1989 — 1993 |
Aronin, Neil Difiglia, Marian |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Signal Transducing Proteins in the Basal Ganglia: the Subcellular Localization, Regulation and Action of G-Proteins in Neostriatal Neurons @ University of Massachusetts Medical School
The neostriatum is a brain region integrally involved in body movement. Advances in neostriatal research have revealed much about its complex neural inputs and intrinsic organization, including the identification and localization of chemical messengers that activate or inhibit other neurons. The molecular events in neostriatal cells which process the effects of the chemical messengers are poorly understood, but are important to understand how these cells convey information. One of these fundamental cellular processes--called signal transduction of chemical messengers--uses a class of proteins, GTP-binding proteins, to mediate and direct the flow of information by chemical messengers. The overall objective of this study is to examine the cellular localization, regulation and action of specific GTP-binding, transducing proteins in the rat neostriatum. Investigating where signal transducing proteins are localized subcellulary in neostriatal neurons, how these proteins are activated, and what intraneuronal processes they affect should advance the knowledge of cellular mechanisms involved in neuronal targeting of information in the neostriatum.
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0.904 |
1996 — 1998 |
Difiglia, Marian |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Receptor Antibodies and Oligonucleotide Probes Preparation @ Massachusetts General Hospital
The recent cloning of multiple receptor subtypes for neurotransmitters, such as glutamate, GABA, dopamine, and acetylcholine has made possible the opportunity to develop probes to examine the neuronal localization and distribution of receptor mRNA and protein for these receptors in the basal ganglia. The overall goal of this core will be to develop well- characterized antibodies and oligonucleotide probes for use by the investigators in this program project. This core will serve the needs of investigators in projects 1, 2, 3 and 4. The development of these probes, together with the ones already in hand, is essential to achieving the goals of individual projects and in providing uniformity across projects so that more meaningful comparisons between studies can be made.
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0.996 |
1996 — 2002 |
Difiglia, Marian |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Localization and Functional Properties of Huntingtin in Striatal Cells @ Massachusetts General Hospital
Huntington's disease; neural degeneration; nerve /myelin protein; cellular pathology; gene expression; gene mutation; corpus striatum; neocortex; phenotype; excitatory aminoacid; protein structure function; neurogenesis; neurons; brain mapping; cell type; laminin; tissue /cell culture; electron microscopy; immunofluorescence technique; epitope mapping; immunocytochemistry; human tissue; histopathology; laboratory rat; transfection;
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0.996 |
1998 — 2001 |
Difiglia, Marian |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Huntington and Vesicle Transport @ Massachusetts General Hospital
Huntington's disease causes motor and cognitive dysfunctions, the degeneration of striatal and cortical neurons in the brain, and death of its victim within 15-20 years. The genetic mutation is an expanded region of polyglutamines at the N-terminus of huntingtin. The function of wild-type huntingtin and the mechanism of HD pathogenesis caused by mutant huntingtin are unknown. We have observed an abnormal accumulation and transport of huntintin in affected neurons of the HD brain. Similar patterns of mutant huntintin accumulation appear in clonal striatal cells transfected with cDNAs encoding huntingtin with an expanded polyglutamine region. Published studies and our preliminary observations suggest that wild-type huntingtin may function in receptor- mediated endocytosis. Mutant huntintin, like wild-type huntingtin, associates with clathrin-enriched membranes. Our overall hypothesis is that mutant huntintin causes neuronal dysfunction through its direct effects on receptor-mediated endocytosis and by its abnormal accumulation and transport. We propose a series of studies in clonal striatal cells to explore wild-type huntingtin's association with endosomes (Aimsl), to analyze the consequences of polyglutamine expansion in huntintin on endocytic function (Aim 2), and to evaluate the subcellular compartments that accumulate mutant huntingtin and contribute to cell death (Aim 3). Our studies will include techniques in confocal immunofluorescence microscopy, immunogold/electron microscopy, subcellular membrane fractionation, immunoisolation and Western blot. The results will identify the subcellular processes involved in HD pathogenesis and will lead to a rational strategy for treatment of this devastating disorder.
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0.996 |
2000 |
Difiglia, Marian |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Core--Imaging @ Massachusetts General Hospital
Huntington's disease (HD) is caused by a elongation of a glutamine stretch in the novel huntingtin protein. It is not clear how this genetic defect leads to the selective pathology of HD. Laser confocal immunofluorescence light microscopy has become an essential tool for evaluating the subcellular localization of single or multiple antigens by immunofluorescence. This approach is essential to understanding huntingtin and its mutant counterpart in HD cells and tissues, and in experimental models . However, HD Center investigators have limited access to existing imaging facilities, creating a bottle neck that is slowing progress on these critical experiments. We propose to establish a laser confocal imaging core facility dedicated to supporting the needs of HD Center investigators. We propose to establish a laser confocal imaging core facility dedicated to supporting the needs of HD Center investigators. The advances in technology of the new generation of confocal microscopes provide for simultaneous acquisition of multi-color images free from bleed through and with reduced fading. This Imaging Core facility will greatly enhance the efficiency and productivity with which HD Center investigators can obtain high quality cell biological data. This knowledge is critical to achieving the long-term goal of the HD Center, understanding mutant huntingtin, its partners and normal and abnormal cellular functions that ultimately cause HD pathology.
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0.996 |
2000 |
Difiglia, Marian |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Early Subcellular and Molecular Pathology @ Massachusetts General Hospital
The identification of early changes in the HD brain at the cellular and molecular level is critical for understanding pathogenesis and developing treatments for the disease. Studies by us and others in HD brain, HD mice, and with in vitro cellular models of HD show that N-terminal fragments of mutant huntingtin containing the expanded polyglutamine tract can accumulate in the nucleus or cytoplasm of neurons and cause dysfunction and cell death. Affected neurons have nuclear and cytoplasmic inclusions, cytoplasmic vacuoles, and dystrophic neurites, and may be associated with activated microglia. N-terminal huntingtin interacts with proteins involved in diverse functions. Our overall hypothesis is that mutant huntingtin impairs function at multiple sites in neurons. The aims are: 1) To examine the regulation of huntingtin proteolysis and the accumulation of N-terminal mutant huntingtin products in neurons, 2) To determine the distribution of huntingtin and huntingtin fragments in the nucleus and a possible huntingtin interaction with a transcription repressor, 3) To determine huntingtin's association with the actin cytoskeleton and the effects of mutant huntingtin on actin- dependent cellular adhesion, 4) To examine the role of the pro-apoptotic proteins caspase 8 and BAX in mutant huntingtin induced cell death and 5) To explore the basis for microglia activation in HD. The results should help to elucidate the mechanisms underlying neuronal dysfunction and cell death in HD.
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0.996 |
2002 — 2005 |
Difiglia, Marian |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Role of Huntingtin in Vesicle Transport @ Massachusetts General Hospital
DESCRIPTION (provided by applicant): A polygutamine expansion in the N-terminus of huntingtin (N-hn) causes Huntington's disease (HD). There is no effective treatment for HD. Although mutant N-Fin fragments are known to accumulate in HD neurons and cause cell dysfunction in vitro, a mechanism (or mechanisms) that explains the selective loss of striatal and cortical projection neurons remains elusive. Wild type and mutant htt associate with membranes in the endocytic and secretory pathway. Our overall hypothesis is that mutant N-htt 's association with neuronal membranes contributes to early cellular dysfunction in the cytoplasm in HD. One of these membrane compartments includes the autophagosome/lysosomal system, which accumulates full-length or large N-htt fragments of mutant htt. Although htt ostensibly lacks transmembrane domains, it associates tightly with membranes. We speculate that candidate domains in the N-terminus involved in protein-protein interactions promote membrane binding. Little is known about the degradative pathways that form N-htt fragments in vivo. Identifying the sites of protease cleavage in the N-terminus of htt is important for understanding how the protein is regulated. Calpain, a calcium dependent protease, which regulates the function of many proteins involved in membrane/cytoskeleton organization, cleaves htt near its N-terminus and produces long-lived N-htt fragments that are enriched in membrane fractions in brain. We speculate that mutant N-htt products of calpain cleavage undergo a different processing from the wt fragments in neurons that leads to cellular dysfunction in HD. HD mice also show abnormal function of striatal NMDA receptors. One way that mutant Fin might cause the dysfunction of NMDA receptors is by disrupting the assembly of NMDA receptor subtypes at the cell surface. The specific aims are: 1: To understand the role of mutant htt induced autophagy in cell dysfunction, 2: To determine whether htt proteolysis by calpain contributes to HD pathogenesis and 3: To determine the effects of mutant htt on protein transport within the secretory pathway. The results of these studies will provide new insights about the mechanisms of cellular dysfunction in HD and suggest novel therapeutic targets that can reduce the potentially harmful effects of mutant N-htt fragments.
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0.996 |
2010 |
Difiglia, Marian |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Request For Electron Microscope @ Massachusetts General Hospital
DESCRIPTION (provided by applicant): This is a re-submission application for a request for a new transmission electron microscope (TEM) to replace an existing one that was purchased in 1979. Our current TEM has been operating for 28 years. It is failing and is wholly inadequate to serve the research needs of our large active and expanding research community in the Department of Neurology at Massachusetts General Hospital. Many of our researchers work in neurodegenerative diseases including Alzheimer disease, Huntington's disease, and Parkinson's disease. Some of the current exciting topics we work on include the role of autophagy in clearance of disease proteins from neurons, the effects of disease proteins on organization and recycling of neurotransmitter receptors in animal models of disease, membrane permeability changes in traumatic brain injury, and the mechanisms underlying axonal demyelination. These and other research areas will be greatly enhanced by having a new TEM in our department. Some of us have long track records in using electron microscopy to study the nervous system. The lack of a reliably functioning TEM hinders our ability to plan new directions involving EM analysis and in training our young investigators in EM applications. Advances in TEMs have greatly improved especially in capability for self-alignment and digital imaging, which we lack in our TEM. We do not have access to other modern instruments at our institution. Since the first submission, I have received an institutional commitment of $141,000 to be used toward the purchase of a new TEM. We have also improved our application by making the recommended revisions suggested by the reviewers.
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0.996 |
2011 — 2014 |
Difiglia, Marian |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Increase Rab11 Activity as Hd Therapy @ Massachusetts General Hospital
DESCRIPTION (provided by applicant): This application responds to a sponsored program announcement (NIH/CHDI) to validate potential therapeutic targets in Huntington's disease (HD). A major goal in finding treatments for (HD) is to identify early events causing neuronal dysfunction. We have identified an early brain dysfunction in the trafficking of cargoes from recycling endosomes back to the plasma membrane in HD neurons. This deficit is due to interference by mutant htt with nucleotide exchange on Rab11, a small GTPase which functions at recycling endosomes. We identify the impaired Rab11 GEF activity in brain of pre-symptomatic HD knock in mice. Defects in membrane trafficking in neurons cause cell death. Rab11 activity is required for cellular homeostasis and survival. Numerous cargoes involved in cellular homeostasis recycle using a Rab11 dependent pathway. These cargoes can be cell specific. So far we found that the recycling of three important Rab11 dependent cargoes is slowed in HD neurons expressing endogenous full-length mutant htt. One neuron specific cargo affected in HD neurons is the cysteine/glutamate transporter. In HD neurons, EAAC1 dependent uptake of cysteine is impaired leading to insufficient glutathione synthesis, elevated ROS and cell death. Lentivirus delivery of dominant active Rab11 in HD primary cortical neurons rescues these deficits. Our in vitro data provide compelling evidence for the trophic effect of dominant active Rab11 and point to Rab11 as a superb target needing in vivo validation. We will validate Rab11 activity as a novel therapeutic target in two HD mouse models that express full length mutant htt-- HD140Q/140Q knock-in and HD YAC 128 transgenic. We have generated transgenic mice that constitutively express dominant active Rab11 (TgRab11 mice). The Tg mice at 9 weeks show no difference in behavior to littermate controls and will be further studied using in vivo and in vitro studies to rule out side effects. In Aim 1, we will cross the HD mouse models with the TgRab11 mice. Since inducible expression simulates the condition for treatment of patients with HD, we will also cross HD mice with two transgenic lines that together incorporate the elements for a Cre-loxP system and will allow for tightly regulated inducible expression of dARab11 in presymptomatic and symptomatic HD mice. Aim 2 will be to determine if increased Rab11 activity rescues HD phenotypes. We will evaluate in vivo motor behavior and biochemical and anatomical readouts and in vitro endosome recycling and neuronal survival. These studies will demonstrate that raising levels of Rab11 activity increases neuronal survival and ameliorates or delays motor deficits and neuropathology in HD. In vivo validation of increasing Rab11 activity will justify a drug discovery effort to screen for small molecules that raise the activity of Rab11 as a therapy for HD.
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0.996 |