Elias Aizenman, Ph.D. - US grants

Nicotine is an addictive drug that rapidly reaches the brain following inhalation of cigarette smoke. Addiction to nicotine through cigarette smoking afflicts more than 50 million people in the United States, and is responsible for approximately 350,000 deaths every year. Although the effects of nicotine on the central nervous system are likely to be the primary reason for its addictive nature, little is known about the properties of central nicotinic receptors. The experiments described here are aimed at evaluating the physiology and pharmacology of nicotinic receptors on mammalian central neurons. These studies utilize isolated and identified neurons which are maintained in culture. Electrophysiological experiments are performed using patch-clamp technology. The specific aims of this study are: 1. To examine the interaction of agonists with the central nicotinic receptor. 2. To examine the effects of antagonists. 3. To evaluate the unique characteristics of the central nicotinic receptor using a specific snake neurotoxin. 4. To investigate the properties of the receptor-associated ion pore at the single-channel level. The long term objectives of the present proposal are to characterize the functional properties of the central nicotine receptor so that pertinent information may be important in understanding and possibly alleviating nicotine addiction.

The neuropathological outcome associated with abnormal activation of glutamate receptors specific for N-methyl-D-aspartate (NMDA) has been implicated in stroke and in a variety of neurodegenerative disorders. The modulation of NMDA receptor-mediated function by endogenous factors may have profound implications in the outcome of glutamate neurotoxicity. Experiments outlined in this proposal are aimed at evaluating the role of a novel modulatory site on the NMDA receptor which is sensitive to sulfhydryl redox reagents. Since the injurious sequelae accompanying cerebral ischemia/reperfusion injury involve changes in redox equivalents, including the generation of oxygen free radicals, this modulatory site on the NMDA receptor may be intimately involved with the outcome of stroke. The specific aims of these studies are: (1) to establish the actions of oxygen free radicals on the redox modulatory site of the NMDA receptor, (2)to study the effects of the essential nutrient pyrroloquinoline quinone (PQQ) on the redox modulatory site of the NMDA receptor, (3)to study the action of "reactive" disulfide compounds on the NMDA receptor, and (4) to examine the kinetic changes in NMDA receptor function following chemical modifications with sulfhydryl redox reagents. The experiments outlined in this proposal utilized a well-defined culture system of rat cerebral cortex. Electrophysiological studies utilizing patch-clamp technology will be combined with intracellular calcium determinations, radioactive ligand binding, and neurotoxicity assays to achieve the proposed aims. The studies planned here will provide a more accurate picture of the events accompanying ischemia/reperfusion injury in the central nervous system, especially as they pertain to NMDA receptor function. Investigations aimed at these processes could lead to better prevention programs as well as to improved treatments for stroke. In addition, these studies may lead to the discovery of probes that may specifically label the NMDA receptor and aid in its biochemical characterization.

DESCRIPTION: (Verbatim from the Applicant's Abstract) NMDA receptor activation is associated with many critical physiological and pathophysiological processes in the brain. Consequently, several endogenous modulating agents tightly regulate NMDA receptor function. Studies that investigate the molecular mechanisms of NMDA receptor modulation are important for characterizing the structural and functional properties of this protein. These investigations can also lead to the development of novel drugs to treat neuronal disorders in which abnormal NMDA receptor activation has been implicated. In this proposal, we expand our studies of NMDA receptor modulation to a novel form of functional regulation of this ion channel. We have observed that brief focal pulses of light potentiate NMDA receptor-mediated physiological responses in cultured neurons. In order to characterize this phenomenon, we have designed a series of studies to address the following Specific Aims: (1) To define the properties of light modulation of NMDA receptor function; (2) To establish whether light alteration of NMDA receptor function is mediated via a novel modulatory site; (3) To determine the properties of light modulation in recombinant NMDA receptors; and (4) To assess whether light modulation of the NMDA receptor has physiological significance in retinal function. Experiments outlined here utilize well-defined culture systems of rat cerebral cortex and retinal ganglion cells. We will perform electrophysiological measurements in these preparations, as in well as in non-neuronal mammalian cell lines that transiently express recombinant NMDA receptors. Additional studies will be performed on retinal slices. The long-term goals of this research program are to characterize fully NMDA receptor function. Results from these studies could facilitate the design of new therapeutic strategies to block NMDA receptor-mediated excitotoxicity. In addition, the investigations proposed here will focus on a novel form of modulation of NMDA receptor function, which may be important in retinal physiology.

DESCRIPTION (provided by the applicant) Vesicular Zn2+ is known to be toxic to neurons following its translocation from presynaptic release sites into the cytoplasm of postsynaptic cells that are destined to die. In addition to this extracellular source of Zn2+, we have observed that this metal can be released from intracellular metal binding proteins by thiol axidants. We have shown further that intracellularly liberated Zn2+ is a powerful apoptotic stimulus in neurons. Here, using molecular, cellular, and whole animal approaches, we propose to evaluate whether the release of intracellular Zn2+ represents a common feature in neuronal cell death following oxidative stress. The Specific Aims of this proposal are: 1. To determine whether peroxynitrite and nitric oxide trigger a neurotoxic cascade that results from the intracellular release of zinc in vitro. 2. To identify molecular components of the cell death pathway that are associated with the intracellular release of zinc in vitro. 3 To evaluate whether the intracellular release of zinc triggers apoptosis in an axotomy-induced in vivo model of neuronal cell death. Experiments described within these aims will test the hypothesis that the liberation of intracellular Zn2+ represents an important and ubiquitous trigger for cell death in neuronal injury. The long-term goal of this research program is to provide additional therapeutic targets to prevent or minimize neuronal cell death in the large number of neurological disorders associated with oxidative injury.

[unreadable] DESCRIPTION (provided by applicant): Methamphetamine abuse continues to increase in the US at alarming rates. Chronic use of this drug can lead to severe neurological and psychiatric impairments as well as to pronounced neurodegenerative changes in humans. The research proposed here is intended to define the molecular cascade leading to microglia activation and subsequent cellular injury following methamphetamine treatment. In this highly focused research project we wish to test the novel hypothesis that methamphetamine exposure leads to the liberation of the monoamine-derived glutamate receptor agonist 2, 4, 5-trihydroxyphenylalanine quinone (TOPA quinone) from catecholaminergic cells, which, in turn interacts with AMPA receptors in microglia. We further hypothesize microglia exposed to TOPA quinone become activated via an AMPA receptor-mediated process and can subsequently induce further cellular damage via a defined molecular cascade. This is an Investigator-Initiated Small Grant (R03) application requesting limited funds to test a defined hypothesis with focused objectives. This is a small, self-contained research project fitting a description for R03 support outlined in NIH announcement PA-06-180. These studies could provide a basis by which therapeutic agents may be used to prevent neuronal injury following methamphetamine exposure. PUBLIC HEALTH RELEVANCE: The research proposed here is intended to define the molecular cascade leading to microglia activation and subsequent neuronal injury following methamphetamine treatment. These studies could provide a basis by which therapeutic agents may be used to prevent neuronal injury following methamphetamine exposure. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application aims to investigate whether a mechanism used by a virus to block hepatocyte apoptosis can be translated and optimized to block neuronal cell death following injury. Although several signaling cascades responsible for neurodegenerative changes following stroke and related disorders have been well characterized, effective neuroprotective therapies to treat human conditions continue to elude us. Thus, investigations designed to uncover novel neuroprotective strategies, such as those describe here, are potentially of very high significance. Our laboratory is testing the general hypothesis that distinct cell death pathways, triggered by diverse injurious stimuli or composed of unique biochemical signaling cascades, require a set of common conditions to operate optimally. Over the last ten years, we have defined a neuronal pro-apoptotic signaling cascade characterized by a robust enhancement of voltage dependent K+ currents. This phenomenon ensures the completion of cell death programs by providing a venue for the loss of cytoplasmic K+, establishing a permissive environment for protease and nuclease activation. Interfering with the processes responsible for the apoptotic K+ current surge can effectively block neuronal cell death. In mammalian cortical and midbrain neurons, the current surge is mediated by a de novo SNARE-dependent exocytotic insertion of Kv2.1-encoded K+ channels into the cell membrane. Remarkably, a product of the translation and processing of the hepatitis C virus genomic RNA, the non-structural protein 5A (NS5A), was recently shown to effectively interfere with Kv2.1-mediated apoptotic K+ currents in liver cells and inhibit hepatocyte cell death. In preliminary studies we observed that NS5A could also be employed to rescue neurons following injury and that this protein interferes with the neuronal Kv2.1-mediated apoptotic K+ current surge. In this application we intend to (i) investigate the mechanism responsible for NS5A interference with Kv2.1 functional expression, and (ii) define the molecular domains of NS5A necessary for restricting channel function. These latter experiments will establish the minimal NS5A-derived sequences that can be used for the design of novel neuroprotective probes. The overarching goal of our research program is to devise new therapeutic strategies to protect neurons following injury. We are exploring a novel, possibly groundbreaking approach to achieve this goal by constructively harnessing a biological strategy that evolved to block cell death in the liver and translating it towards the generation of novel methods to treat stroke and other forms of neurodegeneration. PUBLIC HEALTH RELEVANCE: The research proposed in this application will help us understand the role of potassium channel function in neuronal cell death. Most importantly, research conducted during this project may reveal novel avenues for developing a new class of neuroprotective drugs to prevent brain damage during stroke and related conditions.

In the auditory system, as sound properties of the auditory environment change, auditory cells in the brain adjust their dynamic range of their responses to match changing background noise. This type of adaptation to sound is extremely important for brain auditory processing, for it provides a mechanism for allowing us to focus on specific sounds in the presence of variable background sound levels. However, the mechanisms underlying this adaptation remain unknown. This project will establish a previously unknown link between the metal zinc in the brain and control of auditory processing in the cerebral cortex of the brain. Thus, findings from this project will advance understanding about mechanisms of adaptations of sound processing and create a new framework for approaching and interpreting the role of the auditory system in the processing of sound. This joint NSF/BSF project will establish a joint US-Israel student exchange program as well as target underserved student populations both in Israel and the US and train them in problem-solving at behavioral, neural and molecular levels of analysis. Broad dissemination of the work will be made possible by active engagement with the International Society for Zinc Biology through a variety of activities.

Zinc is packaged into glutamatergic vesicles by the ZnT3 zinc transporter, and released from synaptic terminals in an activity-dependent manner. Although the basic principles underlying zinc neurotransmission have begun to be deciphered, the sensory stimuli leading to the modulation of zinc signaling as well as the role of zinc in regulating sensory processing remain unknown. The central hypothesis of this project is that sound dependent changes in zinc signaling in the auditory cortex play a role in the sound level adaptation of neuronal input-output functions to match the prevailing stimulus intensity of the acoustic environment. This project aims to: i. Identify the consequences of experience-dependent plasticity of zinc-mediated modulation on input-output functions and receptive fields of layers 2/3 auditory cortical neurons in vivo, and ii. Establish the cellular and molecular mechanisms mediating the experience-dependent alterations in zinc-dependent auditory cortical processing. These studies will thus provide the first analysis of regulation of neuronal zinc and its effects on auditory processing, at the molecular, cellular and systems levels.

DESCRIPTION (provided by applicant): The dopamine transporter (DAT) plays a crucial role in the regulation of brain dopamine (DA) homeostasis. Through re-uptake of DA, DAT serves two important functions: the termination of synaptic transmission at dopaminergic terminals, and the replenishment of vesicular DA pools. In addition to uptake or direct transport, DAT can also function to release DA. This process, which is referred to as reverse transport or efflux, is the mechanism used by potent and highly addictive psychostimulants, such as amphetamine and its analogues, to increase extracellular DA levels in motivational and reward areas of the brain. It has long being recognized that DA neurons release DA through exocytotic and non-exocytotic processes. However, the exact mechanism by which physiological signals or psychostimulants, such as amphetamine, induce DA release through DAT still remains a complex and not completely understood area of research. Thus, examining the basic mechanism(s) that affect reverse transport through DAT is critical for both understanding fundamental aspects of DA regulation and clinical intervention in DA-related brain disorders associated with the therapeutic use and abuse of psychostimulants. The long-term goal of our research program is to identify and characterize signaling mechanisms that control DA release through DAT, and elucidate the molecular actions of psychostimulants. This application is based on our recent discovery that the beta upsilon subunit of G proteins (Gbetagamma) binds DAT and regulates transporter activity. This effect was demonstrated in cultured cells, brain synaptosomes, and in vivo. More importantly, activation of Gbetagamma promotes DAT-mediated DA efflux, whereas inhibition of Gbetagamma attenuates amphetamine-elicited DA efflux in cultured cells. Finally, activation of Gbetagamma enhances whereas inhibition of Gbetagamma reduces amphetamine-evoked locomotor activity in vivo. Based on these preliminary data, the central hypothesis of this proposal is that the interaction between DAT and beta upsilon subunits promotes DA release through DAT and is involved in the actions of amphetamine. In this proposal we will i) identify the Gbetagamma interaction site(s) in DAT and their role in transporter regulation, ii) test the hypothesis that Gbetagamma is involved in DAT-mediated DA efflux, and iii) test the hypothesis that Gbetagamma is involved in amphetamine's actions in vivo. The successful completion of the studies proposed here will provide a detailed characterization of the DAT-Gbetagamma interaction and a clear understanding of its contribution to DAT reverse transport. The fact that amphetamine induces DAT reversal suggests that DA can also be released through DAT under physiological conditions. Therefore, our proposed studies will define not only the role that Gbetagamma subunits play in the addictive properties of amphetamine, but also the contribution of Gbetagamma subunits to DA homeostasis as we grow our current understanding of the molecular details underlying physiological DAT reverse transport. The long-term goal of our research program is to identify novel therapeutic targets that can be used in the treatment of neuropsychiatric disorders, including drug addiction.

Project summary. Zinc is key to many of biological processes including gene expression and enzymatic activity, however, it is toxic at high levels. Exposure of cells to high levels of zinc leads to the loss of enzyme activity, generation of reactive oxygen species (ROS), and activation of apoptosis. Excess zinc is especially toxic to the mitochondria, due to its ability to inhibit several components of electron transfer chain. In many organelles zinc concentration is regulated by a system of transposers; however, such as system has not been shown for the mitochondria. Using a combination of bioinformatics and cell biological assays we have identified ZnT9 (SLC30A9) as a candidate mitochondrial transporter. The overarching goal of this proposal is to test the hypothesis that ZnT9 is a critical regulator of mitochondrial zinc under control and pathological conditions, and that its dysfunction can lead to neuronal injury. We propose that under normal conditions, the mitochondrial proton gradient powers zinc expulsion from the mitochondria, limiting zinc toxicity and providing a neuroprotection function. We think that in damages mitochondria ZnT9 is reversed, accelerating the damage. This is a novel function for a zinc transporter and a new mechanism of neuroprotection as well as neurotoxicity. To test this model, we will pursue two specific aims. Aim 1 of the present project is focused on identifying the role of ZnT9 in mitochondrial zinc fluxes under normal conditions and in damaged mitochondria. Aim 2 will answer whether ZnT9 is cytoprotective under zinc overload and oxidative stress conditions, and whether ZnT9 reversal accelerates neuronal cells death. Our proposed studies will likely establish a new molecular determinant of mitochondrial zinc transport and a previously unrecognized component of zinc neurotoxicity. The completion of the studies proposed in this Exploratory/Development Research Program will likely provide the rationale for future investigations of ZnT9 function and dysfunction in in vivo mouse models and an exploration of the possible link of this transporter to human neurodegenerative disorders.

This year marks the 50th anniversary of John Olney?s seminal work that introduced the concept of excitotoxicity as a mechanism for neuronal cell death. Since that time, fundamental research on the pathophysiological activation of NMDA receptors has played a central role in our understanding of excitotoxic cellular signaling pathways, leading to the discovery of many potential therapeutic targets in the treatment of acute or chronic/progressive neurodegenerative disorders. Despites countless efforts, however, translational strategies aimed at inhibiting or regulating NMDA receptor-mediated excitotoxic injury have repeatedly failed in clinical trials, leaving only very few potential applications viable today. Nonetheless, highly innovative approaches in this important area of research could still yield tangible advances in the field of neuroprotection. We this in mind, we introduce here a previously unrecognized modulator of NMDA receptor-mediated excitotoxicity, namely, the ZnT1 (Slc30a1) zinc transporter. We present preliminary data showing that the interaction between ZnT1 and the highly zinc sensitive NMDA receptor subunit GluN2A strongly dictate the inhibitory, regulatory function of the metal on the receptor. Moreover, we reveal the development of a cell-penetrating peptide designed to specifically reduce the interaction between ZnT1 and GluN2A influences NMDA receptor-mediated synaptic responses. We tailor the proposed work by taking advantage of an endogenous neuronal mechanism of zinc-dependent excitotoxic tolerance, and utilize both in vitro and in vivo experimental approaches to achieve the proposed aims, which are: i) to investigate the role of the GluN2A-ZnT1 interaction in regulating NMDA excitotoxicity in vitro, and ii) to establish the role of ZnT1 upregulation and increased GluN2A-ZnT1 interaction in an in vivo model of ischemic preconditioning. If successful, the work proposed in this Exploratory/Development Research Grant (R21) proposal will define an novel approach to regulate NMDA receptor-mediated excitotoxic injury, with translational potential in future work.