Monica Driscoll - US grants

Dominant mutations in two C. elegans genes, mec-4 and deg-1, induce the deaths of specific groups of neurons. Dying neurons are characterized by cell swelling and morphologically resemble a commonly observed pattern of death termed necrosis. Because in C. elegans cell death mechanisms can be dissected using genetic approaches characterization of this necrotic like death should provide novel insight into this death process. Mec-4 and deg-1 are members of a gene family that appear to encode membrane proteins. These genes (called degenerin genes) mutate in the same way to induce death. mec-4 and deg-1 DNA sequences appear to cross hybridize to DNAs from a number of species, raising the possibility that members of gene degenerin families in other organisms might also be capable of mutation to forms that induce death. The term long objective of the research is to determine how mec-4 (d) induced cell death occurs at the molecular level and to investigate whether similar death mechanisms are utilized in other cell types and in other organisms. Specifically, the work outlined in this proposal will 1) accomplish a structure/function analysis to define domains and amino acids required for mec-4 function, 2) identify suppressor mutations that block mec-4(d)-induced death to identify proteins that participate in the death process, and 3) isolate degenerin family members from different species. %%% Elucidation of mechanisms of cell death are important for understanding development, homeostasis and pathology.

The elucidation of mechanisms involved in necrotic cell death is important for understanding development, homeostasis and pathology in the nervous system. In the leech, dominant mutations in two genes, mec-4 and deg-I, induce the death of specific groups of neurons. Dying neurons are characterized by morphological changes commonly observed in cells undergoing a necrotic type of cell death. Mec-4 plays a key role in the degeneration of six touch receptor neurons, by directly killing cells or by generating a signal that activates a "death program". Light and electron microscopy will be utilized to document the process of degeneration of the touch cells. Antibodies against the mec-4 protein will be used to follow the fate of this protein in normal and dying touch cells. The simplicity of the leech nervous system offers advantages for dissecting cell death mechanisms using genetic, molecular and cellular approaches. These studies promise to contribute to our understanding of how mutant proteins can induce neuronal cell death.

IBN-9511710 Driscoll, Monica A. All living organisms have the ability to sense and respond to mechanical stimuli. Mechanotransduction is critical to such diverse processes as hearing, touch and gravitaxis in plants and animals. Mechanotransduction in all species studied to date appears to be mediated by ion channels that open or close in response to mechanical stimuli. The genes encoding these channels have been identified but an examination of the subunit structures, composition and assembly has yet to be undertaken. Dr. Driscoll has been instrumental in identifying these novel channels in a simple biological model system and she proposes to continue her studies with these interesting proteins. In the proposed studies, she will determine how these channels assembled and which subunits are linked together into functional units. Additionally, since tension must be applied to the channel for it to open or close, the implication is that it is somehow anchored into the cell. Further studies will identify the normal constituents of the cellular skeleton that serve as the anchor point for these channels. Through these studies, Dr. Driscoll will gain unique insights into the biochemical nature of the mechanisms by which cellular distention is involved in changing cellular function. In addition, since mechanical forces are involved in changing cellular function in mammals as well as simple organisms, new insights may be gained about the mechanisms in which sonic or tactile information are transduced in humans thereby opening new avenues of clinical significance.

This action funds a facilitation award for an NSF Minority Postdoctoral Fellow who has a physical disability thus requiring the assistance of a technician. The research and training plan supported under the fellowship will use the nematode C. elegans to identify mutations that suppress the induction of late onset neurodegeneration by cell death. This work requires rearing the organism and manipulating it in ways that are beyond Mr. Royal's ability. The technician will perform the laboratory work for the experiments designed and interpreted by Mr. Royal, working in concert with the sponsoring scientist.

"This award is funded under the American Recovery and Reinvestment Act of 2009
(Public Law 111-5)."

Habituation is a form of non-associative learning in which repetition of a stimulus induces a progressive diminution of the behavioral response. The learning underlying habituation is a fundamental process of biological systems that is conserved from protozoans to humans. While habituation is well characterized at behavioral level, the molecular mechanisms which induce this form of learning have not been fully defined. In particular, it is not well understood how potassium channels, which are key players in controlling cellular excitability, contribute to this fundamental behavior. These studies are aimed at understanding the cellular and molecular mechanisms that induce habituation to mechanical stimulation, in the metazoan Caenorhabditis elegans. The principal investigator's laboratory discovered a novel gene, MPS-1, which is an accessory subunit of voltage-gated potassium channels and which possesses kinase activity. They have preliminary data that show that 1) MPS-1 forms a complex with voltage-gated K+ channel KHT-1 in the touch-sensing neurons of C. elegans and 2) that the kinase activity of MPS-1 specifically controls habituation in C. elegans through phosphorylation of KHT-1. In this project they propose to use a combination of genetic, electrophysiological measurements in dissected C. elegans neurons, confocal microscopy, optical measurements of calcium transients and animal model (i.e. mps-1 knock out worms, expression of MPS-1 variants in mps-1 knock outs etc.) studies to resolve the role of potassium currents in determining habituation to tap in C. elegans.

This project might significantly advance our understanding of electrical mechanisms underlying habituation behavior because: 1) a new mechanism of K+ channel regulation will be elucidated; 2) habituation is an universal behavior exhibited by virtually all biological organisms including protozoans. Most importantly, trainees in the laboratory will receive first-rate guidance in a wide range of techniques, including but not limited to electrophysiology, genetics, biochemistry, microscopy and will be consequently very successful.

In summary, the proposed research continues the commitment of the PI to provide training to all levels of students in the theory and practice of scientific research.

? DESCRIPTION (provided by applicant): Exercise is arguably the most potent approach we can take to defer physical decline associated with aging and to protect against late onset diseases such as diabetes, cancer, and Alzheimer's disease. Molecular understanding of how exercise benefits translate into healthy aging is thus of definitive medical interest. We study fundamental processes relevant to healthy aging in the 959-celled nematode C. elegans. Recently we made a fascinating discovery-C. elegans can exercise (swim) to exhibit training benefits, and appear to gain benefits by molecular pathways conserved in humans. Our initial model development opens up a new research area for understanding how tissue-specific and organism-wide health benefits are induced by exercise, and creates a novel paradigm for identifying exercise mimetic drugs that might promote healthy aging. To really harvest the potential of this model, we need to measure the strength of the tiny C. elegans. We collaborated to develop a strength test in which trained animals thread through a matrix of deformable pillars, and the extent of pillar deflection is used to calculate force. Our NemaFlex force detection device is the quantitative foundation with which we expect to break new ground in understanding exercise impact on healthy aging. Here we propose required development to enhance assay throughput and pursue applications that will not only anchor this technology as an essential component of C. elegans exercise evaluation but also accelerate studies on exercise biology and healthy aging in this powerful model. Aim 1 is to develop a novel high throughput tool for direct strength evaluation in C. elegans. This aim will generate an essential tool for analysis of C. elegans strength at multiple life stages, define the exercise regimen that will become the anchor protocol in the field, and reveal features of training in this model. Aim 2 is to use NemaFlex to evaluate exercise mimetic drugs & to facilitate focused pilot genetic screens. This aim will establish critical proof-of-principle for genetic and drug discovery using the NemaFlex. Aim 3 is to initiate dissection of the functional and molecular relationship between exercise and healthy aging, grounded in NemaFlex force measures of training benefits. To begin, we will test how optimized strength training tracks with a broad spectrum of healthspan indicators that decline with age, we will investigate impact of cessation of training on aging quality, and we will ask if exercise mimetic drugs extend healthspan in the absence of training. Our goals will create novel technology that for the first time permits facile quantitativ analysis of exercise adaptations in the powerful C. elegans genetic model. Accomplishment of our tractable aims will anchor a new subfield of genetic investigation of exercise and healthy aging that may influence design of interventions that broadly promote health and defer aging.