropeptide is a potentially potent chemical messenger with unique target- responses, they will characterize which motor nervous system neurons are likely to express individual peptides. The results obtained using this simple system will enhance our understanding of the neurochemical basis of locomotion and its coordination. Moreover, studies on the basic neurobiology of nematodes may provide a rational basis for the design of controls on these parasites found in man, domesticated animals and cultivated plants.

We have isolated and sequenced a large family of 20 novel endogenous neuropeptides in the nematode, Ascaris suum. Preliminary experiments have shown that each of the peptides tested so far has potent activity on the neuromuscular system of Ascaris. Some causes paralysis, others exaggerate locomotory movements. Different peptides affect the physiological properties of different neurons in the motornervous system in a variety of ways. The peptides fall into at least 4 subfamilies based on their amino acid sequences. We have shown that one of these subfamilies, comprising 6 peptides, is derived from a precursor protein encoded by a single mRNA coding sequence, strongly reinforcing the concept of subfamilies. Our long-term aim is to understand the role of these peptides in the control of locomotion in Ascaris. In this proposal we plan to use electrophysiological techniques to characterize the signalling pathways mediated by each peptide, and to use molecular biological techniques to determine the genetic organization of this family of peptides. Our hypothesis is that there is a direct correspondence between the subfamilies of peptides defined three ways: by amino acid sequence, by physiological action, and by co-expression on a transcript. We predict that subfamilies exist because each subfamily is the product of a distinct transcript that organizes the co-expression of peptides having similar biological activity.

The locomotory system of nematodes is the target of many currently used anthelminthics, since disruption of locomotory behavior interferes with the normal processes by which many nematodes maintain their position within the host. The problem of genetic drug resistance is making many of the available drugs less effective, so it is important to develop new generations of anti-nematdde drugs. The proposed research aims to describe new intercellular signaling systems that are important in the motornervous system that controls locomotion. Our strategy is based on our previous work in which we isolated and sequenced a large family of 29 novel endogenous neuropeptides in the parasitic nematode, Ascaris suum. Preliminary experiments have shown that almost all of the peptides tested have potent activity .on the neuromuscular system of Ascaris. Some cause paralysis, others exaggerate locomotory movements. Neuropeptides, therefore, play an important role in the overall activity of the motornervous system. There is every indication that there are many more peptides than the ones we have isolated so far. There is no indication that the currently available peptides are the most potent, so we propose to isolate new peptides, concentrating on those that are present in the motornervous system, in order to detect new peptides that have potent physiological activity. This is basic research upon which new generations of anthelminthics can ultimately be developed.

At the University of Wisconsin-Madison and in other universities throughout the state, there are over a thousand faculty performing research within the life sciences. Fundamental to many of these studies is a mass spectrometric-assisted proteomic analysis of the cells, tissues and organs that make up the organisms. The UW -Madison Biotechnology Center Mass Spectrometry/Proteomic Facility was established in 1998 as a centralized facility for the purpose of acquiring mass spectrometers and making them available to the research community on a fee-for-service basis. During the six years since inception, it has proven successful in meeting the proteomic needs of nearly one hundred different academic laboratories in a cost effective, top quality manner. However, a major deficiency for investigators using this facility has been the inability to obtain protein sequence from very small samples of tissue, a common handicap of biological research. A second limitation is the ability to routinely perform quantitative differential proteomic experiments using isotope-assisted tandem mass spectrometry. The MALDI-based tandem mass spectrometer known commonly as a MALDI-TOFTOF is an instrument that is uniquely capable to address both of these problems. Surprisingly, there is not a single MALDI-TOFTOF available in the state. Wisconsin researchers currently must drive/fly to Chicago or Boston to obtain precious instrument time. Lack of ready access to this instrument locally has hindered our researchers in obtaining the critical information needed for converting genomic sequence 'data' into a real understanding of life processes. The purpose of this award is to acquire this instrument and make it available to academic researchers statewide. The instrument will be placed within the existing UWBC Core Mass Spectrometry/Proteomics Facility, ensuring that (i) it will be accessible to a large diverse group of researchers, (ii) that it will heavily used and (iii) that it will be well maintained for optimal sensitivity and overall reliability.

The broader impacts of this project are several-fold. First, the research users represent a large number of different academic disciplines, from Animal/Food Science to Engineering to Prebiotic Chemistry to the 'traditional' Molecular, Cellular and Organismal Biology areas. Six years of prior experience in operating expensive and sophisticated mass spectrometers ensures that the operation of this MALDI-TOFTOF will be well organized, accessible and affordable for the entire community. In addition, the university has strived to ensure that the UWBC core facilities in general, and the Mass Spectrometry/Proteomics in particular, are utilized for education at all levels, including undergraduates and high schools, both locally and in Wisconsin communities with underrepresented groups, such as economically impoverished schools in Milwaukee and Native American Indians in small communities of northern Wisconsin. This instrument will advance discovery and promote teaching and generally enhance the academic infrastructure, not only at UW-Madison, but also, all over the state.

Neuropeptides are important modulators of neuronal activity. They are synthesized by and secreted from neurons to change the electrical properties of their target cells (other neurons or muscle cells). In order to understand the way the nervous system controls behavior, a complete description of the neuropeptides and the precise way they affect their targets is needed. In vertebrate nervous systems (including the human brain) this information is woefully incomplete because the number of neuropeptides is so large, and because the circuits they control are so large and complex. This project will exploit the unique properties of the nematode nervous system; it is extremely simple, containing a total of only 298 neurons. This enormous reduction in complexity should enable us to understand the role of neuropeptides in controlling the activity of the individual neurons within a circuit as they in turn control behavior. Achieving this understanding is our ultimate goal. The proposed research will involve a newly developed method for picking out identified neurons one at a time, and finding their peptide content by mass spectrometry. The amino acid sequence of each peptide can be determined at the same time. This is a powerful method for finding which peptides are found in each individual neuron. After the peptides have been identified and characterized, their biological activity will be determined.

Because of the striking simplicity of the nematode nervous system and its experimental accessibility, this project will allow us to continue to provide the valuable training opportunities that have already been made available to students, both pre-college students and undergraduates, who are entering research for the first time. Previous efforts have already specifically involved the recruitment of women and underrepresented minorities into research, and this project will enable us to extend this important opportunity.

DESCRIPTION (provided by applicant): The research described in this proposal aims at determining the biological role of a family of 8 related endogenous neuropeptides, encoded by 4 different genes in A. suum. The peptide encoded by one gene has been shown to have very potent inhibitory activity on Ascaris suum muscle. We propose to characterize the biological activity of the remaining 7 peptides. We will also identify the cells (identified neurons, or non-neural cells) which make each peptide, and the site of the receptors for each peptide. We will also study the mechanism of action of each peptide by electrophysiological methods. There are preliminary indications that at least one of these peptides may be expressed in the intestine. We will thoroughly explore this hypothesis, since it suggests a potential new target for anti-nematode drugs, and, even more interesting, a different route for administration of drugs that would not necessitate penetration into the interior tissue space of the nematode.