Jonathan Glass - US grants

This award supports cooperative research on Isolation of Iron Binding Protein of Reticulocyte Coated Vesicles between Jonathan Glass of Louisiana State University and Marco Tulio Nunez of the University of Chile. These two scientists have had a long and productive collaboration and in addition, interactions with other researchers in both countries. Because of Glass's expertise in cell biology of iron metabolism, the first two tasks, development of an assay to detect an iron binding protein and its isolation will take place in his laboratory. Rapid progress is expected. Nunez will participate in this phase and then take the assay to Chile where the role of electrochemical gradients on translocation of iron across coated vesicle membranes will be studied. The Chileans have expertise in conduction across lipid bilayers and transport across vesicle membranes not available at LSU. Much of the preliminary work on this project has already been carried out. The collaboration has been productive and it is likely that significant data will be generated. This will add to the small amount of knowledge about iron transport across membranes in reticulocytes. Because the assay development will take place at LSU, work will be carried on in Chile which could not otherwise. This will benefit the university in Chile and the students there while LSU benefits from an experienced Latin American scientist.

9224245 Glass This Americas Award will support collaborative research between Drs. Jonathan Glass, Louisiana State Medical Center, and M. T. Nunez Gonzalez, University of Chile, on the exploration of the cellular mechanism of iron uptake. The focus of this research is on the biochemical analysis of the events required for iron delivery from transferrin, with the ultimate aim of elucidating the mechanism of iron transport. The laboratory on the Chilean side will focus on utilizing rabbit reticulocytes to obtain detailed analyses of iron mobilization via the regulation of vesicular pH by ionic fluxes, while the laboratory of the principal investigator will determine mobilization rates as a function of ionic composition and membrane potential. Results of the research will contribute to a better understanding of the fundamental aspects of human cellular metabolism. ***

neurologic manifestations; HIV infections; AIDS dementia complex; biomedical facility; tissue resource /registry; information systems; in situ hybridization; immunocytochemistry; human tissue; postmortem;

Development of a painful neuroma is a distressing problem often associated with neuroma formation after limb amputation. The pain is perceived in the zone of innervation of the severed nerve although this region may no longer be anatomically present. Mechano-sensitivity and ectopic excitability of the injured axons likely play a major role in the pain process. Surgical resection of the neuroma may provide only transient pain relief until the neuroma re-forms. To prevent neuroma formation, the neuronal cell body in the dorsal root ganglion or anterior spinal cord must be destroyed resulting in Wallerian degeneration of the axon. Studies have shown that certain toxins, including adriamycin, are retrogradely transported by axons to their cell body resulting in death of the neuron (suicide transport). We hypothesize that treatment of the axons that innervate a neuroma with adriamycin will prevent reformation of the neuroma and will lead to permanent pain relief. In this proposal, we plan to test this hypothesis by performing histological and neurophysiological studies in a neuroma model. Specifically, we aim to answer the following questions: Is adriamycin transported by an axon to its cell body? Can a drug dose be determined that will result in a consistent neuronal lesion secondary to retrograde adriamycin transport? Does the axonal transport of adriamycin cause death only in those axons which were exposed to the drug? Does treatment of a neuroma with adriamycin result in the loss of mechano-sensitivity and ectopic excitability in the nerve? Does the use of suicide transport with adriamycin improve the outcome over standard surgical practice for patients suffering from painful neuromas? Using histo-pathology and teased-fiber electrophysiology recording techniques we will determine the response of neuromas to the treatment with adriamycin in a rat model and subsequently in a monkey model. The laboratory studies will optimize our technique laying the foundation for the clinical study in which amputation patients suffering from pain secondary to neuroma formation will undergo an open labelled/ended trial of neuroma resection with the addition of suicide transport of adriamycin. If the results look promising, a blinded, randomized trial will be performed in the future. We believe that treatment with suicide transport using adriamycin will prove to be an effective modality for eliminating neuroma pain such as that which can follow limb amputation.

DESCRIPTION: The focus of this application is on the role of the calpains in diabetic neuropathy. The general hypothesis to be tested is that the calpain/calpastatin system plays a pivotal role in the pathogenesis of axonal degenerations. Specific aim 1 is to identify and measure changes in the calpain/calpastatin system during Wallerian degeneration, using specific antibodies and in vitro protease assays. Specific aim 2 is to test the potential for calpain inhibition to modify the neuropathological changes induced in dorsal root ganglion explant cultures by hyperglycemia. The third specific aim is to test the ability of various permeant calpain inhibitors to prevent or reduce neuropathy in streptozotocin-induced rat diabetic neuropathy

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Glass

This Americas Program award will support travel and related expenses for five US scientists and 2 postdoctoral students, to participate in an international workshop on iron and copper homeostasis, to be held in Pucon, Chile, November 16-19, 1999. Organizers are Dr. Jonathan Glass, Louisiana State University, Shreveport and Dr. Marco T. Nunez, University of Chile. The workshop will take place in conjunction with the Annual Meeting of the Biology Society of Chile, and is designed to bring together scientists with an interest in molecular aspects of iron and copper transport and metabolism. It has been noted that although copper and iron metabolism are intimately related, the scientists of both fields rarely communicate. Therefore, a better understanding of iron-copper interaction would foster knowledge in iron and copper metabolism, and would establish future avenues of collaborative research.

Besides fomenting interactions between scientists, and strengthening international scientific collaboration, the workshop will enable US and Chilean scientists and students to become aware of the newer molecular developments in this area of research. The attendance of leading scientists in the field from both US, and outstanding Latin American laboratories is likely to lead to closer integration of research efforts In addition to the US and Latin American participants, the workshop will congregate a worldwide assembly of leading scientists in the field, including scientists from Europe, Israel and Japan.


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Axonal degeneration, or death of nerve fibers, is the most common pathological finding in the majority of neurological disorders, including stroke, head and spinal cord trauma, and peripheral neuropathy. There is a paucity of data regarding the basic mechanisms of axonal degeneration, and it is unclear whether axonal death in each of these disorders occurs via multiple or common pathways. Experimental studies, however, support the idea that calcium entry and protease activation are important. The long term goal of these studies is to understand the basic mechanisms leading to axonal degeneration using clinically relevant models of disease, and to use these new data to direct strategies for prevention of axonal degeneration and thus preservation of neurological function. The experimental design exploits: 1) the sensory ganglia culture system for manipulating rodent axons in a reproducible in vitro setting, and 2) the WLD mouse, a unique mutant strain whose sole phenotype is resistance to traumatic axonal degeneration. Aim 1 will test the hypothesis that axonal death caused by trauma (axotomy) and exposure to a neurotoxin (vincristine) occurs by a common mechanism involving calcium entry and activation of the protease, calpain. In Aim 2, axons from the WLD mouse which are resistant to axotomy-induced degeneration will be used to further test the hypothesis stated in Aim 1. These axons will be tested for their ability to resist degeneration when exposed to a neurotoxin induced axonal degeneration. Aim 3 will ask when calcium entry and calpain activation occur after nerve injury, and what relationship these events have to axonal degeneration. Aim 3 will ask when calcium entry and calpain activation occur after nerve injury, and what relationship these events have to axonal degeneration. Using state-of-the-art imaging techniques, the proximal and distal portions of transected nerve fibers will be compared since they are exposed to the same injury, but one survives and the other dies. Aim 4 is directed at identifying proteases other than calpain that are present and active in degenerating nerve fibers, with the premise that a directed strategy for preventing axonal degeneration during disease requires an intimate understanding of all of the processes involved. Overall, the achievement of these Aims will provide a new appreciation of the mechanisms of axonal degeneration that will certainly impact on treatment strategies for a number of brain, spinal cord, and peripheral nerve diseases.

The Core Histology facility for this Program will provide the technical and pathological expertise for all tissue preparation for the 5 projects. These projects are heavily dependent on experimental animals with pathological endpoints. High quality preparation is necessary for quantitative studies, as well as for studies requiring identification of structures located deep within tissues. With the Histology Core in place, equipment and supply duplication will be minimized, and economies of scale will be realized. Quality control will also benefit from a central dedicated facility.

[unreadable] DESCRIPTION (provided by applicant): Multiple Sclerosis (MS) is a common neurological disorder characterized by immune attack on myelin and mylein forming cells of the central nervous system. Current treatments are directed at reducing the immune-mediated inflammatory damage to myelin. Recent observations from neuroimaging and pathological research demonstrate, however, that axonal pathology may also play a large role in the clinical deficits of MS patients, suggesting that the protection of axons may be a potentially important addition to MS therapies. The long-term objective of this proposal is to test the hypothesis that the protection of axons through the inhibition of calcium-activated proteases, or calpains, will ameliorate the clinical deficits in a mouse model of MS. This hypothesis is based on a large volume of data demonstrating the importance of calpain activation in axonal degeneration, and our published experience with preventing axonal degeneration and clinical disease in another animal model of neurological disease. In Aim 1 we will use the model of experimental autoimmune encephalomyelitis (EAE) to investigate the relationship of calpain activation to axonal degeneration and clinical disease. We will measured calpain activation using calpain activity assays and immunocytochemical and immunoblot methods to identify calpain-specific spectrin-breakdown products in tissues. In Aim 2 we will treat animals with EAE with our novel ketoamide calpain inhibitor AK295 and measure attenuation of disease by clinical and patholgical methods. In Aim 3 we will test the ability of AK295 to cross the blood-brain barrier (BBB), since any effective calpain inhibitor will need to get into the brain in sufficient quantities. We will also test the BBB permeability of 10 other novel calpain inhibitor compounds taken from our library of compounds, and compare them to AK295 for BBB permeability. Finally, we will attempt to increase BBB permeability by chemically modifying AK295 and other promising compounds by adding choline and nucleosides in order to take advantage of the inherent BBB transporter systems. These studies of calpain inhibition in a model of MS represent a novel approach to the treatment of this common and devastating neurological disorder. Combined with immunomodulatory therapy, axonal protection by calpain inhibition may significantly change the course of disease for people with MS. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this research program is to discover protein biomarkers for Amyotrophic Lateral Sclerosis (ALS). Biomarkers for neurodegenerative diseases are necessary for accurate diagnosis and monitoring of disease progression and response to therapy. Biomarkers also provide important clues about mechanisms of disease pathogenesis, identifying pathways of cellular dysfunction during the course of disease. Success in biomarker discovery is greatly enhanced when investigation is focused on a homogenous disease population where there is a relevant animal model of the disease. ALS caused by mutations in superoxide dismutase 1 (SOD1-ALS) meets these criteria, and is the focus of this proposal. There are two specific aims: 1) We will use state of the art proteomic methods to quantitatively compare 3000 proteins from spinal cords and motor nerves of SOD1-ALS mice and their normal littermates. We will compare mice from presymptomatic, early symptomatic and late stages of disease in order to correlate protein patterns with the progression of disease. 2) We will select a subset of proteins (about 40) as possible candidate biomarkers based on rank order criteria, such as magnitude of change and progression of change with disease. In order to translate the proteomic findings from tissues into biomarkers that might be used in clinical practice, the selected proteins will be specifically investigated in plasma and spinal fluid samples from SOD1-ALS mice and controls. Finally, we will investigate these candidate biomarkers in plasma and spinal fluid samples from people with ALS, including those with SOD1-ALS and those with sporadic ALS. Here we will take advantage of the large clinical research database of families with ALS at the Emory ALS Center. This proposal represents a systematic approach to biomarker discovery using the most modern technology available combined with sophisticated bioinformatics. We will use these tools develop biomarkers for ALS that are essential for identifying pathogenic mechanisms and developing novel treatments. We propose to use state of the art proteomics combined with sophisticated bioinformatics to identify protein biomarkers in Amyotrophic Lateral Sclerosis (ALS). We will screen for changes in about 3000 proteins in spinal cord and nerve tissue from the SOD1-ALS mouse, and then identify a specific subset of proteins as potential biomarkers. These candidate biomarkers will be tested for their sensitivity and specificity for disease in plasma and spinal fluid samples from mice and people with ALS. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Calpain inhibitors have enormous potential as therapeutic agents for neurological diseases. Calpains are calcium-activated cysteine proteases that are major contributors to the process of axonal and neuronal degeneration. Degeneration of axons leads to the disconnection of neurons from their motor or sensory targets, leading to weakness, numbness, pain, and cognitive impairment. Axonal degeneration underlies neurological dysfunction in stroke, head and spinal cord injury, peripheral neuropathies, and multiple sclerosis. We have had success in preventing axonal degeneration and neurological dysfunction in a model of peripheral neuropathy. We propose to use the Molecular Libraries Screening Center at Emory University for high throughput screening of a large library of small molecules (up to 100,000) for calpain inhibitor activity. We have developed a secondary assay for independent analysis of "hits", and plan for follow-up developmental chemistry for optimizine drug design. Thus, the resources of the MLSCN will allow us to identify new calpain inhibitor compounds that may provide important new treatment for patients with a variety of neurological diseases. The degeneration of axons is a pathological feature common to many neurological diseases that causes neurological disability due to disconnection of neurons from their targets. Preventing axonal degeneration is thus protective, and we are proposing to discover small molecules that can be used for this purpose. The target of our small molecule screen is the calcium-activated protease calpain. Inhibitors of calpain will be developed as novel treatments of neurological diseases. [unreadable] [unreadable] [unreadable]

The primary goal of the Emory ADRC Neuropathology Core is to promote and support research on Alzheimer's disease and related neurodegenerative disorders through banking and distribution of well characterized tissue coupled with comprehensive neuropathological assessment. Given that the neuropathology of AD and other dementias continues to develop with the discovery of new causative and risk related genes and the identification of novel structural changes and pathological protein accumulations, the Core will also support new investigative studies based on novel clinical or pathological findings from our patient population or from pursuit of questions that arise from new developments in the field. The Specific Aims of the Neuropathology Core are 1) To reliably harvest nervous system tissue at autopsy from patients followed in our Clinical Core;2) To maintain an active tissue bank to facilitate the acquisition, storage, handling, and distribution of well-characterized autopsy brain tissue, and other biological samples, and to continually refine methods for rapid freezing and fixation/processing of post-mortem brain tissue as needed to better accommodate RNA extraction and proteomic studies;3) To provide comprehensive neuropathologic assessment of autopsy tissues and to provide reliable neuropathologic data for correlation with information generated by the Clinical Core and for use in research studies at Emory, NACC, and other institutions;4) To genotype cases for apolipoprotein.E and other genes linked to neurodegenerative disease, using either blood samples accessioned by the Clinical Core or autopsy cases accessioned by the Neuropathology Core, and to facilitate collaborative investigations using the core's banks of DNA extracts, plasma samples, and buffy coat isolates. The functions ofthe Neuropathology Core are intimately connected with those ofthe other cores and of the ADRC research projects. The Neuropathology Core will work closely with the other Cores to continue to fulfill its mission of providing high quality tissue for research projects at Emory and at other institutions in the United States and abroad.

DESCRIPTION (provided by applicant): This proposal outlines a plan for a Phase 1b clinical trial for the injection of human spinal cord derived stem cells (HSSCs) into the cervical spinal cord of patients with ALS. This trial is a follow up to a trial already underway at Emory University, where 12 ALS patients have been injected with the same HSSCs into the lumbar spinal cord. In order to move this therapeutic approach closer to a clinical trial to determine if t is effective in ameliorating disease, we are proposing to test the safety of HSSC injection into the cervical spinal cord. Motor neurons in the cervical spinal cord innervate the respiratory diaphragm, the loss of which is typically the cause of death in ALS patients. We propose that the protection of these neurons is likely to prolong life by preserving respiratory function. This safety trial will employ progressive dose escalation to determine the maximum tolerated dose that can be used for the long term goal of performing Phase 2 and Phase 3 efficacy trials. There are two specific aims. In Aim 1 we propose to sequentially escalate the dose of delivery as defined by 1) the number of cells/injection, 2) the number of injections into the cord, and 3) either unilateral or bilateral injections. This dose escalation scheme is designed to safely and efficiently test our ability to achieve a pre-defined target therapeutic dose, which can be used in the next phase of testing therapeutic efficacy. Aim 2 of this proposal is to examine several exploratory endpoints that may be used to test the efficacy of this therapy in future Phase 2 and Phase 3 trials. These include measures of respiratory function, diaphragm function, muscle strength, and electrical characteristics of muscle (Electrical Impedance Myography). Successful completion of this Phase 1b trial will allow for testing of this highly innovative approach to the treatment of ALS. The impact will extend beyond patients with ALS since this novel trial will provide data on surgical approach and safety, as well as trial design that will be highly relevant to cellular therapeutics for spinal cord injury, multiple sclerosis, spinal muscular atrophies, as well as other neurological diseases.

The Emory Alzheimer's Disease Research Center (ADRC) Neuropathology Core (NP Core) supports the ADRC by providing comprehensive neuropathological assessment of patients and control subjects followed by the Clinical Core, by banking and distributing brain and spinal cord tissues for research studies, and by providing expert consultation to investigators regarding case selection and experimental design as they relate to the use of human tissues. During this funding period, the NP Core performed brain autopsies on 59 UDS subjects followed by the Clinical Core, and 6 MDS autopsies, and distributed >2200 tissue samples to 74 investigators leading to >180 peer-reviewed publications, chapters, and abstracts using Core resources. Our well-characterized tissue resources enables highly innovative research projects, such as Project 2 which uses unique proteomics methods to investigate novel targets for neurodegenerative disease, and Project 3 that is investigating differences in A¿ species that may explain disease variability. In this application, we will focus on elderly controls and patients with MCI, AD in order to facilitate research into the mechanisms of onset and progression of AD and related neurodegenerative diseases. To further contribute to innovative research in AD and related dementias, the NP Core is leveraging the amyotrophic lateral sclerosis (ALS) expertise of the NP Core leader, who provides new perspectives and resources for the pursuit of pathogenic mechanisms underlying clinically distinct, but pathologically similar diseases, some of which may coexist with AD. Together with the other ADRC Cores, the NP Core will educate students, residents, fellows, and investigators on neurodegenerative disease neuropathology and techniques used to evaluate neuropathological features in human brains and animal models of disease.

SUMMARY/ABSTRACT: NEUROPATHOLOGY CORE The Emory Goizueta ADRC Neuropathology Core (NP Core) supports this ADRC and national/international research projects by providing comprehensive neuropathological assessment of patients dying with AD and related dementias (ADRD) and control subjects, and by banking and distributing brain and spinal cord tissues for research studies. The NP core provides expert consultation to investigators regarding case selection and experimental design as they relate to the use of human tissues, and also collaborates with the REC to train the next generation of ADRD neuropathologists and researchers. During this funding period, the NP Core performed brain and spinal cord autopsies on 53 UDS subjects and distributed >7000 tissue samples to 80 investigators, leading to 120 peer-reviewed publications. Our well-characterized tissue resource enables highly innovative local, national, and international research projects, including the Goizueta ADRC leadership in the discovery of novel targets for neurodegenerative diseases using unique proteomics and proteo- genomics methods. With this application we continue to focus on patients with MCI, AD, and elderly controls in order to facilitate research into the mechanisms of onset and progression of ADRD. Consistent with our minority outreach programs, we also focus attention to collecting tissues from the African American population followed by the Clinical Core; 562 tissue samples from AA were distributed during this funding period. The NP Core is addressing the local and national interest in the related dementias of frontotemporal dementia (FTD/FTLD) and dementia with Lewy bodies (DLB). We leverage the clinical and pathological expertise of the NP Core leader in the overlap of FTD/FTLD with Amyotrophic Lateral Sclerosis (ALS), which are clinically distinct but pathologically similar diseases, some of which may coexist with AD. Emory is a member of the LBD Research Center of Excellence network; the NP core works to collect and distribute these valuable tissues for DLB researchers around the country. Our work in the related dementias provides new perspectives and resources for the pursuit of pathogenic mechanisms in ADRD. Together with the REC and other ADRC Cores, the NP Core will continue to educate students, residents, fellows, and investigators on the neuropathology of neurodegenerative diseases, as well as the techniques used to evaluate neuropathological features in human brains and animal models of disease.