Stuart A. Lipton - US grants

The lines of communication in the nervous system consist of axonal and dendritic processes of nerve cells. The processes of one cell may communicate with those of another across synaptic clefts via chemical messengers that affect ionic currents in the cell membrane. If injury or disease causes the processes of the mammalian central nervous system to die, they normally do not regenerate, and communication is lost. The study of process communication and regeneration of mammalian central neurons would be aided by examining the detailed membrane properties of an identified cell in a controlled in vitro environment. This approach requires that the cells be isolated, unequivocally identified, and cultured. Cultures of solitary rat retinal ganglion cells, identified with specific fluorescent probes, are being developed in this laboratory. Preliminary studies have shown that process regeneration is enhanced by plating these cells on s specific monoclonal antibody against Thy-1 antigen, which is located on retinal ganglion cells. The specific aims of this proposal are-- (i) The study of intercellular communication by (a) characterizing the various ionic currents of the cell membrane using the patch-clamp technique; (b) testing the effects on these currents of neuroactive substances that may modulate intercellular communication and that are putatively found in amacrine cells that synapse on ganglion cells. These compounds include acetylcholine, GABA, glycine, dopamine, and many peptides such as substance P, somatostatin, TRH, and enkephalin. (ii) The study of process regeneration by (a) attempting to localize the binding of anti-Thy-1, which promotes process regeneration, at an ultrastructural level in the retina; (b) using an existing anti-idiotype against the Thy-1 antibody that will act as an analogue of Thy-1 in an attempt to identify a "naturally" occurring ligand that might recognize the Thy-1 determinant and influence process regeneration in the central nervous system; (c) using the knowledge gained of the specific ionic currents to pharmacologically block or enhance each current in order to affect process regeneration; (d) presenting normal target tissue (tectum and lateral geniculate nucleus) or extracts of these tissues to influence process regeneration; (e) attempting to increase the regeneration of optic nerve in vivo in adult rats by using the factors learned in culture in conjunction with medical nerve guides.

Detailed studies of differentiated central neurons of the mammalian visual system would be aided by examining the membrane properties of an identified cell in a controlled in vitro environment. This approach requires that cells be isolated, unequivocally identified, and cultured. Cultures of solitary rat retinal ganglion cells, identified with fluorescent probes, are being developed in this laboratory. Since ganglion cells are the only retinal neurons that project to other areas of the central nervous system, they can be labelled by retrograde transport of fluorescent dyes injected into their projection sites, such as the superior colliculus and lateral geniculate nucleus. To confirm the identify of retrogradely labelled cells, a monoclonal antibody, that among retinal cells is specific for the ganglion cells, can also be used as a marker with immunofluorescent techniques. Following dissociation of the retina with papain and trituration, the labelled ganglion cells are placed in culture. The specific aims of this proposal are-- (i) To characterize the various ionic currents of the cell membrane of solitary retinal ganglion cells. Conventional intracellular and patch-clamp electrodes will be used to record voltage as well as whole-cell and single-channel currents. Each current will be isolated using a combination of voltage-clamp, pharmacological agents, and ionic substitution. (ii) To test the effects of putative neurotransmitters and modulators on these currents. These substances include acetylcholine, GABA, glutamate, glycine, indoleamines, dopamine, and many peptides such as substance P, somatostatin, thyrotropin releasing hormone (TRH), and enkephalin. The long-term goal of this project is to further the understanding of intercellular communication between central neurons in the mammalian visual system.

A better understanding of the cell surface molecules involved in development of the mammalian visual system and in outgrowth/regeneration of processes by visual neurons, such as retinal ganglion cells, would be of fundamental importance. Such knowledge would aid a rational approach to the study and possible treatment of injured retinal ganglion cells and their axons, which form the optic nerve. One cell surface molecule that appears to be involved in neuronal development and regeneration is Thy-1 glycoprotein, which is located on the surface of mammalian retinal ganglion cells. The Principal Investigator has demonstrated that the regeneration of processes (also termed neurites) by rat and mouse retinal ganglion cells is enhanced by specific monoclonal antibodies against Thy-1. Most importantly, in preliminary experiments the P.I. has gathered evidence for a "naturally-occurring" binding site or receptor for Thy- 1, located on astrocytes, that enhances the regeneration of retinal ganglion cell processes; this is not surprising since the endogenous Thy-1 receptor is, in some respects, a structural analogue of exogenous antibodies against Thy- 1. Using anti-idiotype antibodies against Thy-1 antibodies, tentative identification of the Thy-1 receptor has been made using a combination of biochemical and molecular techniques. In these experiments, the anti-idiotype antibodies serve as probes that should structurally resemble antigenic sites on Thy- 1 since both bind to antibodies against Thy-1. The current grant proposes to further characterize this endogenous Thy-1 receptor. The work will primarily use molecular biological methods, but it will also be complemented by parallel biochemical and immunohistochemical studies. The Specific Aims of this proposal are -- 1. To identify and sequence a Thy- 1 receptor cDNA and map its chromosomal location. 2. To examine the synthesis, distribution, and expression of Thy- 1 receptor in vitro and in vivo. 3. To study the effect of Thy- 1 receptor on the outgrowth of rodent retinal ganglion cells in culture using (a) purified receptor, (b) transfected receptor cDNA into cell lines, and (c) antisense oligonucleotides to Thy- 1 receptor to down-regulate its expression on astrocytes. 4. To identify possible Thy- 1 receptors in other species in retina/optic nerve and elsewhere in the brain.

Detailed studies of postnatal neurons of the mammalian visual system would be aided by examining the chemically-gated ion channels of identified cells in a controlled in vitro environment. For this purpose cultures of rodent retinal ganglion cells, identified with specific fluorescent probes, are used in this laboratory. The specific aims of this proposal are -- (i) To study physiologically and pharmacologically the effects of putative neurotransmitters and modulators on solitary mammalian retinal ganglion cells in culture. The effect of various substances on both whole-cell and single-channel currents will be monitored using the patch-clamp technique. If calcium current or calcium-activated currents appears to be affected by an agent, then calcium will be quantitated using imaging techniques with the calcium-sensitive dye, fura-2. Each of the substances to be studied is a likely candidate to affect ganglion cells because of its presence in amacrine and bipolar cells which normally synapse on the ganglion cells in the intact retina. These substances include acetylcholine, GABA, glycine, glutamate, dopamine, and indoleamines, as well as many peptides such as leu-enkephalin, substance P, somatostatin, cholecystokinin, neurotensin, and vasoactive intestinal peptide (VIP). (ii) To identify the transmitters used at chemical synapses onto retinal ganglion cells. The substances found to affect solitary ganglion cells will be tested for their effects on endogenous synaptic activity which occurs in ganglion cells located among clusters of other retinal cells in the cultures. In this manner an attempt will be made to discern the identity of the physiologically-relevant transmitters and modulators. (iii) To quantify trophic effects of the putative neurotransmitters and modulators and their selective blocking agents by monitoring survival and regeneration of processes by retinal ganglion cells in culture. Preliminary experiments have already suggested that nicotinic cholinergic antagonists and opiate analogs influence the regeneration of neurites by retinal ganglion cells. Ionic mechanisms underlying process regeneration may be uncovered in these experiments since the ionic basis for the current induced by each substance will have been studied in the patch clamp experiments. The long-term goals of this project are to further the understanding of synaptic communication between central neurons in the mammalian visual system and to increase neuronal survival and the potential for process regeneration.

Detailed studies of postnatal neurons of the mammalian visual system would be aided by examining the chemically-gated ion channels of identified cells in a controlled in vitro environment. For this purpose cultures of rodent retinal ganglion cells, identified with specific fluorescent probes, are used in this laboratory. The specific aims of this proposal are -- (i) To study physiologically and pharmacologically the effects of putative neurotransmitters and modulators on solitary mammalian retinal ganglion cells in culture. The effect of various substances on both whole-cell and single-channel currents will be monitored using the patch-clamp technique. If calcium current or calcium-activated currents appears to be affected by an agent, then calcium will be quantitated using imaging techniques with the calcium-sensitive dye, fura-2. Each of the substances to be studied is a likely candidate to affect ganglion cells because of its presence in amacrine and bipolar cells which normally synapse on the ganglion cells in the intact retina. These substances include acetylcholine, GABA, glycine, glutamate, dopamine, and indoleamines, as well as many peptides such as leu-enkephalin, substance P, somatostatin, cholecystokinin, neurotensin, and vasoactive intestinal peptide (VIP). (ii) To identify the transmitters used at chemical synapses onto retinal ganglion cells. The substances found to affect solitary ganglion cells will be tested for their effects on endogenous synaptic activity which occurs in ganglion cells located among clusters of other retinal cells in the cultures. In this manner an attempt will be made to discern the identity of the physiologically-relevant transmitters and modulators. (iii) To quantify trophic effects of the putative neurotransmitters and modulators and their selective blocking agents by monitoring survival and regeneration of processes by retinal ganglion cells in culture. Preliminary experiments have already suggested that nicotinic cholinergic antagonists and opiate analogs influence the regeneration of neurites by retinal ganglion cells. Ionic mechanisms underlying process regeneration may be uncovered in these experiments since the ionic basis for the current induced by each substance will have been studied in the patch clamp experiments. The long-term goals of this project are to further the understanding of synaptic communication between central neurons in the mammalian visual system and to increase neuronal survival and the potential for process regeneration.

Detailed studies of the effects of putative neurotransmitters/modulators and their mechanism of action on postnatal neurons of the mammalian visual system would be aided by studying identified cells in a controlled in vitro environment. For this purpose cultures of rodent retinal ganglion cells, labeled with fluorescent probes, are used in this laboratory. The specific aims of this proposal are -- 1. To study physiologically and pharmacologically the direct effects of putative neurotransmitters and modulators on solitary mammalian retinal ganglion cells in culture. Ganglion cells, fluorescently labeled by retrograde transport, will be dissociated from the retinas of rodents, maintained in culture, and recorded from with the patch-clamp technique. The detailed effects of various substances and their antagonists on both whole-cell and single-channel currents will be monitored. These agents include classical chemicals such as acetylcholine, GABA, glycine, and glutamate and its analogs, as well as many peptides that are found in the amacrine cells that normally synapse on the ganglion cells. Examples of these peptides are leu-enkephalin, substance P, somatostatin, cholecystokinin,,neurotensin, and vasoactive intestinal peptide (VIP). The analysis of single-channel events activated by these drugs may yield insight into their molecular mechanisms of action and possible differences between retinal and other tissues. 2. To identify the transmitters used at chemical synapses onto ganglion cells. The substances that influence solitary ganglion cells will be tested for their effects on synaptic activity found in the ganglion cells in more dense cultures during whole-cell patch-clamp recording. In this manner an attempt will be made to discern the identity of the physiologically-relevant transmitters and modulators. For example, the effects of antagonists to the various putative transmitters and modulators will be characterized on spontaneous and evoked postsynaptic currents that occur in cultured ganglion cells that have formed synapses with other retinal cells. 3. To quantify plasticity effects of the putative neurotransmitters and modulators and their selective blocking agents by monitoring outgrowth of processes and survival of retinal ganglion cells in culture. Preliminary experiments have suggested that several agents, such as nicotinic agonists and N-methyl-D-aspartate (NMDA), influence the outgrowth of neurites and the survival of cultured retinal ganglion cells. To test the effect on process outgrowth, drugs will be applied directly to the growth cone of a neurite of an isolated retinal ganglion cell while monitoring growth with computer-enhanced video microscopy. Viability of retinal ganglion cells will also be monitored after incubation with putative transmitters. Ionic mechanisms underlying process outgrowth and survival of retinal ganglion cells may be uncovered in these experiments since the ionic basis for the current induced by each substance will have been studied in the patch clamp experiments. The long-term goals of this project are to further the understanding of synaptic communication between neurons in the mammalian visual system and to increase neuronal survival and the potential for process regeneration following injury or ischemic insult.

AIDS can produce substantial dysfunction in the central nervous system (CNS), including encephalopathy/dementia by affecting the brain, myelopathy by affecting the spinal cord, and visual loss by affecting the retina. These adverse effects can occur in the apparent absence of infectivity of neurons by HIV-1, and even in the absence of superinfection with other opportunistic organisms. Therefore, one possibility is that HIV-1 somehow produces a toxic agent that injures neurons. One molecule that is a candidate as a toxic product of HIV-1 is the envelope protein gpl20, since it is shed from the virus. For the current proposal, 3 tissue culture models of CNS injury will be used, comprised of (i) postnatal rodent retinal ganglion cells, (ii) rodent hippocampal cortex neurons, and (iii) human cortical neurons (obtained from neurosurgical specimens). In preliminary studies on rat retinal ganglion cell and hippocampal neurons, gpl2O, in picomolar amounts and in a dose-dependent fashion, induces a dramatic increase in intracellular [Ca2+]; in retinal ganglion cells this rise in [Ca2+] is associated with neuronal injury and even cell death. Additional preliminary experiments have demonstrated that calcium channel antagonists are capable of totally preventing this form of neurotoxicity caused by the HIV-1 envelope protein. Studies are proposed to test if these toxic effects apply to other types of neurons, including human cortical neurons, and to elucidate further the mode of action of gpl2O on intracellular [Ca2+] using calcium imaging with the dye fura-2, patch-clamp recording, and concomitant viability assays. The specific aims of the proposed study are -- 1 . To monitor the level of free calcium in retinal ganglion cell and cortical neurons in vitro after exposure to HIV-1 envelope protein gpl 20; rodent and human neurons will be used. 2. To assess injury to these neurons after exposure to gpl20 in vitro using a well-developed test of viability, the uptake and cleavage of fluorescein diacetate; to assess the protective effects from gpl 20 neurotoxicity of a variety of calcium channel antagonists by generating dose-response curves. 3. To perform patch-clamp recordings of these neurons at both the whole-cell and single-channel levels to determine, as suggested in preliminary experiments, if gpl20 affects a specific type of calcium channel; to study the mechanism of this effect.

[unreadable] DESCRIPTION (provided by applicant): The unifying theme of this competing renewal program project is a cellular and molecular approach to developmental neurology in an attempt to uncover processes contributing to neonatal brain injury and mental retardation. The continuation of three interrelated projects is planned. They all concern the influence of overstimulation of the N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) leading to neuronal damage during hypoxic-ischemic brain injury and other insults to the brain. Hypoxic-ischemic insults are known to result in mental retardation and developmental delay. This group of program investigators has shown that at least part of this damage to the nervous system appears to be mediated by excessive NMDAR activation that is not adequately treated by currently available therapeutic regimens. This program project developed the first clinically tolerated NMDAR antagonist, memantine, which the investigators showed is an uncompetitive, open-channel blocker. Here by studying NMDAR structure/function, they will develop improved derivatives of memantine. These drugs are known as the NO-memantines, and are more effective neuroprotectants than memantine because they target, via memantine, NO species to a nitrosylation site on NMDARs to further downregulate excessive activity. Additionally, the investigators have cloned and are studying a new family of NMDAR subunits (the NR3 family comprised of NR3A and NR3B) that are also neuroprotective during neonatal development, in some sense mimicking the effect of the drugs. They are studying NR3 using a range of multidisciplinary approaches with (i) recombinant receptors (using site-directed mutagenesis, electrophysiology, and crystallography of subunit proteins in Project I), (ii) cell biology of primary neurons (patch-clamp and neurodegenerative studies in Project II), and (iii) systems biology (electrophysiology and molecule expression of gene-targeted mice in Project III). In addition, novel NO-memantines will be tested in animal models of hypoxic-ischemic brain injury in Project II. The core supports administration, statistics, tissue culture, and crystallography/modeling of NMDAR subunits and functional sites. [unreadable] [unreadable]

AIDS can produce substantial dysfunction in the central nervous system (CNS), including encephalopathy/dementia by affecting the brain, myelopathy by affecting the spinal cord, and visual loss by affecting the retina. These adverse effects can occur in the apparent absence of infectivity of neurons by HIV-1, and even in the absence of superinfection with other opportunistic organisms. Therefore, one possibility is that HIV-1 somehow produces a toxic agent that injures neurons. One molecule that is a candidate as a toxic product of HIV-1 is the envelope protein gpl20, since it is shed from the virus. For the current proposal, 3 tissue culture models of CNS injury will be used, comprised of (i) postnatal rodent retinal ganglion cells, (ii) rodent hippocampal cortex neurons, and (iii) human cortical neurons (obtained from neurosurgical specimens). In preliminary studies on rat retinal ganglion cell and hippocampal neurons, gpl2O, in picomolar amounts and in a dose-dependent fashion, induces a dramatic increase in intracellular [Ca2+]; in retinal ganglion cells this rise in [Ca2+] is associated with neuronal injury and even cell death. Additional preliminary experiments have demonstrated that calcium channel antagonists are capable of totally preventing this form of neurotoxicity caused by the HIV-1 envelope protein. Studies are proposed to test if these toxic effects apply to other types of neurons, including human cortical neurons, and to elucidate further the mode of action of gpl2O on intracellular [Ca2+] using calcium imaging with the dye fura-2, patch-clamp recording, and concomitant viability assays. The specific aims of the proposed study are -- 1 . To monitor the level of free calcium in retinal ganglion cell and cortical neurons in vitro after exposure to HIV-1 envelope protein gpl 20; rodent and human neurons will be used. 2. To assess injury to these neurons after exposure to gpl20 in vitro using a well-developed test of viability, the uptake and cleavage of fluorescein diacetate; to assess the protective effects from gpl 20 neurotoxicity of a variety of calcium channel antagonists by generating dose-response curves. 3. To perform patch-clamp recordings of these neurons at both the whole-cell and single-channel levels to determine, as suggested in preliminary experiments, if gpl20 affects a specific type of calcium channel; to study the mechanism of this effect.

The neurological manifestations of HIV-1 affect between one and two- thirds of adults patients with AIDS. Included in these complications are a form of dementia producing cognitive, motor, and possible visual dysfunction in the absences of viral infection of neurons, in the absence of opportunistic superinfections, and in the absence of HIV-associated malignancies of the CNS. Recent progress has been made in the laboratory investigation of the basis for this form of dementia (termed the AIDS dementia complex, or more recently, HIV-associated motor/cognitive complex). Evidence from a variety of laboratories around the world suggests that at least part of the neuronal loss observed in the brains of patients with AIDS may be related to a final common pathway invoking excessive stimulation of excitatory amino acid receptors such as the N- methyl-D-aspartate (NMDA)subtype of these receptors. These findings are in the mainstream of current neuroscience research which has found that several acute and degenerative neurologic disorders, ranging from stroke to trauma and epilepsy to Huntington's disease, may have a similar basis for neuronal injury. In the face of overstimulation of excitatory amino acid receptors, ion channels permit excessive influx of calcium ions and consequent nerve cell injury. Although the exact mechanism for neuronal injury by calcium overload is still a matter of intense investigation and current debate, many laboratories have found that limiting the influx of calcium ions under these conditions can protect neurons form injury. For example, laboratory investigation using in vitro and in vivo animal models has suggested that blockade of ion channels permeable to calcium ions may prevent nerve cell damage engendered by HIV-infected macrophages or macrophages stimulated by the HIV-1 coat protein, gp120. At least two types of ion channels contribute to this form of injury, L-type voltage- dependent calcium channels and NMDA receptor-coupled channels. For this reason, clinically-tolerated antagonists of these channels are proposed to be studied in conjunction with the best available anti-retroviral therapy, i.e. zidovudine (ZDV) or nucleosides such as dideoxyinosine (ddI). A second study will be used an adjunctive therapy to these anti- retroviral another well-known drug, memantine, which has recently been recognized to be a potent NMDA open-channel blocker capable of attenuating neuronal injury associated with exposure to gp120 by the P.I.'s laboratory.

AIDS can produce substantial dysfunction in the central nervous system, including subcortical areas, cortex and retina. These adverse effects may occur in the absence of superinfection with opportunistic organisms or productive HIV-1 infection of neurons. Therefore, one possibility is that HIV-1 somehow produces a toxic agent(s) that injures neurons, such as retinal ganglion cells and cortical neurons. Along these lines, HIV-1 coat protein gp120 has been found by this laboratory to increase intracellular free calcium ([Ca2+]) and subsequently to injure cultured primary central neurons, including retinal ganglion cells, in mixed neuronal/glial cultures. Studies from our laboratory indicated that the N-methyl-D- aspartate (NMDA) subtype of glutamate receptor is involved in gp120-induced toxicity. Furthermore, from work in our laboratory and in others, it has become evident recently that at least one pathway to neuronal injury is mediated by toxic factors that are secreted by either HIV-infected or gp120-stimulated macrophages. These putative toxic factors include cysteine, platelet-activating factor (PAF), and arachidonic acid and its metabolites. In this proposal, we plan to characterize further these putative neurotoxic factors. First, we will characterize the release of the NMDA-like neurotoxin, cysteine, from gp120-stimulated human macrophages; gp 120-induced release of cysteine has been documented in the preliminary experiments. Second, we will perform patch-clamp recordings and digital calcium imaging to determine if the putative macrophage- released toxic factors cysteine and PAF directly or indirectly activate NMDA receptor channels (or other membrane ion channels). Cysteine apparently acts as an NMDA agonist while PAF is thought to increase glutamate release from neurons. We will also determine if gp120-induced arachidonic acid production from macrophages affects glutamate uptake/efflux in both astrocytes and synaptosomes, thereby increasing the local concentration of glutamate and hence neurotoxicity. Next, with conventional neurotoxicity assays, drugs that have been shown to down- regulate NMDA receptor activity by oxidizing the receptor's redox modulatory site, a site discovered in this laboratory, will be tested to prevent the neuronal injury induced by these toxic factors. These drugs include nitroso-compounds that produce redox congeners of nitric oxide, such as nitroglycerin (NTG) and sodium nitroprusside (SNP). We will also test other clinically tolerated NMDA antagonists whose mechanism of action has been studies by this laboratory. These drugs include the adamantanes, such as memantine and novel congeners, which produce open-channel block of the NMDA receptor-operated ion channel. Finally, PAF antagonists will be used to try to abrogate neurotoxicity in culture to implicate PAF in at least one HIV-related pathway to neuronal injury. These in vitro studies will use rat retinal ganglion cells and cortical neurons as well as human fetal cortical neurons. The long-term objectives of this study are to better understand the pathways and mechanisms to HIV-induced neuronal injury in order to lead to possible treatments of the neurological manifestations of AIDS.

In Project, we will test two types of clinically-safe N-methyl-D-aspartate receptor (NMDAR) antagonists for their ability to prevent hypoxic-ischemic damage relevant to mental retardation. In these studies we will attempt to ameliorate NMDAR-mediated neurotoxicity first in vitro and subsequently in vivo in stroke models with therapy initiated greater than or equal to 2 hours after the insult. First, open-challenge NMDA blockers (adamantanes) will be tested; one of these drugs, memantine, is currently in clinical use in Europe for other disorders such as Parkinson's disease and spasticity. Second to be tested is nitroglycerin (NTG), which generates a redox-related form of nitric oxide that NO-related species is transferred to cysteine sulfhydryl groups of the NMDAR, a reaction termed S-nitrosylation. Additionally, a series of novel compounds, nitro-memantines, combining the features of NO+ transfer with open-channel block, are being developed. These combinatorial drugs will target NO+ to the NMDAR via memantine and thus avoid systemic side effects of NO such as hypotension. These combinatorial drugs will target NO+ to the NMDAR via memantine and thus avoid systemic side effects of NO such as hypotension. These studies with novel and potentially safe NMDA antagonists have important implications for the treatment of mental retardation and developmental disabilities due to overstimulation of glutamate receptors. To try to extend the potential window of treatment after a stroke, we will attempt to intervene therapeutically in apoptotic pathways activated with a delay after excessive NMDAR stimulation. The pathways studied here involve the action of caspases in the intracellular signaling cascade leading to neuronal apoptosis. Inhibition of caspase activity pharmacologically and with a caspase dominant-negative transgenic mouse will be use din conjunction with fluorescent indicators for Ca2+, reactive oxygen species, mitochondrial membrane depolarization, and lipid peroxidation in an attempt to decipher where caspases act in these signaling pathways that lead to hypoxic/ischemic neuronal damage. According, the Specific Aims of Project II are-2. To test drugs that we hypothesize nitrosylate the NMDAR to prevent hypoxic-ischemic neurotoxicity, including nitroglycerin (NTG) and novel nitro-memantine drugs that target NO+ to the NMDAR to avoid systemic side effects such as hypotension. 3. To characterize the action of caspases on intracellular signaling in excitotoxin-induced neuronal apoptosis.

neurosciences; biomedical facility; tissue /cell culture; electron spin resonance spectroscopy; magnetic resonance imaging;

virus; mental disorders; proteins; AIDS; immunology; human tissue; communicable diseases; lymphatic system; biomedical resource;

In many neurological disorders, injury to neurons may be caused, at least in part, by overstimulation of receptors for excitatory amino acids, including glutamate, aspartate and related congeners. These neurological conditions range from acute insults such as stroke, hypoglycemia, trauma, and epilepsy, to chronic neurodegenerative states such as Huntington's disease, AIDS dementia complex, amyotrophic lateral sclerosis, and perhaps Alzheimer's disease. Glutamate is the major excitatory neurotransinitter in the brain and as such its interactions with specific membrane receptors are responsible for many normal neurological functions including cognition, memory, movement, and sensation. In addition, excitatory neurotransmitters are important in shaping the developmental plasticity of synaptic connections in the nervous system. However, in a variety of pathological conditions, including stroke and various neurodegenerative disorders, excessive activation of glutamate receptors may mediate neuronal injury or death. Olney coined the term 'excitotoxicity' for this condition, which may constitute a final common pathway for neuronal injury from diseases of diverse pathophysiology. This form of injury appears to be predominantly mediated by excessive influx of Ca2+ into neurons through ionic channels triggered by activation of glutamate receptors. An important point concerning these glutarnate receptor subtypes is that the N-methel-D-aspartate (NMDA) subtype of glutamate receptoractivated channel permits the influx of Ca2+ as well Na as+9 and overstimulation of this type of receptor is thought to be the predominant mechanism for calcium overload in neurons. Ca2+ influx triggered via NMDA receptor stimulation activates a variety of enzymes including nitric oxide synthase and the consequent production of nitric oxide. When NMDA receptors are excessively stimulated, nitric oxide may be produced in increased quantities. Under these conditions, NO- and 02'- may react to form a toxic substance called peroxynitrite (ONOO-), resulting in neuronal death In contrast, nitric oxide can be converted to a different chemical state that has just the opposite effect, protecting neurons from injury due to NMDA receptor overstimulation. The chemical state is dependent upon the removal or addition of an electron to nitric oxide, a condition that can be influenced by the presence or absence of electron donors, such as ascorbate or the amino acid cysteine. For example, with one less electron, NOmay yield a substance with NO+ (nitrosonium) character. In this form, the NO group appears to be transferred to a regulatory site on the NMDA receptor, termed the redox modulatory site. This site is comprised of sulfhydryl (-SH) groups; the reaction of -S- with NO+ to form -SNO (a process called Snitrosylation) results in decreased activity of the NMDA receptor, thus affording protection from excessive stimulation. Therefore, depending on its chemical state, the NO moiety can lead to neurodestruction or neuroprotection. These findings have lead to therapeutic approaches to decrease NMDA receptor overactivity using drugs with NO+ character, such as nitroglycerin. NMDA Redox Modulatory Site The motivating force behind the work proposed here is to characterize the cysteine residues comprising the redox modulatory site(s) of the NMDA receptor with respect to their interaction with the NO group. Work from our laboratory with recombinant NMDA receptor subunits has shown that two cysteine residues on the NMDARI subunit of the NMDA receptor are involved in redox modulation (cysteine residues 744 and 798; Fig .1, below). In collaboration with the Chait laboratory, we propose to use electrospray ionization mass spectrometry to visualize S-nitrosylatoin of the NMDARI subunit, to determine if intra- or intermolecular disulfide bond formation subsequently occurs, and to correlate these findings with the know regulatory activity of these reactions on the NMDA receptor from our patch-clamp electrophysiological studies.

The neurological manifestations of AIDS affect approximately half of the children infected with HIV-1 and perhaps on-quarter to one-third of adults. Many children with AIDS display delayed milestones and even frank cognitive and motor decline, causing substantial development disabilities and mental retardation. Quite apart from super-infections with opportunistic organisms and malignancy, at least some degree in the CNS in AIDS appears to be most closely associated with toxins released by brain macrophages after they have been infected by HIV-1 or stimulated by its coat protein, gp120. Neurons are injured in this process and may undergo apoptosis, and at least part of their damage is accounted for by the macrophage and possibly astrocyte toxic factors leading to over-excitation of glutamate receptors, especially of the N-methyl-D-aspartate. (NMDA) subtype. Therefore, in Project, our group of investigators studies the effects of clinically-tolerated NMDA antagonists in preventing the neuronal injury engendered by gp120 in the following model systems: (a) in vitro in rodent cerebrocortical cultures, (b) in vivo in a rodent retinal model using intravitreal injection of gp120, (c) in vivo in a gp120- transgenic mouse model, and (d) in vivo in a SCID (subacute combined immunodeficiency) mouse model of AIDS dementia produced by intracerebral injection of HIV-infected human monocytes. Here we propose to use the clinically-tolerated NMDA antagonists developed in Projects. We will also study apoptotic signaling pathways that may be present in AIDS brains, including those mediated by nitric oxide and caspases (similar to pathways triggered by NMDA and hypoxia-ischemia in Project). To accomplish these goals, transgenic and knockout mice that disrupt these signaling pathways will be used for the intravitreal injection of gp120 or will be crossed with gp120-transgenic mice. The resulting gp120-injected or bigenic mice will be analyzed histologically by confocal laser scanning microscopy and by magnetic resonance spectroscopy (MRS) in the Program's CORE facilities to determine if disruption of these signalling pathways ameliorates gp120-induced damage. It is anticipated that these preclinical studies investigating the role of the NMDA receptor and it signaling pathways to neuronal cell injury may lead to new treatment of the neurological manifestations of AIDS. In the previous grant period, this has already been realized to some extent with one of the NMDA receptor antagonist developed by this Program Project (memantine) going into a nation-wide clinical trial for AIDS dementia.

AIDS can produce substantial dysfunction in the central nervous system, including subcortical areas, cortex and retina. These adverse effects may occur in the absence of superinfection with opportunistic organisms or productive HIV-1 infection of neurons. Therefore, one possibility is that HIV-1 somehow produces a toxic agent(s) that injures neurons, such as retinal ganglion cells and cortical neurons. Along these lines, HIV-1 coat protein gp120 has been found by this laboratory to increase intracellular free calcium ([Ca2+]) and subsequently to injure cultured primary central neurons, including retinal ganglion cells, in mixed neuronal/glial cultures. Studies from our laboratory indicated that the N-methyl-D- aspartate (NMDA) subtype of glutamate receptor is involved in gp120-induced toxicity. Furthermore, from work in our laboratory and in others, it has become evident recently that at least one pathway to neuronal injury is mediated by toxic factors that are secreted by either HIV-infected or gp120-stimulated macrophages. These putative toxic factors include cysteine, platelet-activating factor (PAF), and arachidonic acid and its metabolites. In this proposal, we plan to characterize further these putative neurotoxic factors. First, we will characterize the release of the NMDA-like neurotoxin, cysteine, from gp120-stimulated human macrophages; gp 120-induced release of cysteine has been documented in the preliminary experiments. Second, we will perform patch-clamp recordings and digital calcium imaging to determine if the putative macrophage- released toxic factors cysteine and PAF directly or indirectly activate NMDA receptor channels (or other membrane ion channels). Cysteine apparently acts as an NMDA agonist while PAF is thought to increase glutamate release from neurons. We will also determine if gp120-induced arachidonic acid production from macrophages affects glutamate uptake/efflux in both astrocytes and synaptosomes, thereby increasing the local concentration of glutamate and hence neurotoxicity. Next, with conventional neurotoxicity assays, drugs that have been shown to down- regulate NMDA receptor activity by oxidizing the receptor's redox modulatory site, a site discovered in this laboratory, will be tested to prevent the neuronal injury induced by these toxic factors. These drugs include nitroso-compounds that produce redox congeners of nitric oxide, such as nitroglycerin (NTG) and sodium nitroprusside (SNP). We will also test other clinically tolerated NMDA antagonists whose mechanism of action has been studies by this laboratory. These drugs include the adamantanes, such as memantine and novel congeners, which produce open-channel block of the NMDA receptor-operated ion channel. Finally, PAF antagonists will be used to try to abrogate neurotoxicity in culture to implicate PAF in at least one HIV-related pathway to neuronal injury. These in vitro studies will use rat retinal ganglion cells and cortical neurons as well as human fetal cortical neurons. The long-term objectives of this study are to better understand the pathways and mechanisms to HIV-induced neuronal injury in order to lead to possible treatments of the neurological manifestations of AIDS.

tissue /cell culture

The Society for Neuroscience is the major professional organization for scientists who study the nervous system. An important goal of this organization is to encourage scientists in training to undertake research related to diseases of the nervous system. The objective of this grant application is to support teaching workshops that introduce young neuroscientists to current concepts about the etiology and pathogenesis of disorders of the nervous system. For each workshop, about 12 faculty are chosen by the organizing committee. Clinical presentations provide enrollees with an experience of the human dimension of particular diseases. Lectures cover both clinical research and relevant laboratory work. In addition to lectures, enrollees are given a choice of small group workshops that emphasize either specific conceptual or methodological issues. Since its inception, sixteen workshops have been held, usually on the day prior to the start of the Society for Neuroscience Meeting. Topics have included: Stroke, AIDS in the nervous system, Epilepsy, Huntington's and Parkinson's disease, Muscular Dystrophy, Multiple Sclerosis, Prion diseases, Drug Addiction, Pain and Affective Disorders, Stroke and Excitotoxicity, Neuromuscular diseases, Amyotrophic Lateral Sclerosis, Schizophrenia, Migraine, Mental Retardation and developmental disorders. Enrollment generally runs between 100 and 200 attendees. Most enrollees are graduate students or postdoctoral fellows. Current plans are to cover the following topics in the near future: Tourette's Syndrome and Obsessive-Compulsive Disorder, the neurobiology of brain tumors, AIDS Dementia, Peripheral Neuropathy, Pain, Language Disorders, and Affective Disorders. Other topics will be chosen depending on their potential interest to young neuroscientists, their impact on society, and the quality of recent research related to that disease area. We are especially interested in covering diseases of the nervous system that are important clinically, but which are in need of enhanced basic cellular and molecular understanding.

Description (adapted from applicant's abstract): This studies outlined in this application are based on the general hypothesis that "engagement of chemokine receptors by gp120 directly on neurons and astrocytes is insufficient to cause neurodegeneration under pathologically relevant conditions, but may facilitate the deleterious effects of activated microglia/M by modulating, for example, the excitability and calcium levels of neurons." The five specific aims organize five approaches and sets of experiments: 1. To determine if alpha- or beta-chemokines ameliorate gp120-induced neuronal apoptosis in 'mixed' neuronal/glial rodent and human retinal and cerebrocortical cultures. Both R5 and XR gp120s will be used in these cell culture studies. 2. To assess whether gp120-induced injury is predominantly mediated via microglia/M release of neurotoxins, by direct action on neurons or by both mechanisms; these distinctions will be examined by blocking M using threonine-lysine-proline (TKP) tripeptide. 3. To determine whether chemokines directly or indirectly signal neurons via G-protein pathways using fluorescent dyes to study calcium flux, and also to see if chemokine signaling interferes with gp120-induced signaling as well as monitor intracellular signaling via the JNK, ERK and p38 PK pathways. 4. Use of the gp120 transgenic mouse to determine whether chronic intracerebral injection of TKP affords local neuroprotection, thereby implicating M mediation. 5. Use neuronal cultures derived from CXCR4- and CCR5 knockout mice to assess the necessity of these receptors on in vitro gp120-induced toxicity.

The Core has two components. The Developmental Grants Program provides HNRC resources and/or funds small studies to recruit new investigators and generate pilot data for larger proposals in neuroAIDS research. An external Developmental Grants Committee will review proposals semi-annually and award funds up to $25,000 for 1-2 year efforts. Junior investigators will be targeted for funding, but senior faculty without prior HIV research experience also may apply. The Mentored Investigator Program encourages and monitors trainees affiliated with HNRC faculty who pursue research in neuroAIDS. Support in the form of access to HNRC resources, small grants, and regular monitoring of progress will formalize a program which has had considerable success in training pre- and post-doctoral fellows over the past decade.

The Society for Neuroscience (SFN) is the major professional organization for scientists who study the nervous system. An important goal of this organization is to encourage scientists in training to undertake research related to diseases of the nervous system. The objective of this grant application is to support teaching workshops that introduce young neuroscientists to current concepts about the etiology and pathogenesis of disorders of the nervous system. For each workshop, about 12 faculty are chosen by the Organizing Committee after eliciting proposals from the Society at large. Clinical presentations provide enrollees with an experience of the human dimension of particular diseases. Lectures cover both clinical research and relevant laboratory work. In addition to lectures, enrollees are given a choice of attending two of four small group workshops that emphasize either specific or methodological issues and encourage lively discussion. Since its inception, 20 workshops have been held, usually on the day prior to the start of the Society for Neuroscience meeting. Topics have included: Infections in the nervous system, epilepsy, Huntington's and Alzheimer's diseases, muscular dystrophy, multiple sclerosis, prion diseases, drug addiction, pain and affective disorders, stroke and excitotoxicity, neuromuscular diseases, amyotrophic lateral sclerosis, schizophrenia, migraine, mental retardation and developmental disorders, Tourette's syndrome and obsessive-compulsive disorder, and the neurobiology of brain tumors. Enrollment generally runs between 100 and 200 attendees. Most enrollees are graduate students or postdoctoral fellows. Current plans are to cover the following topics in the near future: Genes, free radicals, mitochondria and apoptosis in Parkinson's disease, AIDS dementia, peripheral neuropathy, pain, language disorders, and affective disorders. Other topics will be chosen depending on their potential interest to young neuroscientists, their impact on society and the quality of recent research related to that disease area. We are especially interested in covering diseases of the nervous system which are important clinically but which are in need of enhanced basic cellular and molecular understanding. Society members are encouraged to suggest topics in the SFN Newsletter.

A quarter to a third of HIV-1 infected individuals eventually develop HIV-Associated Dementia (HAD). Neuronal injury and apoptosis may contribute to the cognitive deficits of HAD. In vitro and in vivo, the HIV-1 envelope glycoprotein gp120 produces injury and apoptosis in both human and rodent neurons, possibly via macrophage activation, and this may account for at least a part of the neuronal damage observed in HAD. Microglia, astrocytes and neurons express co-receptors for HIV-1, namely the chemokine receptors CCR5 and CXCR4. Using chemokine receptor knock-out mice, the objectives of this application are to further elucidate the role of microglia/macrophages in HIV-related neuronal damage, and to study the cellular signaling mechanisms affected by HIV-1 gp120 and chemokines in the brain. Such mechanisms may provide future therapeutic targets and thus new treatments for HAD. The following Specific Aims are proposed 1. To assess which chemokines and chemokine receptors are able to mediate or inhibit gp120-induced neuronal injury in mixed neuronal/glial mouse cerebrocortical cultures. 2. To assess if chemokine receptor activation on glial cells (macrophages/microglia or astrocytes), on neurons, or on both are necessary for neuronal apoptosis due to gp120. For the first two specific aims, both macrophage-tropic/R5 (CCR5- preferring) and T-cell tropic/X4 (CXCR4-preferring) gp120s will be tested in these experiments. To accomplish these aims, recombinant gp120 will be incubated in neuronal cultures produced from CXCR4(-/-) or CCR5(-/-) "knock out" mice in the presence and absence of wild-type microglia and astrocytes containing CXCR4 and CCR5 receptors. Alternatively, wild-type neurons will be incubated with microglia (or astrocytes) isolated from mice lacking these chemokine receptors. 3. To investigate in post mortem human brain tissue (from both demented and non-demented HIV-positive patients) if the appearance and concentration of distinct chemokines, such as SDF- 1 and fractalkine, are correlated with tissue damage and cognitive decline. Chemokines levels will be quantified by ELISA of brain extrancts. Additionally, potential signaling pathways will be assessed in microglia, astrocytes and neurons from the chemokine receptor knock-out mice (versus cells from wild-type mice); activation of the JNK, ERK and p38 mitogen-activated protein kinase pathways will be monitored by immunoblotting and immunocomplex protein kinase assays. Neuronal apoptosis due to gp120 or chemokines will be monitored using several approaches.

DESCRIPTION (Adapted from applicant's abstract): Erythropoietin (EPO), a kidney cytokine regulating hematopoiesis, is also produced in the brain after oxidative/nitrosative stress. Hypoxia inducible transcription factor-1 (HIF-1) upregulates EPO following hypoxic stimuli. Here we propose to study preconditioning with EPO to show that it protects neurons in models of ischemic and degenerative damage due to excitotoxins and consequent generation of free radicals, including nitric oxide (NO). We propose to show that activation of neuronal EPO receptors (EPO-Rs) prevents N-methyl-D aspartate (NMDA)- and NO-induced apoptosis by triggering cross talk between the Janus kinase-2 (Jak2) and nuclear factor KB (NF-KB) signaling pathways. EPO-R - mediated activation of Jak2 leads to phosphorylation of the inhibitor of NF-KB (IKB), subsequent nuclear translocation of the transcription factor NF-KB, and NF-KB-dependent transcription of neuroprotective genes. Transfection of cerebrocortical neurons with a dominant -interfering form of Jak2 or an IKB superrepressor blocks EPO-mediated prevention of neuronal apoptosis. Thus neuronal EPO-Rs activate a neuroprotective pathway that is distinct from previously well characterized Jak and NF-KB functions. Moreover, this EPO effect may underlie neuroprotection mediated by hypoxic-ischemic preconditioning. To test this postulate, we will examine the neuroprotective properties of EPO-related molecules in a mouse middle cerebral artery occlusion model of stroke using the intraluminal suture method. The Specific Aims are as follows: To characterize EPO- induced activation of the NF-KB pathway in neuroprotection. 2. To test whether NF-KB activation following EPO exposure in mixed neuronal-glial cerebrocortical cultures occurs primarily in neurons. 3. To investigate the possibility of a direct role of EPO-activated Jak2 in NF-KB signaling. 4. To study the treatment of stroke in a mouse model with EPO-related molecules. As a proof-of principle of the involvement f o EPO signaling, we will also test these drugs as well as the effect of increased endogenous EPO in transgenic mice expressing a truncated WPO-R that is hypersensitive to EPO-induced Jak2 signaling compared to the normal full-length receptor.

DESCRIPTION (provided by applicant): Apoptotic neuronal cell death may play a role in many acute and chronic neurologic disorders. These disorders range from acute stroke, head trauma and epilepsy to more chronic states, such as Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis, HIV-associated dementia, and glaucoma. Moreover, a contributing factor to such damage is excessive excitation of glutamate receptors, particularly (but not exclusively) the N-methyI-D-aspartate (NMDA) subtype of glutamate receptor because of its high permeability to Ca 2+ and subsequent free radical generation. The aim of this proposed research project is to uncover the role of myocyte enhancer factor-2 (MEF2) transcription factors in this excitotoxic/apoptotic process in neurons during ischemic stroke in vivo. MEF2 transcription factors are activated by p38 mitogen-activated protein kinase during neuronal and myogenic differentiation. Recent work has shown that stimulation of this pathway is anti-apoptotic in stem cells but pro-apoptotic in mature neurons exposed to mild excitotoxic or other stresses. Here, preliminary data in vitro show that mild excitotoxic (NMDA) insults to mature cerebrocortical neurons activate caspases-3, -7, in turn cleaving MEF2A, C and D isoforms. Endogenous MEF2 cleavage fragments containing a truncated transactivation domain but preserved DNA binding domain are shown to block MEF2 transcriptional activity via dominant interference. In vitro transfection of constitutively-active/uncleavable MEF2 (MEF2-CA) rescues MEF2 transcriptional activity following NMDA insult and prevents neuronal apoptosis. Conversely, dominant-interfering MEF2 (MEF2-DN) abrogates neuroprotection by MEF2C-CA. Our underlying hypothesis is that these results obtained in vitro can now be applied in vivo using tetracycline (or doxycycline, "dox")-controlled transgenic mice expressing these MEF2-CA and MEF2-DN transgenes. This grant will define a novel pathway to neuronal apoptosis in ischemia via caspase-catalyzed cleavage of MEF2. The Specific Aims are as follows: 1. To characterize anti-apoptotic effects of MEF2-CA in stroke using dox-controlled transgenic mice. 2. To characterize the effect of caspase cleavage fragments of MEF2 as dominant interfering forms that contribute to stroke damage using dox-controlled transgenic mice that express doxycycline-controlled, MEF2 cleavage products. 3. To characterize MEF2 transcriptional activity in vivo after an hypoxic/ischemic (stroke) insult but prior to cell loss using a MEF2-indicator mouse that has been engineered to activate the LacZ gene in accord with the degree of MEF2 transcriptional activity (designated des-mef2-LacZ).

Our laboratory and others have proposed that neurodegeneration as a consequence of HIV-1 infection in the brain is predominantly dependent on the release of toxins by HIV-infected or gp120-activated microglia and macrophages. These toxins produce an excitotoxic injury via oxidative/nitrosative stress, and result in neuropathological changes, including dendritic injury, synaptic damage, and neuronal apoptosis. In attempting to prevent this type of HIV-related neurotoxicity, we discovered that erythropoietin (EPO), normally thought to be a kidney-generated cytokine, is made in the brain and is neuroprotective. Additionally, we and others found that insulin-like growth factor-I (IGF-I) is also neuroprotective. Here we report our preliminary data that EPO and IGF-I can prevent neuronal damage due to gp120 in a synergistic fashion in vitro, we demonstrate the likely molecular mechanism for this neuroprotective synergy, and we propose to study this effect in the HIV/gp 120-transgenic mouse as in vivo proof-of-principle to expedite human clinical trials with these clinically-tolerated agents. Specific Aims: 1. To assess the possible neuroprotective effect of EPO in the HIV/gp120 transgenic mouse model. Hypothesis Tested: EPO protects neurons from HIV-related neuronal damage via a phosphoinositide (PI) 3 kinase/Akt (protein kinase B)-mediated transduction pathway acting, at least in part, through glycogen synthase kinase 3beta (GSK3beta) to prevent the hyperphosphorylation of tan. In contrast to EPO, gp120 decreases Akt phosphorylation. 2. To assess the possible neuroprotective effect of IGF-I in the HIV/gp120 transgenic mouse model. Hypothesis Tested: IGF-I also protects neurons via the PI3 kinase/Akt-mediated transduction pathway acting, at least in part, through GSK3beta to prevent the hyperphosphorylation of tau. 3. To assess the possible synergistic neuroprotective effect of EPO+IGF-I in the HIV/gp120 transgenic mouse model. Hypothesis Tested: EPO+IGF-I act synergistically to activate the anti-apoptotic PI3 kinase/Akt signaling pathway, and thus provide a synergistic degree of neuroprotection.

Botulinum toxins (BoNTs) are among the most potent toxins known, thus defining it as a Class A bio-terrorism threat by NIAID. Intoxication with BoNT leads to flaccid paralysis, respiratory arrest, and death by blocking acetylcholine release at neuromuscular junctions. BoNTs mediate paralysis by cleaving molecules found in synaptic terminals, synaptosomal-associated protein of 25 kD (SNAP-25), vesicle-associated membrane protein (VAMP), or syntaxin. Early treatment with humanized antibodies can prevent progression and death by BoNT intoxication. However, many surviving patients exhibit a persistent or chronic fatigue syndrome. Unfortunately, not much is known about the cellular and molecular mechanism of this fatigue syndrome. A prime hypothesis here is that this disabling fatigue syndrome is caused by cell injury and death of motoneurons and that different BoNT serotypes have differential effects in mediating this newly recognized neurodegenerative disorder. Thus the goal of this project will be to gain new insight into how BoNTs mediate motoneuron cell death. To achieve this goal, primary motoneurons will be analyzed using approaches such as cell biology, immunocytochemistry, time-lapse deconvolution microscopy, electron microscopy, and gene and peptide transfer. Among the specific questions that will be addressed are: (1) How does BoNT/C but not BoNT/A induce damage to synaptic endings and neurites, eventually resulting in motoneuron degeneration? (2) What are the real-time changes in synaptic endings and neurites, cytoskeleton, and mitochondria linked to BoNT-induced motoneuron cell death? (3) Does cleavage of SNAP-25, syntaxin, or synaptobrevin/VAMP suffice to induce motoneuron damage and cell death initiated by BoNTs? Results obtained here will in general lead to a better understanding of the mode of action of BoNTs. Most importantly, new insights gained here will set the foundation to treat and prevent long-term, devastating neurological effects linked to BoNT-mediated intoxication after a bioterrorism attack.

DESCRIPTION (provided by applicant): Evidence is accumulating that damage and apoptotic death of neurons plays a role in a large number of neurologic disorders, including HIV-associated dementia (HAD), Alzheimer's disease, multiple sclerosis, stroke, and glaucoma. A contributing factor to these diseases is the activation of matrix metalloproteinases (MMPs) in the extracellular matrix. We hypothesize that this leads to a previously unrecognized extracellular signaling pathway contributing to neuronal cell injury and death. However, the mechanism of activation of MMPs in these disease processes, and specifically in HAD, remains a mystery. Here we will study whether nitric oxide (NO)-related molecules, another factor in neurodegeneration in HAD, can trigger the activation of MMPs and thus initiate an extracellular proteolytic cascade contributing to neuronal damage and apoptosis. Specific Aims: 1. To characterize the chemical nature of NO-activation of MMPs (-2 and -9) in vivo in the gp120-transgenic mouse model of HIV-related CNS damage and in human postmortem samples of HAD brain. Hypothesis tested: NO-activation of MMPs occurs via S-nitrosylation followed by further oxidation of the critical thiol group to irreversible sulfinic and sulfonic acid derivatives during HAD. 2. To determine if NO-activation of MMPs leads to laminin degradation and subsequent neuronal cell injury and death in vitro and in vivo. Hypothesis tested: NO generation and subsequent activation of MMPs can contribute to neuronal cell injury and death by an extracellular proteolysis cascade involving, at least in part, laminin degradation. In the future, the work proposed here may lead to new therapeutic targets based on the novel extracellular signaling pathway involving NO-related molecules and the MMPs that will be studied.

Overstimulation of glutamate receptors, especially the N-methyl-D-aspartate receptor (NMDAR), has been associated with neuronal cell injury and death following hypoxic-ischemic, neurodegenerative, and trauma-related insults that can lead to mental retardation and developmental disabilities. Previously, this Program Project Grant demonstrated that the NMDAR antagonist, Memantine, was a clinically-tolerated yet effective neuroprotectant because of its unique mode of action as an uncompetitive, low-affinity, rapid off-rate, open-channel blocker. Subsequently, Memantine, was shown to be clinically useful but only of moderate benefit. In Project II, we will test new, more effective NMDAR antagonists, the NO-Memantines, that are being developed in collaboration with Project I. These agents combine the channel blocking effect of Memantine with S-nitrosylation (targeted delivery of the NO group to critical cysteine thiols on the NMDAR to downregulate excessive activity). We will test these drugs for their ability to prevent hypoxic-ischemic neuronal damage. In these studies, we will attempt to ameliorate NMDAR-mediated neurotoxicity first in vitro and subsequently in vivo in rodent stroke models in a developmental fashion, in both neonates and in young adults. We will also perform extensive safety studies to ensure that we develop clinically-tolerated drugs since the NMDAR plays roles in both normal and abnormal brain activity. Additionally, we will study the effect on primary neurons of a new family of NMDAR subunits that was recently cloned by this P01, designated NR3A/3B. These subunits in some sense mimic the NMDAR antagonist drugs since expression of NR3 subunits in combination with the classical NR1/NR2 subunits decreases NMDAR activity, and may thus offer neuroprotection in early development when NR3A expression is predominant. Our proposed studies with novel NMDAR antagonists have important implications for the treatment of hypoxic-ischemic brain injury and various neurodegenerative disorders. Accordingly, the Specific Aims of Project II are the following: 1. To characterize the effects of NO-Memantines on excessive NMDA-evoked currents vs. physiological NMDAR-mediated synaptic activity (excitatory postsynaptic currents or EPSCs). 2. To test the ability of NO-Memantines to prevent NMDAR-mediated neurotoxicity in vitro and in vivo. 3. To study normal and pathophysiologically relevant actions of NR3A and 3B subunits in primary neurons in culture using patch-clamp recording techniques and neurotoxicity experiments.

The COREs will provide scientific support and statistical services exclusively for members of this program project. Dr. Stuart A. Lipton is responsible for overall scientific and administrative aspects of the CORE and will oversee budget, progress, and financial reports and ensure the scientific merit of the work performed under the program project grant. The scientific aspects of the CORE will be also overseen on a dayto- day basis by Dr. Dongxian Zhang. The CORE is divided into an Administrative/Statistical CORE (see B, below) and a Neuroscience Research CORE Facilities for Tissue Culture and for NMDAR Crystallography/Modeling (see C, below). In the Administrative/Statistical CORE, service is provided to all three projects for expertise of a trained statistician (5% time), who serves the needs of the projects as they design the specifics of their experiments and analyze their data. In the Neuroscience Research CORE, service is provided to all three projects for the following: (1) technical assistance in preparation of cells for tissue culture, and/or for (2) analysis of the effects of NMDAR antagonists in protecting the brain from hypoxic-ischemic insults. All projects will share in the services of this Neuroscience CORE

Our laboratory and others have proposed that neurodegeneration as a consequence of HIV-1 infection in the brain is predominantly dependent on the release of toxins by HIV-infected or gp120-activated microglia and macrophages. These toxins produce an excitotoxic injury via oxidative/nitrosative stress, and result in neuropathological changes, including dendritic injury, synaptic damage, and neuronal apoptosis. In attempting to prevent this type of HIV-related neurotoxicity, we discovered that erythropoietin (EPO), normally thought to be a kidney-generated cytokine, is made in the brain and is neuroprotective. Additionally, we and others found that insulin-like growth factor-I (IGF-I) is also neuroprotective. Here we report our preliminary data that EPO and IGF-I can prevent neuronal damage due to gp120 in a synergistic fashion in vitro, we demonstrate the likely molecular mechanism for this neuroprotective synergy, and we propose to study this effect in the HIV/gp 120-transgenic mouse as in vivo proof-of-principle to expedite human clinical trials with these clinically-tolerated agents. Specific Aims: 1. To assess the possible neuroprotective effect of EPO in the HIV/gp120 transgenic mouse model. Hypothesis Tested: EPO protects neurons from HIV-related neuronal damage via a phosphoinositide (PI) 3 kinase/Akt (protein kinase B)-mediated transduction pathway acting, at least in part, through glycogen synthase kinase 3beta (GSK3beta) to prevent the hyperphosphorylation of tan. In contrast to EPO, gp120 decreases Akt phosphorylation. 2. To assess the possible neuroprotective effect of IGF-I in the HIV/gp120 transgenic mouse model. Hypothesis Tested: IGF-I also protects neurons via the PI3 kinase/Akt-mediated transduction pathway acting, at least in part, through GSK3beta to prevent the hyperphosphorylation of tau. 3. To assess the possible synergistic neuroprotective effect of EPO+IGF-I in the HIV/gp120 transgenic mouse model. Hypothesis Tested: EPO+IGF-I act synergistically to activate the anti-apoptotic PI3 kinase/Akt signaling pathway, and thus provide a synergistic degree of neuroprotection.

DESCRIPTION (provided by applicant): The neurological manifestations of HIV-1 affect approximately one-quarter of adults with AIDS. In addition to super-infections with opportunistic organisms and malignancy, damage in the CNS in AIDS appears to be most closely associated with toxins released by brain microglia/macrophages that have been infected by HIV or stimulated by the viral coat protein, gp!20. Neurons themselves are most likely not infected, but undergo dendritic and synaptic injury and may succumb to apoptosis. At least part of this damage is accounted for by macrophage and possibly astrocyte toxic factors leading to overexcitation of glutamate receptors, especially of the W-methyl-D-aspartate subtype (NMDARs), as shown previously by this grant. Also, this grant helped develop the first clinically-tolerated NMDAR antagonist, Memantine, which led to phase 2 clinical trials for HIV-associated dementia. Here we propose to develop a class of NMDAR antagonists that are superior to Memantine. These new drugs are termed NO-Memantines and are being developed to prevent neuronal injury engendered by gp!20 in the following model systems: (a) in vitro in rodent cerebrocortical cultures, (b) in vivo in a rodent retinal model using intravitreal injection of gp!20 into the eye, and (c) in vivo in a gp120- transgenic mouse model. Additionally, our group has cloned and characterized a new family of NMDAR subunits, called NR3, which when combined with conventional NR1 and NR2 subunits, lead to decreased NMDAR activity, in a sense mimicking the effect of Memantine. Here we propose to study the effect of NR3 on gp!20-induced neuronal damage in similar models to those used for drug testing. We have produced NR3 knockout and transgenic mice to breed with gp!20-transgenic mice for this purpose. The resulting mice will be analyzed histologically by confocal/deconvolution microscopy and behaviorally for evidence of amelioration of gp!20-induced damage. This molecular approach using NR3 subunits will be used to validate the effect of drugs that inhibit excessive NMDAR activity to protect neurons from gp120-induced damage. It is anticipated that these preclinical studies investigating the role of the NMDAR in neuronal cell injury may lead to new treatments for the neurological manifestations of AIDS.

molecular /cellular imaging

Our laboratory and others have proposed that neurodegeneration as a consequence of HIV-1 infection in the brain is predominantly dependent on the release of toxins by HIV-infected or gp120-activated microglia and macrophages. These toxins produce an excitotoxic injury via oxidative/nitrosative stress, and result in neuropathological changes, including dendritic injury, synaptic damage, and neuronal apoptosis. In attempting to prevent this type of HIV-related neurotoxicity, we discovered that erythropoietin (EPO), normally thought to be a kidney-generated cytokine, is made in the brain and is neuroprotective. Additionally, we and others found that insulin-like growth factor-I (IGF-I) is also neuroprotective. Here we report our preliminary data that EPO and IGF-I can prevent neuronal damage due to gp120 in a synergistic fashion in vitro, we demonstrate the likely molecular mechanism for this neuroprotective synergy, and we propose to study this effect in the HIV/gp 120-transgenic mouse as in vivo proof-of-principle to expedite human clinical trials with these clinically-tolerated agents. Specific Aims: 1. To assess the possible neuroprotective effect of EPO in the HIV/gp120 transgenic mouse model. Hypothesis Tested: EPO protects neurons from HIV-related neuronal damage via a phosphoinositide (PI) 3 kinase/Akt (protein kinase B)-mediated transduction pathway acting, at least in part, through glycogen synthase kinase 3beta (GSK3beta) to prevent the hyperphosphorylation of tan. In contrast to EPO, gp120 decreases Akt phosphorylation. 2. To assess the possible neuroprotective effect of IGF-I in the HIV/gp120 transgenic mouse model. Hypothesis Tested: IGF-I also protects neurons via the PI3 kinase/Akt-mediated transduction pathway acting, at least in part, through GSK3beta to prevent the hyperphosphorylation of tau. 3. To assess the possible synergistic neuroprotective effect of EPO+IGF-I in the HIV/gp120 transgenic mouse model. Hypothesis Tested: EPO+IGF-I act synergistically to activate the anti-apoptotic PI3 kinase/Akt signaling pathway, and thus provide a synergistic degree of neuroprotection.

The Research Development Core will bring new investigators into the NIEHS Center for Neurodegeneration Science (CMS) team, studying in particular environmental stressors contributing to Parkinson's disease, by offering support for the initial development of novel lines of investigation that complement the ongoing research projects of this Center. Below, we describe the process of soliciting, reviewing, and awarding and monitoring the progress of these pilot projects. Importantly, to be eligible to obtain one of these pilot awards, the applicant will have no current affiliation with the proposed NIEHS Center. We plan to award three grants per annum, each at $25,000 for a total of $75,000 as suggested in the RFA. We will particularly embrace promising new directions of junior trainees as well as established faculty who wish to enter this field in order to link environmental sciences with neurodegenerative conditions, particularly with the Parkinson's disease (PD) focus proposed for this Center. We believe that the multidisciplinary research environment, well-organized and highly integrated infrastructure, and the rich longitudinal specimen and human data bank found in Core E (Neuropathology/Behavior Core) will foster services that cannot be found anywhere else in Southern California, creating an inviting environment for investigators to enter the field. The new investigators will participate in our weekly laboratory meetings and monthly sessions for the entire Center, as described in detail in the grant Introduction and in Core A (Administrative Core).

DESCRIPTION (provided by applicant) Mutations in the PINK1 gene are linked to an autosomal recessive early onset familial form of Parkinson's disease (PD). The molecular and physiological functions of PINK1 that generate pathological abnormality of PD-associated PINK1 mutants are largely unknown. Therefore, this group has developed a genetic model of PD in Drosophila to study the in vivo role and genetic interactions of PINK1 with known and new potential contributors to this disease. It has been recently shown that inactivation of Drosophila PINK1 (dPINKI) using RNAi results in progressive loss of dopaminergic (DA) neurons and in ommatidial degeneration of the compound eye, which is rescued by expression of human PINK1 (hPINKI). Moreover, expression of human superoxide dismutase 1 (SOD1) suppresses neurodegeneration induced by dPINKI inactivation, and treatment of dPINKI RNAi flies with antioxidants (e.g., vitamin E) significantly inhibits ommatidial degeneration. Thus, PINK1 may normally prevent neurons from undergoing oxidative stress, a potential mechanism by which a reduction in PINK1 function leads to PD-associated neurodegeneration. Therefore, in this proposal it is hypothesized that PINK1 plays a critical role in maintaining survival of dopaminergic neurons via a regulated pathway involving protection against oxidative stress. PD-pathogenic PINK1 mutants impair the functional pathway and therefore lose the ability to protect neurons from oxidative stress. In this study, efforts will focused on utilizing the newly-developed PD fly model (published in PNAS) to first investigate the genetic mechanisms and interactions that influence the severity of the PD pathogenic phenotype produced by PINK1 mutations under different (oxidative) stress conditions. The fact that wild-type human PINK1 but not disease-associated PINK1 mutations can reverse PD-associated pathologies in our fly model provides us with the opportunity to efficiently screen in a whole animal system for genetic and chemical modifiers that are likely relevant to finding therapeutics for this disease. New genetic factors as well as chemical compounds will be screened that function to alter (e.g., ameliorate or aggravate) the neurodegenerative phenotype observed in our PD fly model.

This core will serve all projects by providing administrative support, organization of External and Internal Advisory Groups, overall leadership via the Program Director, Dr. Lipton, and also statistical support to help plan and analyze all experiments. For example, power analyses will be performed to help investigators decide on how large an in vivo experiment to plan, how many animals to use, and what endpoints are realistic in terms of obtaining statistically significant results.

We recently discovered that various Parkinson-related proteins, including parkin, DJ-1, and protein-disulfide isomerase (PDI), are S-nitrosylated and then further oxidized (published in PNAS, Science, and Nature). S-Nitrosylation (chemical transfer of an NO group to a critical cysteine thiol) affects protein function - in the case of parkin, regulating its E3 ligase activity; for PDI contributing to protein misfolding; and for DJ-1, possibly affecting DJ-1 dimerization or its interactions with PINK1 and thus the stability of the parkin/PINK1/DJ-1 complex (as shown by Dr. Z. Zhang in Project 1 of this NIEHS Center Grant Application). These S-nitrosylation reactions appear to contribute to the pathogenesis of Parkinson's disease. Here, we will further study these nitrosylation reactions with the Proteomics Core (Core B), seek structural evidence for their basis with the Structural Core (Core C), and perform high-throughput screening with our Chemical Library Core (Core E) to develop novel drugs that prevent these nitrosylation reactions and that are therefore potentially neuroprotective. These drugs will be tested subsequently within this project in secondary screens for neuronal survival on primary neurons in vitro here in Project 3, and in mouse transgenic (tg) models of PD in conjunction with the Neuropathology and Animal Behavior Core (Core D).

ELECTROPHYSIOLOGY CORE 1. MAIN OBJECTIVES AND NEW DIRECTIONS The Electrophysiology Core will be a new facility. Currently, there is no unified Electrophysiology Core Facility to link neuroscientists on the La Jolla Torrey Pines Mesa. Therefore, this Electrophysiology facility will constitute a new Core to foster collaborations among neuroscientists in the San Diego area. Understanding how the nervous system works requires electrophysiological techniques to gain insights into how synaptic activity is regulated and modulated. The Electrophysiology Core of this Neuroscience Blueprint Initiative aims to make available electrophysiology as a research tool to a wide number of neuroscientists on the Torrey Pines Mesa. In the past several years, the Co- Directors of this Core, Dr. Stephen F. Helnemann and Dr. Charles F. Stevens made major contributions to understanding the functions of the two major excitatory transmitter systems, the nicotinic acetylcholine and glutamate receptor systems. Their work provided major insights into mechanisms of synaptic transmission and synaptic plasticity. Both researchers have contributed their knowledge and effort to a series of collaborations with neuroscientists who do not have direct expertise or the experimental set-up for electrophysiological analyses. While these collaborations accomplished significant research advancements, investigators always depended on whether the host laboratory could accommodate additional experiments in terms of instrument availability and personnel. With the increasing need for electrophysiology by developmental and systems neurobiologists, it is timely to fill the need for additional capacity in electrophysiology. To accomplish that and to further increase collaborative ventures between neuroscientists on the Torrey Pines Mesa, a new core facility for electrophysiology will be created as part of the Neuroscience Blueprint Initiative. Dr. Stephen Heinemann and Dr. Charles Stevens will serve as Co-Directors for this core and are committed to contribute to the design of electrophysiological experiments by the core users. The Electrophysiology Core has two goals: First, to provide expertise and instrumentation for electrophysiological experiments by neuroscientists at the Salk Institute, UCSD, Burnham Institute for Biomedical Research (BIMR) and The Scripps Research Institute;and second, to enhance neuroscience through dialogue and collaboration between the scientists of these institutes. The Electrophysiology Core adds a new research dimension that will provide the tools and know-how for functional and activity-dependent studies designed to acquire knowledge of the activity-dependent changes occurring in neurons either intrinsically or in response to external signals. The research projects proposed by the major users require studies of neuronal activity in brain slices and/or primary neuron cultures. Moreover, this core will inter-phase with the cores for Viral Vectors/siRNA and for Molecular Imaging cores to test fluorescently tagged wild type and mutant proteins in neuronal functions after ectopic expression in brain slices and primary cells.

VIRAL VECTOR AND siRNA CORE FACILITY 1. MAIN OBJECTIVES AND NEW DIRECTIONS The Viral Vector/siRNA Core will be a new facility. Currently, there is no unified Core facility to link neuroscientists on the La Jolla Torrey Pines Mesa for these functions. Therefore, this facility will constitute a new Core to foster the use of viral vectors for payload delivery into neural cells, including RNAi, among neuroscientists and will produce multiple collaborations in an interdisciplinary manner in the San Diego area on the La Jolla Torrey Pines Mesa. The primary core will be located at The Salk Institute because of the presence of a prominent virologist/geneticist there, Dr. Inder Verma, who will Direct the Core with the assistance of Edward Callaway, who will act as Co-Director. A satellite facility will be located at the Burnham Institute for Medical Research (BIMR) headed by Mark Mercola, who will serve as a Co-Director for the core. This satellite is particularly important for seamless interface with the other cores located there that will use the viral vectors, e.g., the Chemical Library Screening/High Throughput Cell Analysis Cores and the Imaging Core. The mission of the Viral Vector and siRNA Core Facility (WS) is to provide a robust means of gene manipulation to Neuroscience investigators. Over the last decade, methods for the production of a variety of viral vectors for stable and non-toxic delivery of genetic material to postmitotic neurons have become relatively routine. However, the laboratories that would most benefit from the use of these vectors often have expertise in neuroanatomy, neurophysiology, or behavior, but not virology. This disparity between expertise and need greatly limits the use of these valuable vectors within the San Diego Neuroscience community. Furthermore, even those laboratories that have the technical capability to produce one or another type of vector would benefit from a core facility which maintains proven protocols, technical expertise and experience, and all the necessary plasmids required to implement the full range of possibilities afforded by modern virology. The San Diego neuroscience community presently has the expertise to establish and maintain a state-of-the art facility, but piecemeal collaboration amongst a limited set of laboratories does not fully realize the potential of this community. Establishing a facility to make a broad range of vectors including adeno-associated virus (AAV), lentivirus, and herpes simplex virus (HSV) amplicon vectors will have fundamental impact on the culture of Neuroscience in San Diego. This core will make possible experiments that would be otherwise impossible and will foster collaborations amongst molecular neuroscientists who routinely use genetic methods, and other neuroscientists who focus on behavior, anatomy or physiology. Viral vectors are now fundamental tools at the cutting edge of experimental neuroscience. This is because they harness the proven power of molecular and genetic techniques by complementing and extending the capabilities afforded by the production of transgenic mice. Amongst the most prominent advantages of viral vectors are: 1) the short time required to test a genetic manipulation (e.g. gene knockdown with siRNA) compared to the generation of a transgenic mouse line;2) the ability to target gene expression to particular brain regions where specific promoters are not available for targeting;3) the ability to initiate manipulation in adult animals;4) the ability to use genetic technologies in species where transgenic methods are impractical (e.g., monkeys). Nevertheless, each of the available viral vector technologies have limitations, such that no single vector will meet the needs of every possible experimental goal. We have selected three types of viral vectors, AAV, lentivirus, and HSV amplicons, because these are all proven to be highly effective and they are a complementary set. Limitations of one vector can often be obviated by the use of another. These vectors all have in common, however, that they can efficiently transduce nearly every neuron in the vicinity of a brain injection and they can yield stable gene expression for months or years without toxicity (Naldini et al., 1996;Blomer et al., 1997;Xiao et al., 1997;Rabinowitz and Samulski, 1998;Sandier et al., 2002;Davidson and Breakefield, 2003; Kootstra and Verma, 2003). In addition to supporting the construction and use of viral vectors, the core will also provide reagents and expertise in gene knockdown by si/shRNA. Gene attenuation has been central to experimental biology for the past century and has increased exponentially with the advent of reverse genetics or the use of targeted attenuation of gene activity to reveal function. It is anticipated that gene attenuation studies performed at a large, genome or proteome-wide scale, will be increasingly important to assign gene function now that the sequences of many experimental organisms are complete. Moreover, gene function will point to targets that will be interrogated further using other technologies embodied in the other Neuroscience Cores, including Chemical Library Screening and Structural Biology;thus, this core is an important component of an integrated approach to Neurosciences. Currently, there is no si/shRNA and viral vector facility or core service available to Neuroscientists. Individual laboratories contract with commercial entities to produce siRNA oligos or construct si or shRNA vectors internally and viral vector technology is represented at a very sophisticated level in a few laboratories. The Cancer Center at BIMR is establishing a facility that will distribute a commercial library of siRNA oligonucleotides against human genome targets, but this will be available only to Cancer Center members, and efficacy of transfection of siRNA oligonucleotides into many cell lines, especially primary cell lines, is limited. The core facility will offer 1) AAV, lenti and HSV vector support, 2) access to a commercial siRNA oligonucleotide library for screening, 3) access to a shRNA lentiviral library to the human kinome, 4) . shRNA lentiviral libraries to other targets will be offered as libraries become available;in addition, the core will offer: 5) tagged human cDNA clones representing the entire kinome in a 3rd generation lentiviral vector and 6) a negotiated preferred price structure for purchasing siRNA oligonucleotides from a commercial vendor. Kinases have been chosen for the first shRNA and expression lentiviral libraries to be offered by the facility because they constitute one of the largest and most important of protein families, accounting for ~2% of genes in human and other eukaryotic genomes (Manning et al., 2002a;Manning et al., 2002b). By phosphorylating substrate proteins, kinases modify the activity, location, and affinities of up to 30% of all cellular proteins, and direct most cellular processes, particularly in signal transduction and coordination of complex pathways. In fact, it is difficult to think of any pathway that is not modulated by kinases, making them, as a group, a lynchpin of cell biology, and promising that a set of high quality knockdown reagents would provide insight into the control of almost any cellular process. The human genome contains 518 kinase genes, of which over 150 have already been implicated in human disease (Manning, 2005). The WS facility will be housed at the Salk Institute with a satellite facility at the BIMR. The purpose of the BIMR satellite is to have access to the robotic liquid handling capacity of the Chemical Library Screening (CLS) to amplify and array the libraries. The Salk facility will be under the direction of Inder Verma, PhD and Ed Callaway, PhD as co-director. Dr. Verma's laboratory has dedicated the last 30 years to understanding the molecular underpinnings that convert a normal cell to a cancer cell. He has developed expertise in large-scale manipulation of lentiviral vectors for the expression of cDNAs and si/shRNA. Dr. Callaway is an expert in cortical neural function and organization and his lab has expertise in the production and use of AAV, lentiviral and HSV amplicon vectors for gene delivery. The lab also has extensive experience in using these vectors in vivo in monkeys, ferrets, rats and mice.

STRUCTURAL BIOLOGY CORE 1 . MAIN OBJECTIVES AND NEW DIRECTIONS The use of Structural Biology on the La Jolla Torrey Pines Mesa has increased tremendously over the past 5 years. For example, BIMR has added 4 new faculty members as major users of its Structural Biology Facility and is directed by crystallography Robert Liddington. However, presently this Core is part of the Burnham NIH-funded Cancer Center with little or no access to neuroscientists on the La Jolla Torrey Pines Mesa. Additionally, two faculty at The Salk are also heavily invested in crystallography but heretofore were not organized as a Core and thus have little interaction with neuroscientists. Moreover, the Head of NMR at UCSD, Stan Opella, is world-renowned in this area and has an NMR facility that would be made available to neuroscientists on this grant as a satellite core. Hence, the structural core will function with headquarters at BIMR and satellite facilities at The Salk Institute and UCSD. This Structural Core will foster collaborative interactions by allowing equal access to neuroscientists. BIMR has made major investments in instrumentation for structural biology, including the acquisition of three new NMR spectrometers and upgrades for the X-ray diffraction set up. UCSD has installed a state-or-the-art 900 MHz spectrometer, which will be available for collaborations of neuroscientists. Scientific progress has been impressive in the recent past by these facilities, including 6 papers in the journals Cell, Science and Nature that utilized structural data from the facilities. However, Neuroscientists have had minimal, if any, access to the faculty or instrumentation because they are located within the Cancer Centers at BIMR, TSRI, UCSD, and The Salk. The current grant will rectify this by making the facility and the faculty available to Neuroscientists as a Core Unit. In summary, the Structural Biology Core comprises two facilities: Crystallography and NMR. Scientific, logistical, and budgetary issues provide justification for this merger of services. The primary application of Crystallography and NMR overlap, and most research projects require both Crystallography and NMR. The merged facility serves the needs of users with a broad range of expertise. There are several neuroscience investigators who are not mainstream structural biologists but who have a desire to learn structural techniques. The facility provides appropriate training and direction for these people. Other "hardcore" structural groups require much less support, and the facility will primarily supply the necessary hardware and technical support to achieve the scientific goals. One manager (who is free to this grant) oversees both techniques within the Structural Biology Facility and provides the necessary training (in conjunction with the faculty supervisors);technicians paid for by the current grant will coordinate the efforts at the three centers (BIMR, Salk, UCSD, and facilitate TSRI neuroscientist access as well). Future plans include a protein expression facility in this Core.

STRUCTURAL BIOLOGY CORE 1 . MAIN OBJECTIVES AND NEW DIRECTIONS The use of Structural Biology on the La Jolla Torrey Pines Mesa has increased tremendously over the past 5 years. For example, BIMR has added 4 new faculty members as major users of its Structural Biology Facility and is directed by crystallography Robert Liddington. However, presently this Core is part of the Burnham NIH-funded Cancer Center with little or no access to neuroscientists on the La Jolla Torrey Pines Mesa. Additionally, two faculty at The Salk are also heavily invested in crystallography but heretofore were not organized as a Core and thus have little interaction with neuroscientists. Moreover, the Head of NMR at UCSD, Stan Opella, is world-renowned in this area and has an NMR facility that would be made available to neuroscientists on this grant as a satellite core. Hence, the structural core will function with headquarters at BIMR and satellite facilities at The Salk Institute and UCSD. This Structural Core will foster collaborative interactions by allowing equal access to neuroscientists. BIMR has made major investments in instrumentation for structural biology, including the acquisition of three new NMR spectrometers and upgrades for the X-ray diffraction set up. UCSD has installed a state-or-the-art 900 MHz spectrometer, which will be available for collaborations of neuroscientists. Scientific progress has been impressive in the recent past by these facilities, including 6 papers in the journals Cell, Science and Nature that utilized structural data from the facilities. However, Neuroscientists have had minimal, if any, access to the faculty or instrumentation because they are located within the Cancer Centers at BIMR, TSRI, UCSD, and The Salk. The current grant will rectify this by making the facility and the faculty available to Neuroscientists as a Core Unit. In summary, the Structural Biology Core comprises two facilities: Crystallography and NMR. Scientific, logistical, and budgetary issues provide justification for this merger of services. The primary application of Crystallography and NMR overlap, and most research projects require both Crystallography and NMR. The merged facility serves the needs of users with a broad range of expertise. There are several neuroscience investigators who are not mainstream structural biologists but who have a desire to learn structural techniques. The facility provides appropriate training and direction for these people. Other "hardcore" structural groups require much less support, and the facility will primarily supply the necessary hardware and technical support to achieve the scientific goals. One manager (who is free to this grant) oversees both techniques within the Structural Biology Facility and provides the necessary training (in conjunction with the faculty supervisors);technicians paid for by the current grant will coordinate the efforts at the three centers (BIMR, Salk, UCSD, and facilitate TSRI neuroscientist access as well). Future plans include a protein expression facility in this Core.

STEM CELL CORE 1. MAIN OBJECTIVES AND NEW DIRECTIONS The Stem Cell Core will be a new facility. Currently, there is no unified Stem Cell Core Facility to link neuroscientists on the La Jolla Torrey Pines Mesa. Therefore, this Stem Cell facility will constitute a new Core to foster stem cell projects and collaborations among neuroscientists in the San Diego area. Interest in human embryonic stem cells (hESCs) has grown steadily since the report of the first hESC line in 1998. hESCs are attractive because of their potential as cell therapy, either directly replacing cells or by using them as vehicles for gene therapy (Muller et al. 2006). But the cells themselves are intrinsically interesting, because of their unique qualities of pluripotence and self-renewal, and these qualities have led to a resurgence of interest in fundamental cell biology. hESC are the perfect tools to study events that occur during human neurogenesis, both to understand normal development and to identify what goes wrong during neurological disease. The versatility of hESC means that from the same population one can generate multiple cell types that can then be used to dissect complex cellcell interactions. For example, by differentiating hES cells into motor neurons, skeletal muscle cells, and astroglia, one can do mix-and-match experiments to identify the vital components of degeneration in ALS. By generating human/mouse chimeric brains using transplanted human neural stem cells or neuronal precursors derived from hESC, it will be possible to ask how human neurons react to the amyloid plaques that develop in transgenic models of Alzheimer's disease. Additionally, hESCs will eventually be used for transplantation therapies in regenerative medicine. This Core facility proposes to use only approved Presidential ES cell lines. The funds requested are particularly important because Prop 71 which was to fund stem cell work in California has not become a reality, and it is currently not clear if these funds will become available in the future. In addition to hESCs, this core will allow neuroscientists on the La Jolla Torrey Pines Mesa access to methods regarding the use of murine (m)ES cells. Additionally, the Stem Cell Core has expertise with neural progenitor cells (NPCs) derived both from hES and mES cells. Moreover, NPCs from fetal human and mouse brains (obtained by workers at BIMR and UCSD), and adult rodent NPCs (from the laboratory of Fred Gage at The Salk Institute) will be available through this Stem Cell Core. The origins of all of these cells and cell lines have received both Institutional approval and Presidential approval, where necessary for hESC lines. The purpose of the Stem Cell Core will be to provide training and fundamental analysis tools for stem cell research throughout the La Jolla Torrey Pines Mesa. Currently, while there is an NIH-sponsored Stem Cell Center at the Burnham Institute for Medical Research (BIMR), it is aimed at specific projects and is insufficient to provide support for local neuroscientists who would like to use it. With this grant, we propose to take advantage of the BIMR's experienced Stem Cell Center faculty and staff and expand the capacity of the core laboratory to make the facilities available to all neuroscientists in the La Jolla Torrey Pines Mesa area (UCSD, Salk, BIMR and TSRI). The Stem Cell Core will be directed by Dr. Alexey Terskikh (BIMR, who trained with Prof. Irving Weismann, a well-known stem cell expert at Stanford. Co-directors of the facility will be stem cell experts Drs. Evan Snyder and Jeanne Loring (at BIMR), and Fred (Rusty) Gage and Juan Carlos Belmonte at The Salk Institute. The overall objective of the Stem Cell core is to lower the barrier for neuroscientists to involve stem cells in their research projects, by providing training and expert advice from experienced stem cell biologists. This will improve communication among scientists in a variety of neuroscience subspecialties to foster interdisciplinary collaborations and novel approaches. By sharing common techniques and accumulated knowledge about the fundamental biology of human ES cells and somatic (adult) stem cells, there will be a considerable savings in time, effort, and cost. The Stem Cell Core will play an important role in ensuring that the users are kept up to date with the standardization of hESC methods called for by the international stem cell scientific community (Andrews et al. 2005;Loring 2005). The principal focus of this core is to provide stem cell-specific technologies that will lead to new insights in neuroscience and aid in development of clinical applications for stem cells. For example, correlation of the characteristics of stem cells in culture and their behavior after transplantation will help in decisions about which cells to use and what quality control measures will be necessary for clinical use. Core support will include culturing of ES and other stem cells, karyotyping, SNP genotyping, microarray analysis, immunohistochemistry for stem cell and differentiation markers, assays for pluripotence and differentiation, and access to BIMR's extensive databases of stem cell information. In addition, the stem cell core faculty will offer advice for experimental design, data analysis, interpretation of results, and assistance with manuscripts and grant applications. Stem cell core faculty will also serve as mentors for postdocs and young faculty members, to help them publish and obtain grant support.

The Salk Institute is a world-renowned independent organization conducting peer-reviewed, laboratorybased research funded primarily through the NIH. The Salk ranks eight among NIH funded medical institutes and also has an illustrious and well-known neuroscience effort headed by Drs. Stephen Heinemann, Charles Stevens, Fred Gage, Greg Lemke, Dennis O'Leary, Terrence Sejnowski, and others. The President of The Salk Institute, Richard Murphy, formerly performed neuroscience research himself and strongly endorses this grant (email letter appended at end of this Administrative Core).

DESCRIPTION (provided by applicant): The overall goal of this inter-institutional grant Is to foster new interdisciplinary collaborations among neuroscientists in La Jolla institutions by allowing access to scientific cores to which they were previously denied or had little access, and by creating new cores to promote state-of-the-art technology. To accomplish this goal, we have chosen among the very best existing core facilities, and have chosen top scientists to run new cores at the institutions supporting neuroscientists on the La Jolla Torrey Pines Mesa, composed of the Burnham Institute for Medical Research (BIMR), The Salk Institute, The Scripps Research Institute (TSRI), and the University of California, San Diego (UCSD). These institutions share one of the top-rated neuro- science graduate programs in the country and have committed considerable institutional funds towards these cores. This work is aimed at developing new treatments for neurological disorders. Specific Aims include: 1. To obtain Core support for Neuroscientists in La Jolla to enable collaborative research in Systems Neurobiology. Examples of Cores for this discipline are Behavioral testing, Electrophysiology, Neuropathology, Stem Cells, Bioinformatics/Systems Biology, Viral Vectors/siRNA, and Cell Imaging/Histology. 2. To obtain Core support for Neuroscientists to enable collaborative Developmental Neurobiology research. Examples of Cores for this discipline are Electrophysiology, Cell/Molecular Imaging, Stem Cells, Proteomics, and Viral Vectors-siRNA Libraries. 3. To obtain Core support for Neuroscientists studying Neurodegenerative Disorders and developing Neuroprotective Therapies. Cores to enable collaboration in this discipline include Proteomics, Structural Biology-crystallography, High-throughput Cell Analysis, Chemical Library Screening, Behavioral testing, Gene Analysis/cDNA Microarrays, Bioinformatics, Neuropathology, Viral Vectors- siRNA, and Stem Cells. 4. To obtain Core support for Neuroscientists working the area of Stem Cells and Regenerative Medicine. Cores needed for collaborative work in this area include Stem Cells, Proteomics, Gene Analysis/cDNA Microarrays, Bioinformatics, Neuropathology, Electrophysiology, High-throughput Cell Analysis Chemical Screening, and Behavioral testing.

PROTEOMICS CORE 1. OVERVIEW AND MAIN OBJECTIVES The mission of the Neuroscience Proteomics Facility is to provide identification, characterization, and detailed analysis of proteins, peptides, carbohydrates and drug-like small molecules. This facility will allow access to and thereby foster collaborations among Neuroscientists on the La Jolla Torrey Pines Mesa (comprised primarily of Burnham, Salk, Scripps, and UCSD neuroscientists). The facility will leverage the existing infrastructure and knowledge-base of the Burnham Institute for Medical Research's (BIMR's) existing Proteomics Center as well as state-of-the-art mass spectrometry (MS) at The Scripps Research Institute (TSRI) and The Salk Institute, which constitute Satellite Centers located across the street from BIMR on North Torrey Pines Road in La Jolla (and within half a mile from UCSD). Presently this Core Facility at BIMR is part of the Burnham's NIH-funded Cancer Center with little or no access to neuroscientists on the La Jolla Torrey Pines Mesa. Under the present application for an NIH Blueprint Grant for Neuroscience Center Cores, all modern proteomic technologies will be available to neuroscience users of the Facility including comprehensive proteomic analysis of complex proteomes using Multi-dimensional Protein Identification (MUDPIT), activity-based enzyme profiling with chemical mass tags, Product Terminal Isotope Coding for the identification of substrates for proteases (products of proteolysis), phospho-peptide and protein identification using precursor ion scanning, neutral loss scanning and MS3 scanning, and automated high-throughput MALDI-TOF/TOF analysis of protein and peptide mixtures. The Facility also performs the more routine mass analysis of intact recombinant proteins and synthetic peptides and glycopeptides, protein identification from gel slices/spots using peptide mass fingerprinting and peptide sequencing. In the spring of 2006 the Director of the Facility will also apply for funds to purchase an FTMS which will provide the highest mass accuracy and resolution, broad fragmentation capability (such as CID, ECD and IRMPD) and the ability to perform de novo peptide and protein sequencing (top-down proteomics) and directly characterize the most complex post-translational modifications in intact proteins, without prior digestion and processing of the sample. A primary objective for this award is to integrate the proteomics efforts of the La Jolla Neurobiology community into the existing Proteomics Center at the Burnham Institute for Medical Research. This will involve the purchase of additional instrumentation (an LTQ Ion Trap in Year 1), and the hiring of additional personnel. By amalgamating the neuroscience Proteomics Shared Resource with that of the current Center (which includes the Proteomics Facility for the Burnham's Cancer Center as well as the NTCNP Center on Proteolytic Pathways), we will be able to capitalize on the considerable instrumentation and expertise of the existing Center for the benefit of the Neuroscience community. The current grant application will facilitate equal access to these facilities by Neuroscientists, who do not have access currently because the facility is in the Cancer Center. The integration of the Neuroscience proteomics effort into the existing Proteomics Center will provide a substantial jump-start for the Neuroscience proteomics effort. In addition to the current equipment at the Burnham Institute for Medical Research (BIMR) listed above, which is under the direction of Dr. Jeff Smith, satellite facilities are also available at The Salk Institute (under the direction of Wolfgang Fischer) and at The Scripps Research Institute (TSRI), under the direction of noted proteomics authority John Yates, III.

NEUROPATHOLOGY CORE 1. MAIN OBJECTIVES AND NEW DIRECTIONS The Experimental Neuropathology Laboratory (ENL) at the Neurosciences and Pathology Departments at UCSD is primarily focused at better understanding the mechanisms of synaptic pathology and at developing new experimental therapies for Alzheimer's Disease (AD), Parkinson's Disease (PD), HIV associated dementia (HAD), and other neurodegenerative conditions. This laboratory is headed by Dr. Eliezer Masliah, who has collaborated with a number of neuroscientists on the La Jolla Torrey Pines Mesa and elsewhere, but these facilities have not been previously available to all neuroscientists as a Core. Dr. Masliah will serve as Director of the proposed New Neuropathology Core, which will be located at UCSD. We propose, via this grant proposal and in the spirit of the NIH Blueprint mission, to make this facility into a Core and to therefore make it readily available to other neuroscientists in the region. This new Core will foster further collaborations and enhance research into neurodegenerative disorders in the San Diego area. In recent years, with establishment of more sophisticated animal models of neurological disorders and the considerable growth of the neurosciences groups interested in neurodegenerative diseases at UCSD, the Burnham Institute for Medical Research (BIMR), The Scripps Research Institute (TSRI), and The Salk Institute, an increased demand for core services focused at characterizing neuropathological alterations in human and animal models has developed. Furthermore, interest has grown not only from the fields of AD, PD and HAD, but also in stroke, trinucleotide repeat disorders (such as Huntington's disease), taupathies, prion diseases, amyotrophic lateral sclerosis (ALS), and multiple sclerosis, just to mention a few. This has created a considerable need for the formation of a consolidated and unified Core Neuropathology Facility that will provide support and help coordinate efforts to all of these different programs. In this context, the overall objective of the Neuropathology Core is to provide to investigators in the La Jolla Neurosciences community a set of technical and neurobiological resources that allows analysis of the degenerative processes associated with neurological conditions in human brains and animal models from a comprehensive and dynamic perspective. The Core will allow the acquisition and analysis of Neuropathology related data with standardized protocols. CORE RESOURCE OBJECTIVES 1) Provide an array of techniques including histopathology, immunocytochemistry, microinjection, confocal microscopy, stereology, electron microscopy, digital imaging and 3D reconstruction to facilitate quantitative analysis of neuronal injury that will facilitate studies of the pathogenesis of degenerative, inflammatory and demyelinating CNS disorders. 2) Provide technical assistance on state-of-the-art neuropathological approaches such as twophoton confocal microscopy and immuno-electron microscopy. 3) Provide scientific consultation on molecular pathogenesis and targets of neurological disorders. 4) Support preliminary studies in human tissues and animal models that utilize neuropathology data. 5) Provide mentorship for students and junior faculty in the theory and techniques of neurobiology and neuropathology. 6) Collaborate with the other Cores of the program to disseminate information on neuropathology related methods of analysis.

CELL IMAGING AND HISTOLOGY CORE 1. MAIN OBJECTIVES AND NEW DIRECTIONS Under the NIH Neuroscience Blueprint Core Center Grant, the Cell Imaging/Histology Core will be built as an expansion of an existing Cancer Center core at the Burnham Institute for Medical Research (BIMR). As described elsewhere in this grant application, historically, Cancer Center cores have not allowed access to neuroscientists on the La Jolla Torrey Pines Mesa. This Neuroscience Center Grant will provide equal access and foster novel Neuroscience-specific imaging tools and histology-related applications for the Neuroscience community. The Core will also advance the molecular and cellular understanding of neural development, function and pathology, and serve as an initiator for collaborations in the Neuroscience community throughout San Diego. Current neuroscience research widely employs genetic manipulations to address the workings of specific molecules in model organisms and neural cultures. The observation of the molecular changes implemented in these systems critically depends on histological/immunohistological and advanced molecular imaging techniques. The mission of the Neuro-lmaging and Histology Core is to provide state-of-the-art imaging and histological tools to the Neuroscience community on the Torrey Pines Mesa. Currently, Neuroscientists at the Salk Institute and BIMR have only limited access to expertise and instrumentation in neuronal imaging and histology of the nervous system. The Salk Institute operates a standard single photon confocal microscope (Leica TCS SP2 AOBS) on a shared basis that is suited for molecular imaging and extensively used by Salk researchers. However, the capabilities of this instrument are limited, in particular when it comes to imaging of cellular or molecular events in thick tissue preparations, or to live tissue preparations. The Burnham Institute's existing Cell Imaging and Histology Facility has available within its well-equipped Core, the instrumentation, expertise and capacity for multiphoton, confocal imaging that will accommodate this void. Neither the existing Salk nor the BIMR facilities offer services geared specifically to Neuroscience applications. The current BIMR service for histology is well versed for the preparation and analysis of tumors and large organs, but cannot accommodate tasks geared towards analyzing specific structures within the complex architecture of the nervous system. The goal for this Neuroscience Blueprint Initiative is to overcome these limitations and establish an open access Core Facility for neuroscientists on the Torrey Pines Mesa. The Burnham Institute's Cancer Center Cell Imaging Facility has experienced significant expansion during the past year and invested in three new confocal microscopes as part of its institutional commitment to expand use and capabilities. With these new instruments, the imaging capabilities at BIMR are significantly improved and additional imaging capacity is now open. Recent techniques in molecular imaging have experienced significant advancement with the development by Dr. Roger Tsien of UCSD of fluorescent dyes that allow the tracing of molecules within living cell. Dr. Tsien will bring the imaging tools he has developed to this Neuroscience Blueprint Core and participate in "spying" on the molecules of the nervous system as the Co-Director. Thus, users will gain easy access to the tools developed by Dr. Tsien through this Core. For the histology section of the Core, specialized on-site expertise in the anatomy and histology of the nervous system will be added to the existing Cancer Center BIMR Histology facility. The Core will accommodate the preparation of brain sections to view specified anatomical structures in the developing and adult nervous system. Moreover, the Core will operate a state-of-the-art laser microdissection system that will allow excising specified cells or nuclei for microarray and proteomic profiling. Thus, the Imaging/Histology Core will interphase with the Gene Analysis and the Proteomics Cores of this application. Electron microscopy (EM) is not included in this Imaging/Histology Core as Neuroscience researchers will have access to excellent ultrastructural analyses of the nervous system through the Neuropathology Core in this grant, which is directed by Dr. Eliezer Masliah (see Neuropathology Core Description in this application). Through the Neuropathology Core, Neuroscientists on the Torrey Pines Mesa also have priority access to the National Center for Microscopy and Imaging Research (NCMIR) Facility directed by Dr. Mark Ellisman of UCSD. The NCMIR Facility supports scanning-, transmitted-, and high voltage electron microscopy. The mission for the Cell Imaging and Histology Core is to extend molecular and cellular imaging capabilities and histology services to the Neuroscience community on the Torrey Pines Mesa. The main Core objectives are: 1) Provide high-resolution microscopic techniques in combination with molecular probes to obtain information about the location, movement, and activities of molecules within cells; 2) Provide histological services to reveal the anatomical structures and cellular components of the developing and adult nervous system, and for the laser dissection of cell populations for gene array and proteome analysis. 3) Offer consultation, train users and assist with the analysis of the data generated within the Core. 4) Foster collaborations between neuroscientists through information exchange and interphase work with the other Cores of this Blueprint Initiative.

HIGH-THROUGHPUT CELL ANALYSIS CORE 1. MAIN OBJECTIVES AND NEW DIRECTIONS Many of the major advances in tumor biology and immunology were made possible by the ability to rapidly discriminate and isolate discrete cell populations. The Burnham Institute for Medical Research (BIMR) High Throughput Cell Analysis (HTCA) facility was established in 2002 to provide high-speed cell sorting, analytical cytometry and high-throughput microscopy provides this capability to members of the NIH-funded BIMR Cancer Center. Currently, this facility is not available to La Jolla Torrey Pines Mesa neuroscientists;therefore, the current lack of general access to modern cytometry, especially image-based high-throughput microscopy with advanced image algorithms design support, as provided in this Core, imposes restrictions on the range of experimental design by neuroscientists in San Diego. Moreover the HTCA Core interacts extensively with the Chemical Screening Library (CSL) Core (see separate write up of that core) in screening for lead compounds and for drug actions in cell-based assays. The HTCA Core facility will offer analytical cytometry using two user operated benchtop cytometers (Becton-Dickenson FACSCanto 6-color and BD FACSort 3-color cytometers), a FACSVAntage SE with Digital Option (FACSDiVa) high-speed sorter operated by a skilled specialist, and a Beckman-Coulter Eidaq 100 automated high-throughput microscope. The director of the facility (Dr. Mark Mercola) offers expert consultation and periodic workshops on cytometry. In addition, affiliated faculty at BIMR and UCSD offer advice in imaging algorithms (see below). The current facility has supported a large number of BIMR Cancer investigators at a reduced charge-back rate and offered limited support to outside Cancer users at an external rate. Scientific progress has been impressive, with numerous publications in top-tier journals. The present application will make this facility available to Neuroscientists as a Core Unit. As mentioned above, Facility staff work closely with the Chemical Library Screening facility, located contiguously at the BIMR, to support the design and optimization of high-throughput cell-based screening assays.

The proposed Salk Microarray core overcomes limitations of the Illumina microarray system, in that currently there are only arrays available for human and mouse gene expression analysis. Thus, to enable global gene expression analysis for other model organisms used in the neurosciences, as well as a variety of microarray approaches beyond gene expression assays^ a satellite core facility at the Salk will provide Affymetrix-based array analysis. The Salk facility is currently a core in their Cancer Center, and increasing its capacity will provide previously unavailable access to La Jolla area neuroscientists. The Salk Microarray facility, begun in 1999, has also evolved, first making in-house spotted arrays and then later adding the Affymetrix platform for manufactured arrays. While Affymetrix arrays have become the predominant approach for analysis of gene expression in the Salk core, an active effort and capacity for custom printing of spotted arrays and their analysis has been maintained in this facility. The Salk facility also provides service and training for real-time PCR analysis.

GENE ANALYSIS CORE 1. MAIN OBJECTIVES AND NEW DIRECTIONS The mission of the proposed Gene Analysis Resource is to provide routine services such as DNA sequencing and mouse genotyping, but the major focus is more cutting-edge analysis of gene expression through real-time quantitative PCR and whole genome microarray expression analysis. The BIMR core facility, directed by Craig Mauser, is currently a part of the Burnham NIH-funded Cancer Center, but the current capacity of Q-PCR and microarray analysis is just sufficient to serve the needs of the Cancer Center. Thus, the La Jolla neuroscierice community has very limited if any access to these services. The microarray and Q-PCR services provided by the core located at the BMIR will provide unique and valuable resources to the La Jolla neuroscience community. The BIMR core performs Microarray analysis using the Illumina BeadStation platform, which has been found to produce outstanding quality data for global gene expression analysis. This analysis is far more reproducible than spotted arrays, and can be performed at about half the total cost of the Affymetrix or Agilent-based core facilities in La Jolla, or through the Neuroscience Microarray Consortium sites. In addition to the array technology, an important component of the BIMR gene analysis core is its close integration with the Informatics core. This informatics resource provides assistance in array data analysis, not only for array data that has been generated in the core, but comparison of these data to other datasets. For Gene expression analysis, both the core and satellite will provide full-service analysis, where investigators provide RNA and receive data and assistance in its informatic and statistical analysis. In addition to providing access to both Illumina and Affymetrix microarray facilities, creation of a La Jolla neuroscience microarray user group associated with the array cores will enhance the most challenging part of microarray analysis: the downstream informatic and intuitive analysis required to convert vast amounts of data into neuroscience knowledge. Thus, beyond data sharing, it is anticipated that sharing information on analytical approaches and resources will significantly accelerate progress from microarray analysis in this community. The Real-time quantitative PCR (Q-PCR) service that the Gene Analysis core will provide is not unique to the BIMR, but its focus and expertise on SYBR green based Q-PCR analysis is distinct from other La Jolla area resources. Of the >1 4,000 Q-PCR reactions performed by the BIMR Cancer Center core in the last year, >95% of the analyses were with SYBR green. The alternative hybridization probe (Taqman) based Q-PCR methods in wider use are an excellent way to quantitate gene expression, and the Salk core has expertise in this approach. However, the expense of the hybridization probes makes it impractical to use this method to query the expression of a large number of genes. With the proper controls, SYBR green-based Q-PCR is nearly as sensitive and specific as taqman, but more rapid, and the materials needed to analyze each gene cost almost an order of magnitude less. Thus, for many of the proposed approaches (e.g. microarray follow-up, gene family profiling, hypothesis testing, tissue sample profiling) the BIMR Q-PCR core will provide important support for neuroscientists quantitating gene expression. In addition to providing expert service, both core facilities will provide training for investigators who wish to perform their own QPCR. Management of the core will be performed by Dianne Foster, who is the manager of the current BIMR Cancer Center Gene Analysis core. A technician dedicated to meeting the microarray and Q-PCR needs of neuroscientists will be added to the BIMR core facility. Similarly, an additional technician at the Salk facility will be dedicated to providing gene expression analysis and other microarray-based analysis for La Jolla area neuroscientists. This will effectively increase the capacity in these vital areas, as it is personnel and not instrumentation that is limiting capacity. In addition to ongoing work in the analysis of gene expression, it is anticipated that follow-up from the new research areas enabled by the neuroscience Cores will require a substantial increase in microarray/Q-PCR analysis by neuroscientists. Examples of this include identifying affected pathways and inferring mechanism from newly identified biologically active chemical compounds, and profiling of developmental markers in stem cells differentiated along neural pathways. Overall, the Gene Analysis core will provide investigators with a backbone of routine services, as well as new access to increasingly important advanced facilities for analysis of gene expression.

A primary objective for this award is to integrate the proteomics efforts of the La Jolla Neurobiology community into the existing Proteomics Center at the Burnham Institute for Medical Research. This will involve the purchase of additional instrumentation (an LTQ Ion Trap in Year 1), and the hiring of additional personnel. By amalgamating the neuroscience Proteomics Shared Resource with that of the current Center (which includes the Proteomics Facility for the Burnham's Cancer Center as well as the NTCNP Center on Proteolytic Pathways), we will be able to capitalize on the considerable instrumentation and expertise of the existing Center for the benefit of the Neuroscience community. The current grant application will facilitate equal access to these facilities by Neuroscientists, who do not have access currently because the facility is in the Cancer Center. The integration of the Neuroscience proteomics effort into the existing Proteomics Center will provide a substantial jump-start for the Neuroscience proteomics effort. In addition to the current equipment at the Burnham Institute for Medical Research (BIMR) listed above, which is under the direction of Dr. Jeff Smith, satellite facilities are also available at The Salk Institute (under the direction of Wolfgang Fischer) and at The Scripps Research Institute (TSRI), under the direction of noted proteomics authority John Yates, III. For example, when required for larger proteins or for very precise measurements of mass, the recently-installed Finnigan LTQ/FT instrument at TSRI will be made available to neuroscientists. A technician at TSRI will be designated to specifically interact with and meet the needs of the Neuroscience Community in this regard. Additionally, at the satellite proteomics facility at The Salk Institute, The Bruker Ultraflex Mass Spectrometer has an interface for sequencing peptides on SELDI CHIPs initially characterized by smaller Chiphergen systems located in individual laboratories at the BIMR, Salk, or TSRI. Hence, the three facilities at BIMR (hub facility) and Salk and TSRI (hub facilities) will work together to allow each other's equipment to be used for maximal efficiency. Drs. Smith, Yates, and Fischer will meet monthly to discuss usage, prioritize projects, and insure that neuroscientists have access.

BIOINFORMATICS AND SYSTEMS BIOLOGY/DATA MANAGEMENT CORE 1. MAIN OBJECTIVES AND NEW DIRECTIONS The Bioinformatics and Systems Biology/Data Management Core is an existing Core in the Cancer Center at BIMR. Similar cores exist at The Salk Institute and UCSD. In all three cases neuroscientists have limited or no access to these cores and have to rely of informal, personal links for bioinformatics and data management support. This NIH Neuroscience Blueprint Core Center Grant will make these facilities available to Neuroscientists on the La Jolla Torrey Pines Mesa. The Core will be headquartered at BIMR with satellite facilities, linked electronically, at The Salk and the UCSD Supercomputer facility. Director Dr. Adam Godzik is located at BIMR and Co-Director Gerhard Manning at The Salk Institute (both Godzik and Manning are members of the UCSD Supercomputer Facility and thus have access at UCSD as well). The mission of this Core is three-fold. First, the Facility will provide support and training for a set of over 100 publicly available informatics programs that can be used by neuroscientists. Second, the Facility will create a virtual environment for archiving, analyzing, and sharing data on specific proteins that are studied in individual laboratories that are part of this NIH Neuroscience Blueprint Center Core Grant. Similar to the "Cancer Molecule Pages Database" being developed by the Burnham Cancer Center, the "Neuroscience Molecule Pages database" will display many important aspects of individual proteins (sequence and structural information, involvement in biochemical pathways, and links to key papers published on the protein) involved in neurological diseases. Third, and most significantly, the Facility will generate integrated tools for archiving, analyzing, and disseminating information generated in four of the other Neuroscience Center Core Facilities: High Throughput Cell Analysis Facility, the Gene Analysis Facility, the Proteomics Facility and the Chemical Library Screening Facility. Each of these Facilities will generate massive volumes of data. The role of the Informatics Facility will be to establish automated methods of collecting and analyzing the data;and to generate query protocols (automated where possible) for probing the data sets to identify features of significance. It is the intent of the Facility to create a relational database where investigators can make simultaneous queries across data from all four Facilities, across data archived from individual laboratories, and information in the public domain (PubMed, USPTO, PDB etc.).

ELECTROPHYSIOLOGY CORE 1. MAIN OBJECTIVES AND NEW DIRECTIONS The Electrophysiology Core will be a new facility. Currently, there is no unified Electrophysiology Core Facility to link neuroscientists on the La Jolla Torrey Pines Mesa. Therefore, this Electrophysiology facility will constitute a new Core to foster collaborations among neuroscientists in the San Diego area. Understanding how the nervous system works requires electrophysiological techniques to gain insights into how synaptic activity is regulated and modulated. The Electrophysiology Core of this Neuroscience Blueprint Initiative aims to make available electrophysiology as a research tool to a wide number of neuroscientists on the Torrey Pines Mesa. In the past several years, the Co- Directors of this Core, Dr. Stephen F. Helnemann and Dr. Charles F. Stevens made major contributions to understanding the functions of the two major excitatory transmitter systems, the nicotinic acetylcholine and glutamate receptor systems. Their work provided major insights into mechanisms of synaptic transmission and synaptic plasticity. Both researchers have contributed their knowledge and effort to a series of collaborations with neuroscientists who do not have direct expertise or the experimental set-up for electrophysiological analyses. While these collaborations accomplished significant research advancements, investigators always depended on whether the host laboratory could accommodate additional experiments in terms of instrument availability and personnel. With the increasing need for electrophysiology by developmental and systems neurobiologists, it is timely to fill the need for additional capacity in electrophysiology. To accomplish that and to further increase collaborative ventures between neuroscientists on the Torrey Pines Mesa, a new core facility for electrophysiology will be created as part of the Neuroscience Blueprint Initiative. Dr. Stephen Heinemann and Dr. Charles Stevens will serve as Co-Directors for this core and are committed to contribute to the design of electrophysiological experiments by the core users. The Electrophysiology Core has two goals: First, to provide expertise and instrumentation for electrophysiological experiments by neuroscientists at the Salk Institute, UCSD, Burnham Institute for Biomedical Research (BIMR) and The Scripps Research Institute;and second, to enhance neuroscience through dialogue and collaboration between the scientists of these institutes. The Electrophysiology Core adds a new research dimension that will provide the tools and know-how for functional and activity-dependent studies designed to acquire knowledge of the activity-dependent changes occurring in neurons either intrinsically or in response to external signals. The research projects proposed by the major users require studies of neuronal activity in brain slices and/or primary neuron cultures. Moreover, this core will inter-phase with the cores for Viral Vectors/siRNA and for Molecular Imaging cores to test fluorescently tagged wild type and mutant proteins in neuronal functions after ectopic expression in brain slices and primary cells.

CHEMICAL LIBRARY SCREENING CORE 1. MAIN OBJECTIVES AND DIRECTIONS The mission of the Chemical Library Screening (CLS) Facility Core is to provide new chemical tools and potential drug develop to Neuroscience investigators. The utility of small molecule screening for solving problems in biology is illustrated by a number of recent reports in the literature (see citations below). It is also anticipated that this route of study will provide assay and chemical inputs into the pipeline for the development of new lead molecules for the treatment of neurological disorders. Since high-throughput screening is not widely available at academic institutions, this facility will provide a new and unique approach toward characterizing proteins and signaling pathways involved in many aspects of neural development and neurologic disease. This Core will be located at the Burnham Institute for Medical Research (BIMR) and represents an expansion of an existing Core that was developed by BIMR's Cancer Center, but is not available to neuroscientists at this time. BIMR's contribution to this grant is represented by the institutional purchase of equipment, including the robotic screening facility, high-throughput microscopes, and chemical libraries, valued at well over $2 million. The proposed NIH Blueprint Neuroscience Core Center Grant will facilitate and encourage use of this Chemical Screening Core by neuroscientists on the La Jolla Torrey Pines Mesa at BIMR, The Salk Institute, The Scripps Research Institute (TSRI), and the University of California, San Diego (UCSD). The Core facility will offer chemical libraries, including an integrated robotic line with an automated incubator, plate hotel and tip loader, for biochemical assays with readouts including FRET, fluorescence polarization, and luminescence. Also offered is a complete high content screening line that includes robotics for liquid and plate handling, automated incubator, plate hotel in a clean-room environment for cell-based assays. Image-acquisition capabilities include Beckman Coulter and GE automated microscopes with robotic plate feeding. Sophisticated software packages are used for automated tracking of plates and compounds, cheminformatics, and image analysis. In general, two kinds of screens are performed with the following nomenclature: (i) HTS is high throughput screening, typically used for biochemical screens or plate-reader screens of cell-based assays;(ii) HCS is high content screening, used for image-based screens of cell assays, i.e. type of assays that use image analysis of cells. Expert assistance is available for all aspects of biochemical and cell-based screens, including automation engineering, medicinal chemistry, image acquisition, algorithm engineering and analysis. The CLS facility is located at BIMR within the San Diego Chemical Genomics Center (SDCGC), which also operates as one of the 9 NIH funded extramural centers of the Multiple Library Screening Center Network. The center is directed by Dr. Mark Mercola and managed by Dr. Steve Vasile. Dr. Mercola also directs Assay Development and Assay Implementation units of the SDCGC. Currently, the CLS facility operates as a charge-back basis to support members of the BIMR Cancer Center under the auspices of the BIMR Cancer Center Support Grant (CCSG). Because of heavy demand, it has offered only very limited and occasional service to members of the Cancer research community not at BIMR, and virtually no access to neuroscientists. The primary goal of this application is to provide salary support for personnel within CLS to support and facilitate interdisciplinary, collaborative research projects of neuroscience investigators on the La Jolla Torrey Pines Mesa.

The overall goal of this grant is to foster new interdisciplinary collaborations among neuroscientists in La Jolla institutions by allowing access to scientific cores to which they were previously denied or had little access, and by creating new cores to promote state-of-the-art technology. To accomplish this goal, we have chosen among the very best existing core facilities and have chosen top scientists to run new cores at the institutions supporting neuroscientists on the La Jolla Torrey Pines Mesa, composed of the Burnham Institute for Medical Research (BIMR), The Salk Institute, The Scripps Research Institute (TSRI), and the University of California, San Diego (UCSD). Of note, these four institutions share one of the top-rated neuroscience graduate programs in the country and have committed considerable institutional funds towards the cores described herein. The four institutions are located within a mile of one another on the Torrey Pines Mesa, a geographic area in La Jolla that runs along North Torrey Pines Road. The four collaborating institutions have grants far in excess of the required 15 qualifying Blueprint institute-funded neuroscience research projects (see Tables 2 and 3, below). The organization of this grant proposal includes the Budgets and the BioSketches of the Major Users (located prior to this section), followed by the description of the Administrative Core, which you are currently reading. This section includes lists in tabular format describing the (i) proposed dozen Scientific Cores (Table 1), (ii) Major Users (Table 2), and (Hi) Other Users (Table 3). The Administrative Core write-up is followed by details of each of the Scientific Cores. Following each Scientific Core, the BioSkectches of the directors (and co-directors) of the proposed Core can be found. Please note that examples of Core Usage by Neuroscientists are described in each Core write-up, but a more complete list of core usage by both Major Users and other Users with qualifying grants can be found in Tables 2 and 3 of this Administrative Core (see below). In addition, junior and developing investigators without qualifying grants will be encouraged to use the Cores to further neuroscience research, as provided for below.(see specific aims)

BIOINFORMATICS AND SYSTEMS BIOLOGY/DATA MANAGEMENT CORE 1. MAIN OBJECTIVES AND NEW DIRECTIONS The Bioinformatics and Systems Biology/Data Management Core is an existing Core in the Cancer Center at BIMR. Similar cores exist at The Salk Institute and UCSD. In all three cases neuroscientists have limited or no access to these cores and have to rely of informal, personal links for bioinformatics and data management support. This NIH Neuroscience Blueprint Core Center Grant will make these facilities available to Neuroscientists on the La Jolla Torrey Pines Mesa. The Core will be headquartered at BIMR with satellite facilities, linked electronically, at The Salk and the UCSD Supercomputer facility. Director Dr. Adam Godzik is located at BIMR and Co-Director Gerhard Manning at The Salk Institute (both Godzik and Manning are members of the UCSD Supercomputer Facility and thus have access at UCSD as well). The mission of this Core is three-fold. First, the Facility will provide support and training for a set of over 100 publicly available informatics programs that can be used by neuroscientists. Second, the Facility will create a virtual environment for archiving, analyzing, and sharing data on specific proteins that are studied in individual laboratories that are part of this NIH Neuroscience Blueprint Center Core Grant. Similar to the "Cancer Molecule Pages Database" being developed by the Burnham Cancer Center, the "Neuroscience Molecule Pages database" will display many important aspects of individual proteins (sequence and structural information, involvement in biochemical pathways, and links to key papers published on the protein) involved in neurological diseases. Third, and most significantly, the Facility will generate integrated tools for archiving, analyzing, and disseminating information generated in four of the other Neuroscience Center Core Facilities: High Throughput Cell Analysis Facility, the Gene Analysis Facility, the Proteomics Facility and the Chemical Library Screening Facility. Each of these Facilities will generate massive volumes of data. The role of the Informatics Facility will be to establish automated methods of collecting and analyzing the data;and to generate query protocols (automated where possible) for probing the data sets to identify features of significance. It is the intent of the Facility to create a relational database where investigators can make simultaneous queries across data from all four Facilities, across data archived from individual laboratories, and information in the public domain (PubMed, USPTO, PDB etc.).

The administrative structure places the responsibilify for this NINDS P30 Core Grant largely in the hands of the Program Director and Steering Committee; the steering committee is composed of the Program Director (Dr. Lipton at SBMRI) plus Pis of two of the qualifying projects (Huaxi Xu of SBMRI and Al La Spada of UCSD) and the directors of each Core (Dongxian Zhang/Electrophysiology-Optogenetics, Marc Mercola/HT Library Screening, Eliezer Masliah/Neuropathology). This group represents a wide range of neuroscience interests and senior leadership. Additionally, Dr. Stephen F. Heinemann, head of the Neurobiology Laboratory at the Salk Institute and Adjunct Professor at UCSD and SBMRI (and a member of the SBMRI Scientific Advisory Board), will serve as an Advisor to the group. Dr. Heinemann has served as president of the Sociefy for Neuroscience (SfN), and Dr. Lipton as head of the Education Committee of the SfN (which ran all of the courses at the annual meeting), and therefore both have nationally-recognized organizational and leadership skills. Additionally, the head of Finance at SBMRI, Antony Peake, will oversee the use of funds. Decisions regarding Core Usage are outiined under each Core and will be administered by each Core Director in consultation with this Steering Committee; the Core Directors, as part of the Core Steering Committee, will meet every month. At these meetings, coordination of ongoing neuroscience research among the Cores and area institutions will be discussed, as well as access of other neuroscience projects to the Cores as needed.

Project I - Zhang & Chen Program Director/Principal Investigator (Last, First, Middle): Lipton, Stuart A. PROJECT SUMMARY (See instructions): The N-methyl-D-aspartate subtype of gluatmate receptor (NMDAR) is essential for normal function of the central nervous system (CNS). However, excessive activation of NMDARs, particulariy of extrasynaptic as opposed to synaptic receptors, mediates, at least in part, neuronal or synaptic damage in many neurological disorders, such as hypoxic-ischemic brain injury and, as recently suggested, in Down syndrome. Blockade of excessive NMDAR activity must be achieved without interference with its normal brain function. We have taken two approaches for clinically-tolerated pharmacological and genetic intervention on NMDARs. One approach is to use Memantine but also NO species to further down regulate the NMDAR by S-nitrosylation. The structural determinants on NMDARs for the action of Memantine and NO-like species will be characterized further under the auspices of this grant. Another approach is to utilize the inhibitory effect of a novel family of NMDAR subunits, composed of NRSA and NRSB, to downregulate NMDARs by affecting channel permeability, in a sense mimicking the effect of the NMDAR antagonist drugs that are also being developed here. We will study the role of the MS domain of NRS subunits that downregulate activity of NMDARs and also design NRS ligand-binding domain (LBD)-based screening assays to discover new compounds that modulate NRS-containing receptors. These agents will be useful for characterizing NRS-containing receptors, and possibly for neuroprotection. Accordingly, the Specific Aims of this proposal are as follows: 1) To study the effect of S-nitrosylation/redox modulation of the loose linker region between the amino-terminal domain (ATD) and the LBD of NMDARs by electrophysiology; 2) To develop LBD-derived screening assays to screen for ligands selective for the NRS subunit of the NMDAR. These ligands will be further characterized and refined by secondary assays, chemical modification, and co-crystallization; 3) To study the inhibitory effect of peptides derived from the out vestibule (MS) region of NRS subunits on NMDAR permeability.

Project II - Lipton Program Director/Principal Investigator (Last, First, Middle): Llpton, Stuart A. PROJECT SUMMARY (See instructions): Since NMDA-type glutamate receptors play an important role in both normal and abnormal function of the CNS, it is important to develop a drug that has minimal effects on normal NMDAR function, such as synaptic transmission and plasticity, but has significant effects during pathological conditions of excessive stimulation. This Team of Investigators was the first to show that Memantine, a charged adamantane drug that is a low affinity NMDAR antagonist, is clinically tolerated because of its unique mechanism of action in the NMDAR channel, termed Uncompetitive, Fast Off-rate {UFO; Lipton, 2007). This work greatly contributed to the FDA approval of Memantine for Alzheimer's disease. In the past five years, we developed drugs with improved efficacy over Memantine, termed NitroMemantine drugs. The new drugs take advantage of additional sites on the NMDAR for S-nitrosylation (transfer ofthe NO group to critical Cys residues), which we discovered decrease excessive receptor activity, in combination with the channel blocking effect of Memantine. We found that NitroMemantine compounds decrease hypoxic-ischemic disease in the newborn and adult to a greater extent than Memantine. These drugs avoid systemic side effects of NO by targeting the NO group to the NMDAR via attachment to the Memantine moiety (as shown in Project I). Of importance, redox-active adduct of NitroMemantine (the nitro group) is an alkyl nitrate and therefore lacks the toxicity of true nitric oxide (NO) since it has one less electron than NO' and consequently has different chemical reactivity. Our studies with novel/safe NMDAR antagonists have important implications for the treatment of various forms of mental retardation and developmental disabilities due to overstimulation of extrasynaptic glutamate receptors that injure synapses, as we recently published. Here, we propose to develop these novel NMDAR antagonists in vitro and in vivo in animal models to treat cognitive dysfunction in Down syndrome by carrying out the following specific aims. 1) To characterize the contribution of synaptic vs. extrasynaptic NMDAR activity in p-amyloid (AB)¿mediated neuronal damage related to Down syndrome. 2) To restore synaptic vs. extrasynaptic 'balance' in the hippocampal autapse in vitro model using the novel NMDAR antagonist NitroMemantine vs. Memantine in order to prevent B-amyloid (AB)¿mediated neuronal damage. For these experiments, we will use induced pluripotent stem cell (iPSC)-derived and fetal brain-derived neurons from Down syndrome patients. 3) To treat the cognitive deficits associated with Down syndrome in vivo in the Ts65Dn mouse model by normalizing the balance between synaptic and extrasynaptic activity with NitroMemantine treatment.

DESCRIPTION (provided by applicant): Cyanide poisoning is a potential bioterrorist agent, is life threatening, and acute exposure results in hypoxic brain injury, cardiopulmonary failure, and death within minutes to hours. Such acute toxicity is treatable by various antidotes, but success depends on rapid administration and effective penetration of the various tissues. However, even with treatment, acute or chronic cyanide intoxication in humans can induce a delayed neurological syndrome, including dystonia. Typically, these patients show Parkinsonian symptoms after weeks to months, with progressive rigidity and predominant features of flexed upper limbs and extended lower limbs. CT and MRI examinations of these patients have consistently revealed lesions in the basal ganglia, including the globus pallidus and putamen. Damage in the human brain has been confirmed via autopsy. Similar observations have been reported following both acute and chronic cyanide exposure. Multiple mechanisms are thought to underlie cyanide-induced neuronal damage, including inhibition of Cytochrome C oxidase (CcOX) and generation of reactive oxygen species (ROS) in response to cyanide- induced lipid peroxidation. Particularly harmful to neurons is dysfunctional glutamate transport and ionic pump failure, contributing to excitotoxic neuronal cell death by overstimulation of NMDA-type glutamate receptors (NMDARs). Furthermore, cyanide induces chemical reduction of the redox site(s) of the NMDAR, which were originally discovered in our laboratory, and result in additional activation of NMDAR-operated ion channels, Ca2+ influx, and consequent neuronal damage; these mechanistic considerations account for the fact that anti- oxidants and NMDAR antagonists can prevent cyanide-induced neuronal damage. As both toxic effects of cyanide by inhibition of CcOX and potentiation of NMDARs converge on oxidative stress, one possible strategy for the development of neuroprotective drugs for cyanide poisoning is to search for low-molecular-weight compounds that can counter oxidative damage. Here, as potential countermeasures against CNS toxicity by cyanide, we propose to use carnosic acid (CA), an electrophilic compound from rosemary extract, which crosses the blood-brain-barrier to exert effects by up-regulating endogenous anti-oxidant enzymes via the Nrf2 transcriptional pathway. We hypothesize that such electrophilic compounds have an advantage over antioxidant molecules for counteract the toxic effect of cyanide in brain because their action is more sustained and amplified by transcription-mediated signaling pathways. We will test this hypothesis by investigating the neuroprotective activity of CA in culture neurons exposed to cyanide and in a mouse model of cyanide poisoning. Specific Aims of this proposal are as follows: Specific Aim 1. To investigate neuroprotective effects of CA in an in vitro model of cyanide poisoning. Specific Aim 2. To examine neuroprotective effects of CA in an in vivo mouse model mimicking the effects of cyanide ingestion.

PROJECT SUMIVIARY (See instructions): The Administrative and Statistical Core (CORE A) is overseen by the PI, Dr. Lipton. This Core organizes and handles supply ordering, equipment maintenance, budgets, preparation of grant reports and manuscripts, scheduling of weekly Program Meetings, scheduling relevant seminars, and other administrative duties of the Program Project. Since ordering is centralized for all members of the Program Project, an economies of scale will be achieved by insuring that duplicate orders do not occur between the various projects and cores, and also that the various projects and cores can share similar orders. Additionally by ordering in bulk for the entire Program Project, discounts can often be obtained. The Statistical Services ofthe Core will be provided by Professor lan Abramson, a Ph.D. statistician who is expert in this area and housed in the nearby Department of Mathematics at UC San Diego, where the P.I., Dr. Lipton, also has a co-appointment. The statistical component of the Core will provide statistical assistance with experimental design, data evaluation, and manuscript preparation. Statistial services are extremely important to several aspects of the planned experiments analyzing the effectiveness of NMDA receptor antagonists and related molecules in ameliorating cognitve dysfunction in animal models (and eventually humans) with Down syndrome (DS). Power analyses performed by the Statistician are useful in planning how many animals will be required for a given experiment in order to have adequate probability of detection of an expected difference. Project I will use the Statistician to analyze data for significance. Projects II and III will use Power Analyses to plan experiments in terms of how many animals need to be tested in each paradigm. All three projects will order supplies by the Administrative Core and use the secretarial/managerial sen/ices provided.

DESCRIPTION (provided by applicant): The unifying theme of this revised P01 Grant renewal is a cellular and molecular approach to intellectual and developmental disabilities in an attempt to uncover processes contributing to neuronal synaptic damage, particularly in Down syndrome (DS). Three inter-related projects are planned. They all study overstimulation of the N-methyl-D-aspartate subtype of glutamate receptor (NMDAR), leading to synaptic damage and cognitive dysfunction in children. Here we show that oligomers of AP protein, as found in DS with or without Alzheimer's disease, can trigger excessive stimulation of extrasynaptic NMDARs, contributing to loss of thin dendritic spines, with resulting compromise of synaptic function and cognitive ability. This P01 Grant is credited with developing the first clinically-tolerated NMDAR antagonist, Memantine, which we showed is an uncompetitive, open-channel blocker with a relatively fast off-rate, accounting for its clinical tolerability. We then developed new, more effective drugs, called NitroMemantines, for the treatment of neonatal hypoxic- ischemic brain damage. NitroMemantines act on NMDAR channels (like Memantine) but also donate NO species to react at nitrosylation sites on the NMDAR to further downregulate excessive activity better than Memantine. Additionally, we plan to develop novel drugs based on structure-function relationships of the NR3 family of NMDAR subunits, which were discovered under the auspices of this PO1 grant. Project I will study the basis of NitroMemantine action and develop new NMDAR antagonists based on NR3 structure- function. Project II will test NitroMemantine vs. Memantine to prevent synaptic damage and cognitive deficits in DS using human fetal and IPS cell-based models in culture and the mouse Ts65Dn model in vivo. Project III will complement Project II by taking a genetic rather than a pharmacologic approach to downregulating excessive NMDAR activity or its downstream effects. Accordingly, Project III will test genetic models of altered NR3 genes or the downstream takusan family of genes for neuroprotection in similar in vitro and in vivo models of DS as in Project 11. The CORE supports administration, statistics, tissue culture, and crystallography/modeling of NMDAR subunits and functional sites, all critical to the proposed Projects.

DESCRIPTION (provided by applicant): The number of HIV (human immunodeficiency virus)-positive individuals has risen over recent years mainly because combination antiretroviral therapy (cART) is helping these patients live longer. However, the prevalence of HIV-Associated Neurocognitive Disorders (HAND) has also risen since cART drugs in general do not effectively cross the blood-brain-barrier. To address this issue, we propose to evaluate a new type of treatment targeted for HAND. In patients with HAND and in animal models of the disease process, we and others have accumulated evidence that neurons are damaged. Additionally, emerging evidence from our laboratory and others suggests that adult neuroprogenitor cells (aNPCs) are also decreased in the brains of patients with NeuroAIDs. Active neurogenesis normally occurs throughout life in the dentate gyrus in the hippocampus. Newly generated neurons are incorporated into the neural network, contributing to certain types of learning and memory. Intriguingly, hippocampal neurogenesis is significantly altered in several neurodegenerative diseases in addition to HAND. Recently, we reported that the HIV-envelope glycoprotein gp120, which is associated with HAND pathogenesis, inhibits proliferation of adult neural progenitor cells in vitro and in vivo in the dentate gyrus of the hippocampus in a NeuroAIDS rodent model, the HIV/gp120- transgenic mouse. As potential treatment, we and our collaborators have synthesized new peptide inhibitors of chemokine receptors (CXCR4 and CCR5, which are co-receptors for HIV/gp120), and found that these novel synthetically and modularly modified (SMM)-chemokines block the inhibitory effect of gp120 on neural progenitor proliferation in vitro. Here, we propose to investigate the effectiveness of SMM-chemokines in vivo, beginning with new, very selective and non-toxic CXCR4 antagonists. In particular, we propose to investigate whether transnasal (also called intranasal) application of SMM-chemokines can prevent the decrease in proliferation of adult neural progenitors in mouse models of NeuroAIDS. Notably, we have recently shown that transnasally administered-peptides are preferentially delivered to the brain, and other peptides have been approved in humans by the FDA for brain delivery in this fashion. We list the following Specific Aim: (1) To investigate whether transnasal application of SMM-chemokine analogs protects adult neurons and restores the proliferation of adult hippocampal progenitors in HIV/gp120-transgenic mice. Note that in future work, we plan to test for 'generalization' of the effect of the SMM-chemokine analogs by investigating whether transnasal application of SMM-chemokine analogs also protects neurons and restores the proliferation of adult hippocampal progenitors in a second NeuroAIDS model, consisting of NOD/scid-IL- 2Rgcnull mice that are reconstituted with human hematopoietic CD34+ stem cells and then infected with HIV-1. This model, as developed by our collaborator Dr. Howard Gendelman and colleagues, while more difficult to utilize, closely mimics human HAND progression and cognitive dysfunction.

DESCRIPTION (provided by applicant): This R01 grant aims to identify new molecular pathways and treatments to prevent mitochondrial-based injury and dopaminergic (DA) neuronal cell death in Parkinson disease (PD). As tools, we will take advantage of the potential interplay of genes mutated in PD and exposure to environmental risk factors, such as certain pesticides, that might contribute to disease in part as mitochondrial toxins. Although several epidemiological studies have suggested an association of pesticides, particularly the combination of paraquat (PQ) and maneb (MB), to the etiology of PD, evidence for a direct role of their effect on human DA neurons remains poorly studied. One reason for this is the inability to effectively model the disease in human cells, in part due to the nature of PD manifestations (i.e., late onset and slow progression of pathology), and in part due to complications arising from epistatic effects of the patient's genetic background that might influence the outcome after exposure. To overcome these problems, we are using a human iPSC model of PD in which the control and mutant cells are genetically identical (isogenic) except for a single pathogenic poin mutation in the ?-synuclein locus (A53T). This model gives us an unprecedented opportunity to examine the vulnerability of human A9-type DA neurons after pesticide exposure with regard to genetic background. We mount preliminary data that decreased activity of MEF2, a transcription factor involved in both neurogenesis and neuroprotection, may play a contributory role in PD pathogenesis due to environmental or genetic insult. We find that the pesticides PQ, MB, or rotenone affect mitochondrial function in DA neurons, producing excessive nitric oxide (NO) and reactive oxygen species (ROS). NO/ROS lead to aberrant S-nitrosylation/oxidation of MEF2 (forming SNO-MEF2 and SOH-MEF2). These posttranslational modifications impair MEF2 transcriptional activity. We identify potential downstream cellular events resulting from nitrosylation/oxidation of MEF2, including a decrease in the transcriptional co-activator molecule PGC1?, whose gene is regulated by MEF2 and is a key regulator of mitochondrial function. We will next attempt to rescue hiPSC-derived neurons from PD-related cell death by (a) preventing nitrosylation/oxidation of MEF2 via genetic modification of MEF2, or (b) boosting PGC1? activity. Finally, in our HTS Center, as potential therapeutics we will identify small molecules tht increase MEF2 activity or prevent its oxidation. Thus, these studies will elucidate molecular events linking genetic and environmental risk factors in PD, and we will use this information to develop novel therapeutic targets for drug screening for the treatment of PD in the human context (by using hiPSC-derived DA neurons). As Specific Aims we plan: Aim 1. To characterize environmental risk factor- induced vulnerability in isogenic hiPSC A53T-?-synuclein vs. WT DA neurons (and vs. non-DA neurons). Aim 2. To elucidate pathways for this susceptibility involving SNO-MEF2 of SOH-MEF2. Aim 3. To screen for novel agents that protect from this genetic or environmentally-induced neuronal injury in PD.

? DESCRIPTION (provided by applicant):Emerging evidence suggests the heuristic model that both HIV/AIDS and several forms of drug abuse affect the brain via free radical damage involving the generation of reactive oxygen species (ROS, producing oxidative stress) and reactive nitrogen species (RNS) such as nitric oxide (NO, resulting in nitrosative stress). For example, both HIV- associated neurocognitive disorder (HAND) and methamphetamine appear to produce neuronal damage via oxidative and nitrosative stress (Cadet and Krasnova, 2007). However, the cellular and molecular targets of this free radical stress remain largely unknown. These unknown protein targets are a major challenge for the field and need to be identified in order to develop both Biomarkers of disease and to allow screening for new therapies to prevent corruption of these targets by free radical stress. Here, I propose an innovative approach using newly emerging Mass Spectrometry (MS) techniques to identify the posttranslational modifications (PTMs) of proteins resulting from such nitrosative- and oxidative-induced redox stress and hence identify new protein targets affected by AIDS and drug abuse. Heretofore, it has not been possible to identify the full range of proteins undergoing PTMs due to nitrosative and oxidative stress, but new MS techniques should allow this identification. These newly identified target proteins are expected to serve as Biomarkers of the disease process and also should allow us to direct future therapeutic intervention by discovering new pathogenic pathways.

The overall goal of this interinstitutional NINDS P30 Core Grant is to foster new interdisciplinary collaborations among neuroscientists in La Jolla institutions by allowing access to scientific cores to which they were previously denied or had little access, and by promoting new cores to promote state-of-the-art technology; these cores were started under the prior auspices of the NIH Blueprint Core Grant (P30 NS057096) that was awarded to the PI, Dr. Lipton, for the past five years. Since the entire Blueprint Core Grant Program has been terminated by NIH, NINDS Program Officials asked us to submit this NINDS P30 Core Grant. To accomplish our goal, we have chosen among the very best core facilities on the Blueprint Core Grant, and have chosen top scientists to run these cores at the Sanford-Burnham Medical Research Institute (SBMIR) and the Unviersity of California San Diego (UCSD) in order to assist NINDS neuroscientists in La Jolla. The cores proposed here represent (i) High-Throughput (HS) Library Screening (at SBMRI), (ii) Neuropathology (at UCSD), and (iii) Electrophysiology (at SBMIR with Advisors at Salk), in addition to (iv) an Administrative Core (centered at SBMRI). These Core Facilitites will be available to neuroscientists at all four institutions on the La Jolla Torrey Pines Mesa, composed of SBMRI, UCSD, Salk, and The Scripps Research Institute. These institutions share one of the top-rated neuroscience graduate programs in the country and have committed considerable institutional funds towards these cores. This work is aimed at developing new treatments for neurological disorders. The Specific Aims, which will be facilitated by all 3 Scientific Cores and the Administrative Core, are as follows: 1. To obtain Core support for Neuroscientists studying Neurodegenerative Disorders. The Cores will also help develop Neuroprotective Therapies. These disorders, represented in the Qualifying NINDS Projects of Major USERS, include Alzheimer's, Parkinson's, ALS, and Huntington's disease. 2. To obtain Core support for Neuroscientists studying Neoplastic Disorders of the brain. These disorders, represented in the Qualifying NJNDS Projects of Major USERs, include glioblastoma multiforme and medulloblastoma tumors. 3. To obtain Core support for Neuroscientists studying Developmental Disorders. Disorders include abnormal neuronal migration, and are represented in NINDS Qualifying Projects of Major USERs.

The overall goal of this interinstitutional NINDS P30 Core Grant is to foster new interdisciplinary collaborations among neuroscientists in La Jolla institutions by allowing access to scientific cores to which they were previously denied or had little access, and by promoting new cores to promote state-of-the-art technology; these cores were started under the prior auspices of the NIH Blueprint Core Grant (P30 NS057096) that was awarded to the PI, Dr. Lipton, for the past five years. Since the entire Blueprint Core Grant Program has been terminated by NIH, NINDS Program Officials asked us to submit this NINDS P30 Core Grant. To accomplish our goal, we have chosen among the very best core facilities on the Blueprint Core Grant, and have chosen top scientists to run these cores at the Sanford-Burnham Medical Research Institute (SBMIR) and the Unviersity of California San Diego (UCSD) in order to assist NINDS neuroscientists in La Jolla. The cores proposed here represent (i) High-Throughput (HS) Library Screening (at SBMRI), (ii) Neuropathology (at UCSD), and (iii) Electrophysiology (at SBMIR with Advisors at Salk), in addition to (iv) an Administrative Core (centered at SBMRI). These Core Facilitites will be available to neuroscientists at all four institutions on the La Jolla Torrey Pines Mesa, composed of SBMRI, UCSD, Salk, and The Scripps Research Institute. These institutions share one of the top-rated neuroscience graduate programs in the country and have committed considerable institutional funds towards these cores. This work is aimed at developing new treatments for neurological disorders. The Specific Aims, which will be facilitated by all 3 Scientific Cores and the Administrative Core, are as follows: 1. To obtain Core support for Neuroscientists studying Neurodegenerative Disorders. The Cores will also help develop Neuroprotective Therapies. These disorders, represented in the Qualifying NINDS Projects of Major USERS, include Alzheimer's, Parkinson's, ALS, and Huntington's disease. 2. To obtain Core support for Neuroscientists studying Neoplastic Disorders of the brain. These disorders, represented in the Qualifying NJNDS Projects of Major USERs, include glioblastoma multiforme and medulloblastoma tumors. 3. To obtain Core support for Neuroscientists studying Developmental Disorders. Disorders include abnormal neuronal migration, and are represented in NINDS Qualifying Projects of Major USERs.

The overall goal of this interinstitutional NINDS P30 Core Grant is to foster new interdisciplinary collaborations among neuroscientists in La Jolla institutions by allowing access to scientific cores to which they were previously denied or had little access, and by promoting new cores to promote state-of-the-art technology; these cores were started under the prior auspices of the NIH Blueprint Core Grant (P30 NS057096) that was awarded to the PI, Dr. Lipton, for the past five years. Since the entire Blueprint Core Grant Program has been terminated by NIH, NINDS Program Officials asked us to submit this NINDS P30 Core Grant. To accomplish our goal, we have chosen among the very best core facilities on the Blueprint Core Grant, and have chosen top scientists to run these cores at the Sanford-Burnham Medical Research Institute (SBMIR) and the Unviersity of California San Diego (UCSD) in order to assist NINDS neuroscientists in La Jolla. The cores proposed here represent (i) High-Throughput (HS) Library Screening (at SBMRI), (ii) Neuropathology (at UCSD), and (iii) Electrophysiology (at SBMIR with Advisors at Salk), in addition to (iv) an Administrative Core (centered at SBMRI). These Core Facilitites will be available to neuroscientists at all four institutions on the La Jolla Torrey Pines Mesa, composed of SBMRI, UCSD, Salk, and The Scripps Research Institute. These institutions share one of the top-rated neuroscience graduate programs in the country and have committed considerable institutional funds towards these cores. This work is aimed at developing new treatments for neurological disorders. The Specific Aims, which will be facilitated by all 3 Scientific Cores and the Administrative Core, are as follows: 1. To obtain Core support for Neuroscientists studying Neurodegenerative Disorders. The Cores will also help develop Neuroprotective Therapies. These disorders, represented in the Qualifying NINDS Projects of Major USERS, include Alzheimer's, Parkinson's, ALS, and Huntington's disease. 2. To obtain Core support for Neuroscientists studying Neoplastic Disorders of the brain. These disorders, represented in the Qualifying NJNDS Projects of Major USERs, include glioblastoma multiforme and medulloblastoma tumors. 3. To obtain Core support for Neuroscientists studying Developmental Disorders. Disorders include abnormal neuronal migration, and are represented in NINDS Qualifying Projects of Major USERs.

Project Summary The systemic diseases, metabolic syndrome (MetS) and Type 2 diabetes mellitus (T2DM), increase risk for Alzheimer's disease (AD). The molecular mechanism for this association remains poorly defined. Here, in preliminary studies we show in human and rodent tissues that elevated glucose, as found in MetS/T2DM, and oligomeric ?-amyloid (A?) peptide, thought to be a key mediator of AD, coordinately increase neuronal Ca2+ and nitric oxide (NO) in an NMDA receptor-dependent fashion. The increase in NO results in nitrosative stress and consequent S-nitrosylation of insulin-degrading enzyme (IDE) and dynamin-related protein 1 (Drp1), thus inhibiting insulin and A? catabolism as well as hyperactivating mitochondrial fission machinery. Consequent elevation in A? levels and compromise in mitochondrial bioenergetics result in dysfunctional synaptic plasticity and synapse loss in cortical and hippocampal neurons. The NMDA receptor antagonist memantine attenuates these effects to some degree, and we posit that the new, improved NMDA receptor antagonist, NitroMemantine, will manifest an even greater beneficial effect. Our preliminary studies show that the redox-mediated posttranslational modification of S-nitrosylation affects brain protein function, thus linking A? and hyperglycemia to cognitive dysfunction in MetS/T2DM and AD. We propose novel studies that do not merely extend our prior findings but instead offer innovative insights by determining a comprehensive compendium of the S-nitrosoproteome and its pathological downstream effects in these diseases. Accordingly, our Specific Aims are as follows: Aim 1: To characterize the aberrant S-nitrosoproteome in rat cerebrocortical neuronal cultures, cortico- hippocampal slices, and in hiPSC-derived neurons in in vitro models of Alzheimer?s disease (AD) and type 2 diabetes mellitus (T2DM)/metabolic syndrome (MetS). Aim 2: To determine in vivo if brains of db/db (leptin receptor-deficient) mice, as a model of T2DM/MetS, when crossed with J20-hAPP (human amyloid precursor protein overexpressing) AD transgenic (tg) mice, manifest more synaptic and neuronal damage than either mouse model alone. High fat/high glucose fed (to induce the T2DM/MetS phenotype) AD tg mice will also be tested in this manner. Aim 3: To determine (a) if treatment with NitroMemantine protects from synaptic and neuronal damage in T2DM/AD genetic mouse models to a greater degree than memantine; and (b) if non-nitrosylatable mutant knockin mice for IDE, Drp1 or new targets are protected from T2DM/AD-mediated synaptic injury.

SUMMARY This collaborative R01 application between a neuroscience lab (led by Stuart Lipton at Scintllon Inst./UC San Diego) and a chemistry lab (led by Steve Tannenbaum at MIT) will identify the redox posttranslational modification of proteins called S-nitrosylation by developing a more effective and integrated Mass Spec-based platform to screen for the S-nitrosoproteome and resulting alterations in protein function that contribute to the pathogenesis of Alzheimer?s disease (AD). Our hypothesis is that entire biochemical pathways critical to neuronal function are affected by aberrant S-nitrosylation of multiple proteins, these aberrant redox reactions (which are located, at least in part, downstream of Aß insult) contribute to the pathogenesis of AD, and the reactions occur in both sporadic and familial cases of the disease. Chemical and functional analysis of S- nitrosylated proteins will be assessed by biochemical assays, and by imaging of cells and tissues, including human AD brain and various in vitro and in vivo models of AD, ranging from transgenic mice to hiPSC-based model systems. We will also use site-directed mutagenesis and CRISPR/Cas9 techniques to generate DNA constructs or genes encoding proteins that that cannot be S-nitrosylated (thus forming non-nitrosylatable proteins). Accordingly, our Specific Aims are as follows: AIM #1. To determine the S-nitrosoproteome in human AD brain and transgenic mouse models. We will validate our recent S-nitrosoproteome findings in the CK-p25 mouse model of AD (published in PNAS, 2016) and determine if it generalizes to human AD brain and other transgenic mouse models of AD, e.g., hAPP-J20 and Tg2576. AIM #2. To use hiPSC-derived cerebrocortical neurons generated from human AD patients or WT exposed to oligomeric Aß (as a model of sporadic AD) as an in vitro model system to study the S-nitrosoproteome and how it affects biochemical pathways. This approach will allow us to study the functional effect of SNO-proteins in AD in a human context. AIM #3. To screen the effects of various S-nitrosoproteins in hiPSC-based models for impact on potential biological functions, e.g., effect on synaptic loss or neuronal cell death. This will be accomplished by generating non-nitrosylatable constructs of proteins (e.g., substituting Ala for Cys) by replacing the underling gene by CRISPR/Cas9 technology. For selected gene products that manifest profound effects of S- nitrosylation on synaptic functions and neuronal cell survival in hiPSC-based models, the non-nitrosylatable version of the gene can also be created in mice using CRISPR/Cas9 to mechanistically test its effect in vivo.

COVID-19-Related Administrative Supplement to NIA RF1 AG057409 under PA-18-591 and NOT-AG-20-022 PROJECT SUMMARY Epidemiological studies of pandemic COVID-19 suggest that aged populations, especially those with Alzheimer?s disease and related dementias (AD)/ADRD, are particularly vulnerable. We therefore propose an Administrative Supplement for work in line with the Division of Neuroscience at NIA, namely, ?studies aimed at discovery and development of novel drugs, as well as repurposing and repositioning existing drugs, for preventing and treating COVID-19, particularly drugs that are specific for COVID-19 related CNS targets and CNS mechanisms related to or driving the viral-mediated pathophysiology.? Specifically, we will test drugs developed in the parent RF1 award by screening them for anti-viral activity to fight the infection and treat potential CNS ramifications in AD/ADRD and aged populations. Intriguingly, aminoadamantane drugs (e.g., amantadine, rimantadine, and memantine) were first discovered as anti-viral agents because they can block the ion channel found in the envelope of viruses such as influenza, but also found in the SARS-CoV family. The PI, Dr. Lipton, subsequently found that these aminoadamantanes had activity in the CNS by blocking excessively-activated NMDAR-associated ion channels, and Lipton?s work eventually led to FDA approval of memantine for use in AD. Recently, the Lipton group designed and synthesized aminoadamantane nitrate drugs under the auspices of the parent RF1 Award to be used to inhibit excessively-activated NMDARs to a much higher degree than memantine by adding a nitro-based warhead to an aminoadamantane in order to S-nitrosylate (via covalent reaction of NO) and thus further inhibit receptor activity in a targeted fashion. As a seemingly amazing coincidence, it was recently reported that the SARS-CoV family of viruses are susceptible to NO, in part by inhibiting their replication cycle. However, the delivery of NO or a NO-related species to an already ill patient could have severe consequences, such as lowering the blood pressure dramatically. Hence, in this proposal we develop a novel targeted delivery of NO-related species directly to the SARS-CoV-2 virus by using the aminoadamantane moiety that binds to the envelope ion channel and has a nitro-based warhead that it then delivers directly to the virus. Another critical feature of the current proposal is that we use these same aminoadamantane nitrate compounds to protect the brain from injury potentially engendered by the virus via inhibition of excessive NMDAR activity. Importantly, up to 37% of patients hospitalized for severe COVID- 19 reportedly display neurological sequelae. Mechanistically in this regard, in the face of severe viral infections, including coronaviruses, excessive levels of glutamate are released (or not taken up) by astrocytes, leading to glutamate-related neurotoxicity (excitotoxicity). In our parent RF1 grant, we reported similar findings in AD, i.e., that Aß-stimulated astrocytes release glutamate onto neurons. Therefore, this Administrative Supplement will test the top ?hits? of aminoadamantane nitrates capable of inhibiting SARS-CoV-2 in additional screens for their ability to prevent viral-related damage to neurons in the brains of AD/ADRD and aged populations.

COVID-19-Related Administrative Supplement to DP1 DA041722 under PA-18-591 and NOT-DA-20-047 PROJECT SUMMARY The worldwide pandemic of the 2019 novel coronavirus, or COVID-19, has led the research community to believe the possibility that it could affect some populations with substance use disorders or HIV particularly hard. Therefore, we propose new work here that is ?in scope? with our parent NIDA DP1 grant (DP1 DA041722) that would potentially address the pandemic, at least in part, by developing an anti-viral drug to fight the infection. We propose, as listed in NOT-DA-20-047, to perform ?research to develop therapeutic approaches for comorbid SARS-CoV-2 infection.? In the parent DP1 grant, we are studying the nitric oxide (NO)-related posttranslational modification of proteins, which we previously named S-nitrosylation, in patients with HIV-associated neurocognitive disorder (HAND) and drug use, particularly methamphetamine. During the course of these studies, we developed a novel series of therapeutic agents in the class of compounds called aminoadamantane nitrates, with the lead drug designated NitroSynapsin, that have shown activity in protecting neurons in the context of HIV/methamphetamine abuse as well as in the context of Alzheimer?s disease and other neurologic disorder. Our novel approach concerns the fact that this family of agents that we developed may also show activity at the ion channel in the envelope of the SARS-CoV-2 virus, the causative agent of the COVID-19 pandemic. The mechanism of action (MOA) that we propose against SARS-CoV-2 is best summarized as follows: Compounds in the aminoadamantane family are generally known to block ion channels in envelope viruses, including SARS-CoV-2, which causes COVID-19 respiratory disease. Moreover, nitric oxide (NO) and related compounds have been reported to inhibit this class of viruses. We reasoned in a novel fashion that the targeted delivery of NO-related species to the virus would avoid systemic side effects of NO-like drugs. For this purpose (but originally for use in the brain), we had devised a series of aminoadamantane nitrates, with the aminoadamantane moiety acting as a ?guided missile? to enter the viral envelope channel and then deliver a ?warhead? of a nitro group directly to the virus to disrupt viral activity. Accordingly, we propose to rapidly test our drugs in an ongoing screen against SARS-CoV-2 viral activity in our Calibr Drug Development Core Facility at The Scripps Research Institute in La Jolla, California.

We propose a novel target and new drug to treat Alzheimer?s disease (AD) via the Nrf2 transcriptional pathway. Our drug candidates for this target are currently at the stage of active hit-to-lead optimization, as described below. As background, AD is a condition with loss of synapses and neurons in the brain, characterized by the presence of amyloid beta (A?) plaques and tau tangles, which cause damage to synapses and neurons at least partially via oxidative stress. Our prior studies have indicated that carnosic acid (CA), a component compound in the herb Rosemary, can protect neurons and synapses from damage caused by oxidative stress by activating the Nrf2 transcriptional pathway. Our Preliminary Data show that CA treatment ameliorates various behavioral and histological deficits in AD transgenic mutant mice, while showing no significant side effects. Activation of the Keap1/Nrf2 (kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2) pathway upregulates transcription of phase II antioxidant and anti-inflammatory proteins. We and others have shown that this can be a valuable therapeutic strategy in several neurodegenerative diseases. Here, we further test this approach in mouse models of AD using carnosic acid (CA), which we have shown in our publications to activate the Keap1/Nrf2 pathway. CA is known to be clinically tolerated because of its presence in herbs like Rosemary. Conversely, in humans with AD, a decreased expression pattern of Nrf2 in hippocampal neurons and astrocytes, as well as a significant decrease in nuclear Nrf2 levels in frontal cortex, have been reported. The Keap1/Nrf2 pathway can be activated by an electrophilic compound when it reacts with a specific thiol on Keap1, releasing Nrf2 in the cytoplasm to enter the nucleus where it binds to the antioxidant responsive element (ARE) on the promoters of phase II genes. An important issue clinically with regard to electrophilic drugs (such as dimethyl fumarate, curcumin, and Bardoloxone) is that they not only react with Keap1 to activate Nrf2, but they also non-specifically react with other thiol groups, which may explain both their actions and side effects. Our alternative, innovative strategy to avoid such side effects is to use pro-electrophilic compounds that are activated by the very oxidation in redox-stressed cells that is injurious. The compound CA represents a starting point for such a pro-electrophilic drug (PED). Accordingly, our Specific Aims/Goals are ? Aim/Goal 1. To screen for and characterize the neuroprotective effects of PEDs that activate the Nrf2/ARE transcriptional pathway in an in vitro model of oligomeric Aß-induced oxidative damage and neuroinflammation using hiPSC-derived cortical neurons and cerebral organoids. Aim/Goal 2. To determine the lead PED by assessing neuroprotective effects by neurobehavior and histology in vivo in AD transgenic mouse models (hAPP-J20 and 3x Tg mice) and optimize formulation of the lead. Toxicity testing will also be performed.

SUMMARY This R35 leadership application is relevant to Milestone 6D of the goals/milestones of the NIH Alzheimer?s disease and Alzheimer?s disease related dementias (AD/ADRD) Summits??Initiate drug discovery efforts to develop novel therapeutic agents.? Our overriding goal in this application is to support the infrastructure the PI is assembling for AD/ADRD drug discovery at Scripps Research, which will continue well after the award is over. Our major planned milestone is aimed to INSPIRE program building and create team building for AD/ADRD Drug Discovery using the very considerable institutional resources at Scripps Research and its Calibr Drug Discovery Center. We are also building one of the nation?s top-ranked Graduate School programs in drug discovery via our mentoring using the faculty of Scripps? #1/#2-ranked chemistry- biochemistry departments in the world. As proof of feasibility, of the six currently FDA-approved medicines for AD, three were developed out of the PI, Dr. Lipton?s laboratory. Via this R35 Leadership Application, Dr. Lipton will mentor others to develop AD/ADRD-related drugs acting at novel targets toward disease-modifying therapy. Specifically, Dr. Lipton will serve as research mentor for New Investigators and Early Stage Investigators (NI/ESI) in AD/ADRD drug discovery. To date, virtually all AD/ADRD therapeutics evaluated in human clinical trials have so far failed to show disease-modifying effects for AD/ADRD. For example, the lack of significant efficacy in clinical trials with A?-centered therapeutics to date demands a new paradigm for development of effective AD therapeutics. To overcome these significant gaps in knowledge and to advance therapies, improvements in the models and strategies by which AD/ADRD researchers function and execute need to occur. One solution to this challenge is to unite the best basic scientists with training in neuroscience, structural biology, bioinformatics and computational biology, with equally expert teams in the pharmaceutical industry trained in high-throughput screening, assay development, medicinal chemistry, chemical biology and pharmacology. With a collective and harmonized team along with significant mentoring efforts for junior faculty (NI/ESI), the PI, Dr. Lipton, has initiated three major efforts to address this unmet medical need, which will be carried out under the auspices of the current R35 Leadership Award application: · Investigation, identification and characterization of potential AD/ADRD therapeutic targets in the context of the central pathophysiological processes in AD/ADRD. · Execution of drug discovery campaigns to develop investigational new drug (IND) clinical candidates iteratively with the target elucidation efforts, while building upon Scripps drug discovery infrastructure to accomplish this. · Continue to mentor junior faculty and build a world-class graduate school around the chemical biology of drug design for AD/ADRD at Scripps (currently ranked #2 by US News for Biochemistry).