Douglas L. Feinstein - US grants

nervous system; genetics; biotechnology; biomedical resource; Invertebrata; Mammalia; Chordata;

The Neurochemistry/Molecular Biology Core will provide biochemical, molecular biological, cell culture services and support, general laboratory safety services (life safety, biosafety, radiation safety), maintenance of equipment, and general services such as laundry, water purification, and sterilization to the projects and other core groups.

DESCRIPTION (Applicant's Abstract) In brain, activation of astrocytes can induce calcium independent nitric oxide synthase (NOS-2) expression, which can contribute to the damage during ischemia, demyelinating disease, and viral infection. The applicants have previously characterized factors which activate astroglial NOS-2 expression, and two methods for potently blocking that expression, namely stimulation of b-ARs, and pretreatment with inhibitors of protein tyrosine kinases. They have also found that heat shock of glial cells reduces NOS-2 expression. Whether these methods of suppression represent distinct mechanisms, or share common features, is not known. Since activation of the transcription factor NFkB is required for NOS-2 expression, it is possible that all three suppressive means interfere with NFkB activation. This grant therefore proposes to test and characterize NFkB activation in astrocytes as it relates to NOS-2 induction. Differential NFkB activation by different inducers and/or in different cell types (e.g. astrocytes versus C6 cells) may explain differences in levels of NOS-2 induction. Since protein phosphorylation plays a key role in NFkB activation, the applicants will test if NE effects are mediated via a protein kinase, and if so if that kinase modifies NFkB activation. They will also test if NE induces IkB expression. They will fully characterize the heat shock response in astrocyte and C6 cells with the purpose of defining which heat shock proteins can block NOS-2 induction, and if that is due to blocking NFkB activation. They will test if heat shock proteins physically bind to the NFkB and/or IkB proteins, if they block IkB phosphorylation and/or degradation, or if they simply block NFkB movement into the nucleus. They will test if NE effects are due in part to induction of heat shock proteins. Some experiments (e.g. heat shock) will be carried out in animals to test if this suppressive method also works in vivo. These experiments will define multiple pathways to block NOS-2 expression that may be useful in preventing pathological NOS-2 expression in brain.

DESCRIPTION (provided by applicant): In brain, activation of glial cells induces nitric oxide synthase (NOS2) expression, which can contribute to damage in disease and trauma, including ischemia, multiple sclerosis, and Alzheimer's disease (AD). Prevention of NOS2 expression could reduce inflammatory damage. The signaling pathways underlying NOS2 expression are not fully characterized, however it is clear that transcription factor NFkB is crucial. We characterized NOS2 expression in rat astrocytes, and now will characterize signals leading to NOS2 expression in human astrocytes and rat and human microglia. Recent studies of NFkB regulation by inhibitory IkB proteins show that NOS2 induction requires persistent, not transient activation NFkB activation. Experiments will test the hypothesis that astroglial and microglial NOS2 expression also requires persistent NFkB activation. Since expression of a stimulatory form of IkB-beta is associated with persistent NFkB activation, we willdetermine if LPS or cytokines induce re-expression of this isoform. We showed two mechanisms toreduce NOS2, incubation with Norepinephrine (NE) or induction of a heat shock response (HSR),whether these methods prevent human glial NOS2 is not known. We propose that mechanisms which these two suppressors both cause an increase inhibitory IkB proteins, which we will test, and if so if those proteins enter the nucleus to inhibit NFkB. Our results suggest that depletion of brain NE levels increases brain inflammation, which is relevant to AD where noradrenergic neurons are lost.We will test this in vivo by chemical lesioning of noradrenergic neurons, and by using knockout mice which lack NE. The responses to injections of amyloid beta into the cortex, and whether the HSR reverses those responses, will be determined. These experiments will provide targets for therapeutic intervention to reduce pathological NOS2 in brain, and help develop a new animal model for examining brain inflammatory in the context of diminished NE levels.

DESCRIPTION (provided by applicant): The demyelinating autoimmune disease experimental autoimmune encephalomyelitis (EAE) is an often-used animal model to study possible causes and therapeutic interventions for Multiple Sclerosis (MS), which involves T-cell activation, migration into the CMS, induction of parenchymal cell inflammatory gene expression, damage to oligodendrocytes and myelin loss, and eventually irreversible axonal damage. One of the key signaling events required for inflammatory gene expression is activation of transcription factors, such as NFkB in glial cells and T cells, and of other such as transcription factor NFAT in T cells. Anti-inflammatory agents which reduce transcription factor activation could therefore be of therapeutic benefit. We and others have shown that induction of a heat shock response (HSR) potently reduced activation of transcription factor NFkB and inflammatory gene expression in brain glial cells, and that a brief period of hyperthermia (42[unreadable]C for 20 minutes) completed prevented the onset and development of EAE in mice. A HSR can also be induced by inhibiting activity of the HSP90 protein. Inhibition of HSP90 has two primary consequences: release of transcription factor HSF1 which in turn activates the HSR;and release and degradation of 'client'proteins, some of which can potentiate inflammatory responses or increase survival of inflammatory cells, such as the protein kinase AKT. In the current proposal, we will characterize the efficacy of novel small molecular weight HSP90 inhibitors to reduce glial (astrocyte and microglial) and T-cell activation, and to reduce clinical and pathological progression in EAE, with the ultimate goal of identifying compounds for further testing in MS patients. This goal will be addressed in 3 specific aims: Aim 1, characterizing the ability and mechanism of HSP90 inhibitors to block glial cell inflammation;Aim 2, determine the efficacy and mechanisms of HSP90 inhibitors to block T-cell activation;Aim 3, use selected HSP90 inhibitors to block clinical and histological symptoms in chronic and relapsing EAE model .

DESCRIPTION (provided by applicant): The current application is for support for the 41st annual ASN meeting to be held at the newly completed Convention Center in Santa Fe, NM, from March 6-10th, 2010. NIH funding for previous ASN meetings has been invaluable for supporting the ASN scientific programs and for enhancing our ability to reach graduate students and postdoctoral researchers. To accommodate the breadth of neurochemistry as well as cellular and molecular neurobiology, and to provide in depth analysis of particular topics, the ASN continues to build its scientific program around four interwoven, but distinct, themes. These themes have been selected to increase our understanding of the cellular and molecular bases of neural development and disease. These themes are: Building the Nervous System, Metabolism and Cell &Molecular Neuroscience, Glial Mechanisms &Injury, and Neuronal Degeneration &Disease. This meeting is an excellent opportunity for interactions because total attendance is approximately 500, which provides extensive opportunities for discussions. The Society has several mechanisms to enhance development of junior investigators at this meeting. We have luncheons with the plenary speakers, which gives attendees a chance to discuss topics with the speakers. The fees for these luncheons are reduced for students and postdoctoral fellows. There is a dinner exclusively for students and postdoctoral fellows, which has been highly noted as an outstanding opportunity to network. There is a luncheon entitled Women in Neurochemistry, which is open to all attendees and junior investigators have reduced fees. There are up to 6 sessions that are formed by selection of abstracts submitted with an emphasis on selecting from graduate students and post doctoral fellows. There are travel awards given to graduate students and post doctoral fellows to assist their finances. There is a job posting site and students can meet with potential future mentors or faculty members during the meeting. Together, and from results of yearly exit surveys we believe that the annual ASN meeting has and will continue to provide an excellent venue for cutting edge neurochemistry and for enhancement of the careers of young investigators. PUBLIC HEALTH RELEVANCE: This proposal requests support for the 41st annual meeting of the American Society for Neurochemistry taking place March 6th to 10th in Santa Fe, New Mexico. The support for this meeting will provide valuable assistance for this Society in publicly presenting current developments in this field. This annual meeting presents the latest information on advances in molecular and cellular neurobiology. Studies on numerous neurological diseases are presented. A Public Forum is held, which is advertised and focused on presenting information to the general public. This year's Public Forum is on the importance of nutrition and exercise for brain health. The support requested will also help keep registration fees low and be used to support events that enhance the careers of young investigators, women, and minorities.

DESCRIPTION (provided by applicant): The current application is for support for the 42nd annual ASN meeting to be held in St Louis, MO from March 19th to 23rd, 2011. NIH funding for previous ASN meetings has been invaluable for supporting the ASN scientific programs and for enhancing our ability to reach graduate students and postdoctoral researchers. To accommodate the breadth of neurochemistry as well as cellular and molecular neurobiology, and to provide in depth analysis of particular topics, the ASN continues to build its scientific program around four interwoven, but distinct, themes. These themes have been selected to increase our understanding of the cellular and molecular bases of neural development and disease. These themes are: Building the Nervous System. Metabolism and Cell &Molecular Neuroscience. Glial Mechanisms &Injury. Neuronal Degeneration &Disease. This meeting is an excellent opportunity for interactions because total attendance is approximately 500, which provides extensive opportunities for discussions. The Society has several mechanisms to enhance development of junior investigators at this meeting. We have luncheons with the plenary speakers, which gives attendees a chance to discuss topics with the speakers. The fees for these luncheons are reduced for students and postdoctoral fellows. There is a dinner exclusively for students and postdoctoral fellows, which has been highly noted as an outstanding opportunity to network. There is a luncheon entitled Women in Neurochemistry, which is open to all attendees and junior investigators have reduced fees. There are up to 6 sessions that are formed by selection of abstracts submitted with an emphasis on selecting from graduate students and post doctoral fellows. There are travel awards given to graduate students and post doctoral fellows to assist their finances. There is a job posting site and students can meet with potential future mentors or faculty members during the meeting. Together, and from results of yearly exit surveys we believe that the annual ASN meeting has and will continue to provide an excellent venue for cutting edge neurochemistry and for enhancement of the careers of young investigators. PUBLIC HEALTH RELEVANCE: This proposal requests support for the 42nd annual meeting of the American Society for Neurochemistry taking place March 19th to 23rd in St Louis, MO. The support for this meeting will provide valuable assistance for this Society in publicly presenting current developments in this field. This annual meeting presents the latest information on advances in molecular and cellular neurobiology. Studies on numerous neurological diseases are presented. A Public Forum is held, which is advertised and focused on presenting information to the general public, this year on Alzheimer's disease. A large part of the support requested will help keep registration fees low and be used to support events that enhance the careers of young investigators, women, and minorities.

DESCRIPTION (provided by applicant): Superwarfarins are modified forms of warfarin (Coumadin) with greater toxicity and longer duration of action. They potently reduce vitamin K levels throughout the body by inhibiting its recycling leading to loss of activity of Vitamin K dependent proteins including prothrombin. Loss of prothrombin leads to systemic anticoagulation, hence the basis for the widespread use of superwarfarins as rodenticides and to reduced embolisms in stroke patients. Superwarfarins were developed following reports of warfarin resistant rodent strains and are now commonly used throughout the world. Reported clinical occurrences of superwarfarin poisoning have reached over 16,000 per year in the USA, with >90% in children under age 6. Although superwarfarin poisoning can be treated it requires long term supplementation with Vitamin K, and any interruption in treatment can lead to recurrence of symptoms. Other VKDPs play important roles in brain development; and reductions in those proteins is associated with increased inflammation, neuronal damage, loss of myelin, and cognitive deficits. In addition, in utero exposure to warfarin has adverse effects on embryonic development, suggesting the in utero exposure to superwarfarins could have disastrous outcomes. The superwarfarin most commonly used today as a rodenticide is brodifacoum (BDF). The estimated fatal oral dose for humans is very low (15 mg), and because it is highly lipophilic, it accumulates in tissues with a biological half-life of over 20 days. Thu, even though there is an existing cure BDF poisoning (chronic Vitamin K treatment), its toxicity, exceptionally long half-life, the relative ease of obtaining it, and the array of potential methods for its harmful distribution (contamination by aerosol dispersal or direct application on plants sol, or food), make BDF a serious potential chemical threat. In this proposal, we will test the therapeutic potential of an already approved FDA treatment, Lipid Emulsion (LE), as a countermeasure against BDF poisoning. The infusion of LE is already used to treat certain toxic drug overdoses, and is thought to act in part by scavenging toxins and movement to the liver where they are metabolized. Acute LE infusion should offer rapid removal of BDF from the body, thereby preventing life threatening reductions in clotting, protect against long-term debilitating CNS sequelae of poisoning; and prevention of in utero exposure. In addition, since intravenous administration of a counter measure may be impractical in a scenario involving mass casualties, we have developed an alternative method of administration, namely intraosseous (IO) which infuses LE into bone where it can rapidly enter the blood stream. In this program, we will optimize the ability of IO LE to eliminate BDF from adult rat tissues. If levels are not sufficienty reduced by a single IO bolus, we will add follow up IV infusions of LE. We will characterize the consequences of in utero exposure to BDF, and optimize treatment of pregnant rats with LE to minimize those later consequences. A major goal of this project is to submit an IND for the use of LE to treat BDF poisoning; since testing in humans is not feasible, in year 4 of this project we will carry out similar studies in rabbits to match the FDA requirements under the animal rule for a new drug. We anticipate that LE will be proven to be an effective treatment for BDF poisoning, thereby providing a mechanism to address the potential damage due to a large scale release of BDF by either intentional or accidental causes.