Eric Klann - US grants

DESCRIPTION (provided by applicant): Oxidative damage is a key feature in the brain of Alzheimer's disease (AD) patients. In one well- studied mouse model of AD, Tg2576 mice that overexpress a double mutant form of amyloid precursor protein (APP), amyloid plaque deposition was shown to be associated with oxidative damage. In addition, these AD model mice exhibited diminished axon transport in vivo, as measured by manganese enhanced magnetic resonance imaging, long-term potentiation (LTP), and impaired hippocampus-dependent memory. We recently have found that scavenging mitochondrial superoxide by overexpression of mitochondrial superoxide dismutase (SOD-2) can prevent the aforementioned abnormalities. Although these are extremely exciting findings, it is not clear how preventing oxidative stress originating from mitochondria can reverse nearly all of the cellular and behavioral phenotypes in the Tg2576 mice. One possibility is that overexpression of SOD-2 prevents increased phosphorylation of the translation initiation factor eIF2?. Phosphorylation of eIF2? results in an inhibition of general protein synthesis, and it was shown recently that eIF2? phosphorylation is elevated in the hippocampus of Tg2576 mice. Importantly, it also was shown that the increased eIF2? phosphorylation exhibited by the Tg2576 mice could be prevented by the anti-oxidant vitamin E. Therefore, we examined the levels of eIF2? phosphorylation in Tg2576 mice that were crossed with the transgenic mice that overexpress SOD-2. Our preliminary data indicate that overexpression of SOD-2 prevents the increased eIF2? phosphorylation in the brains of Tg2576 mice. These exciting preliminary findings have prompted us to hypothesize that preventing increased eIF2? phosphorylation will prevent A?-induced blockade of LTP in vitro and reverse the aforementioned impairments in LTP and hippocampus-dependent memory displayed by the Tg2576 mice in vivo. To test this hypothesis, we will 1) determine whether A?-induced blockade of LTP requires reactive oxygen species (ROS) produced via mitochondria and/or NADPH oxidase, 2) determine whether A? induces ROS-dependent increases in eIF2? phosphorylation via mitochondria and/or NADPH oxidase, 3) determine whether A?-induced blockade of LTP requires eIF2? phosphorylation via PERK, and 4) determine whether genetic reduction of eIF2? phosphorylation prevents impairments in hippocampal LTP and memory displayed by Tg2576 mice. The results of these experiments should provide crucial information concerning whether PERK- eIF2? signaling is a target of oxidative stress in AD. Such information should be useful in developing pharmacological and therapeutic strategies for treatment of not only AD, but also other neurodegenerative diseases that involve oxidative stress and alterations in protein synthesis. PUBLIC HEALTH RELEVANCE: The overall goal of the proposed work in this application is to determine the source and mechanisms responsible for oxidative stress-induced impairments in synaptic plasticity and memory in models of Alzheimer's disease, the most common form of dementia in older individuals. It is estimated that 5.2 million people in the United States are living with Alzheimer's disease and 10 million baby boomers will develop the disease in their lifetime. Current costs of Alzheimer's are estimated to be $148 billion per year. We have proposed experiments to determine specific targets of oxidative stress that impact protein synthesis, and will use pharmacological and genetic approaches to reverse A?-induced impairments in synaptic plasticity in vitro and synaptic plasticity and memory impairments in vivo in mice that model Alzheimer's disease. These studies have the potential to identify several new therapeutic targets for the treatment of Alzheimer's disease.

Several forms of hippocampal synaptic plasticity have been shown to require de novo protein synthesis. N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) is the most widely studied cellular model of learning and memory. One form of LTP, long-lasting late-phase LTP (L-LTP), requires both gene transcription and protein translation. Another form of hippocampal synaptic plasticity, metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) is of particular interest because it requires rapid translation of preexisting mRNA, bypassing the need for transcription. Do similar signaling pathways couple mGluRs and NMDA receptors to the translation machinery during mGluR-LTD and L-LTP, respectively? This appears to be the case for cap-dependent and 5'TOP translation. In the past several years, several laboratories, including my laboratory, have shown that two key signaling pathways regulate cap-dependent and 5'TOP translation during both mGluR-LTD and L-LTP. These findings have generated much excitement because they were the first demonstration of biochemical regulation of translation during hippocampal synaptic plasticity. We plan to address two critical questions to gain a more complete understanding of the translational control mechanisms operating during hippocampal synaptic plasticity. First, is cap-dependent translation similarly regulated during mGluR-LTD and L-LTP? Second, is eIF2a phosphorylation and are uORF-containing mRNAs differentially translated during mGluR-LTD versus L-LTP? These questions will be addressed by utilizing the powerful multidisciplinary combination of electrophysiological recording techniques, Western blot analyses, direct enzymatic assays, subcellular fractionation, immunocytochemistry, and genetically-modified mice to study mGluR-LTD and L-LTP, as well as learning and memory. The results of our experiments will provide important information concerning the signaling mechanisms that underlie not only synaptic plasticity, but also learning and memory processes. Finally, these studies will generate critical information about the biochemical basis of the alterations in synaptic plasticity that occur in fragile X syndrome and tuberous sclerosis complex, mental retardation syndromes that have altered translation.

? DESCRIPTION (provided by applicant): In this competing renewal application, we request funds to continue support for an integrated, multidisciplinary, neuroscience training program at New York University, one that prepares trainees for the intensely collaborative and interdisciplinary nature of modern neuroscience research. Historically, two neuroscience graduate programs existed in parallel at NYU. However, with substantial support from the University over the past seven years, we have reached a new phase of program integration that seamlessly merges neuroscience graduate education at NYU and offers far greater breadth and depth of training than each program offered individually. Our program, which includes 81 training faculty, combines the strengths in systems, cognitive, and computational neuroscience from the Washington Square-based Center for Neural Science with those in cellular, molecular, developmental, translational, and clinical neuroscience at the NYU School of Medicine campus, and it serves as the foundation for the extensive neuroscience community at NYU. Our graduate program is highly competitive at the national level, proven by our success during the previous funding period in recruiting outstanding graduate students as well as a number of new junior and senior faculty. Since our last submission, the Medical School also recruited Richard Tsien to establish the NYU Neuroscience Institute, which has created even greater enthusiasm around the neuroscience program here at NYU. The specific goals of our neuroscience training program are: (1) To provide a rigorous, high-quality, and broad-based graduate education in neuroscience within the context of an interactive, collegial, and cutting-edge research environment; (2) To increase the number of high caliber students that apply to and participate in the program, including active recruitment of underrepresented minorities; (3) To provide students with guidance of a rigorous mentoring system that ushers students through a series of milestones to a doctoral degree typically in 5-6 years; (4) To train students in necessary professional skills, including critical reading, grant writing, oral presentation, leadership, management, and networking; (5) To encourage a broad perspective on the field of neuroscience that encompasses basic, translational, and clinical research; and (6) To prepare students for the variety of scientific career opportunities that will be available to them after graduate school. We request funding for 12 predoctoral students in their first and second years. The increase in slots over the previous funding period is amply justified by the large increase in faculty and student populations. Through our newly integrated graduate program, we provide trainees with a vast and rich intellectual environment, as well as the resources and experience, to confidently pursue their own scientific interests and become independent scientific leaders, who will make future breakthroughs in basic, translational, and clinical neuroscience.

DESCRIPTION (provided by applicant): Angelman syndrome (AS) is a human neurological disorder that is associated with symptoms that include cognitive impairment, motor abnormalities, and epilepsy. In most cases, AS is caused by the deletion of small portions on chromosome 15, which includes the UBE3A gene. The UBE3A gene encodes an enzyme termed ubiquitin ligase E3A (also termed E6-AP), which is one of a family of enzymes that covalently attaches polyubiquitin chains to proteins to signal for their recognition and degradation by the 26S proteasome. A mouse model of AS has been generated and these mice exhibit seizures, impaired motor function, and cognitive deficits that correlate with neurological alterations observed in humans. Hippocampus-dependent learning and memory is impaired in AS model mice, as is long-term potentiation (LTP), a long-lasting form of synaptic plasticity thought to be a cellular substrate for memory. Recently it was reported that AS model mice exhibit mitochondrial dysfunction. Moreover, mitochondria are considered to be one of the primary sources of oxidative stress in cells, and we recently have shown that reduction mitochondrial-derived superoxide can rescue synaptic plasticity and memory impairments in Alzheimer's disease model mice. Taken together, these findings have led us to hypothesize that mitochondrial-derived superoxide contributes to synaptic plasticity and memory impairments in AS model mice. Consistent with this idea, our preliminary data indicate that the levels of mitochondrial superoxide are elevated in the hippocampus of AS mice. Herein, we propose to determine whether reducing levels of mitochondrial-derived superoxide using pharmacological and genetic approaches can 1) rescue hippocampal LTP deficits displayed by AS mice, 2) reverse memory impairments displayed by AS mice, and 3) improve motor performance and reduce audiogenic seizures displayed by AS mice. The results of these studies should provide insight into whether oxidative stress is associated with AS, how it impacts hippocampal synaptic plasticity, hippocampus-dependent memory, and other forms of neurological dysfunction in AS, and whether use of mitochondria-targeted antioxidants could be a viable therapy for treating individuals with AS. PUBLIC HEALTH RELEVANCE: A mouse model of Angelman syndrome (AS) has been generated that exhibits symptoms consistent with the human genetic disorder, including cognitive impairment, motor abnormalities, and epilepsy. We have proposed to determine whether reducing oxidative stress caused by mitochondrial dysfunction can reverse the aforementioned neurological abnormalities in AS model mice. These studies will determine whether reducing mitochondrial-derived oxidative stress is a potential therapy for the treatment of individuals with AS.

DESCRIPTION (provided by applicant): We are requesting partial support for the next three Molecular and Cellular Cognition Society (MCCS) Meetings. The next meeting will be held in the San Diego Convention Center on November 7-8, 2013, immediately prior to the annual meeting of the Society for Neuroscience. The MCCS meeting brings together junior and senior scientists that combine molecular (pharmacology, genetics, transgenics, viral approaches, etc.) and physiological (electrophysiology, optical physiology) and other cellular approaches to study behavior, including learning and memory. The general goal of these studies is to derive explanations of cognitive processes that integrate molecular, cellular, and behavioral mechanisms, as well as to use this information and related animal models in the search for treatments for cognitive, psychiatric and neurological disorders in children, adults and the elderly. These meetings have been organized under the sponsorship and leadership of the MCCS, a society that was founded in 2002 and whose main function is to organize meetings and promote interaction and collaborations among laboratories working in this general area. Although there are a few learning and memory meetings in the USA and abroad, the MCCS meeting is unique because it brings together individuals that integrate molecular, physiological and behavioral approaches in studies of cognition, memory and learning-related disorders. Although the molecular and cellular cognition field is relatively new, it already has had a profound impact on neuroscience research. Currently, this is the only periodic meeting in the field, an invaluable opportunity to exchange information, and develop this young field. The 2010-2012 meetings were highly successful, attracting each year a diverse group of approximately 500 participants from North America, Europe, and Asia, and we have every reason to believe that the 2013-2015 meetings will be equally successful.

Over the last 15 years, several laboratories, including my laboratory, have identified multiple signaling pathways that regulate translation via the translation initiation factors eIF4E and eIF2? during protein synthesis-dependent forms of long-lasting synaptic plasticity and various memory processes in rodents, including the consolidation, reconsolidation, and extinction of auditory threat memory. These findings have generated much excitement because they demonstrate the complex biochemical regulation of translation during synaptic plasticity and memory. Despite this progress, a number of critical and unresolved questions regarding the requirement for de novo protein synthesis in memory consolidation remain unanswered. We plan to focus on auditory threat memory in the amygdala and address three new questions that are critical for a more complete understanding of the role of de novo protein synthesis in memory formation. First, which cell types in the lateral amygdala (LA) and centrolateral (CeL) amygdala require eIF4E-dependent translation for auditory threat memory? Second, which cell types in the LA and CeL require eIF2?-dependent translation for the consolidation and reconsolidation of auditory threat memory? Third, does auditory threat learning induce cell type-specific translation profiles in the LA and CeL? These questions will be addressed by utilizing the powerful multidisciplinary combination of new groundbreaking genetically-engineered mice, electrophysiological recordings, immunocytochemistry, innovative methods to measure de novo protein synthesis in vivo, and cell-type translational profiling. The results of these studies will provide fundamental insights into the molecular events in both excitatory and inhibitory neurons that support consolidation and reconsolidation of auditory threat memory. Moreover, these studies have the potential to provide cell type-specific therapeutic targets for multiple brain disorders that are associated with dysregulated translation.

DESCRIPTION (provided by applicant): There is a general lack of understanding concerning the molecular signaling pathways that are altered in Alzheimer's disease (AD), and whether dysregulation of these pathways contributes to impairments in synaptic plasticity and memory deficits associated with AD. The studies in this competing renewal are focused on the phosphorylation of the translation initiation factor eIF2? and the protein kinases that phosphorylate it. eIF2? has four known protein kinases: the general control non-derepressible-2 (GCN2), the double-stranded RNA activated protein kinase (PKR), heme-regulated inhibitor (HRI), and the PKR-like endoplasmic reticulum (ER) resident protein kinase (PERK). The phosphorylation of eIF2? on serine 51 causes a decrease in general translation initiation, but it also selectively increases the translation of a subset of mRNAs that contain upstream open reading frames (uORFs) in their 5' untranslated region (UTR). Previous studies showed that eIF2? phosphorylation increased in the brains of AD model mice and postmortem brains from AD patients, suggesting that increased eIF2? phoshorylation decreases general translation and upregulates the translation of mRNAs with uORFs in their UTRs in AD. Consistent with this notion, in the previous funding period we found that genetic deletion of PERK prevents decreases in general translation, increased expression of ATF4 (whose mRNA contains a uORF), impairments in synaptic plasticity, and memory deficits in AD model mice. Based on these observations, we have formulated a central hypothesis, which is that elevated eIF2? phosphorylation in AD via activation of multiple eIF2? kinases results in impaired synaptic plasticity and memory deficits due to differential mRNA translation and protein expression. To test this hypothesis, we will 1) determine whether genetic deletion of GCN2 and PKR prevents altered translational control and amyloidogenesis in AD model mice, 2) determine whether genetic deletion of GCN2 and PKR prevents aging-related impairments in synaptic plasticity and memory deficits displayed by AD model mice, and 3) determine the identity of proteins with altered synthesis and expression in AD model mice and in eIF2? kinase mutant mice. These studies will provide important information concerning whether reduction of eIF2? phosphorylation via deletion of GCN2 and/or PKR can correct dysregulated translation, impaired synaptic plasticity, and memory deficits in AD model mice in a manner similar to the deletion of PERK, and whether these eIF2? kinases might be suitable therapeutic targets for AD. Moreover, these studies have the potential to identify additional targets by identifying the proteins with dysregulated translation in the brains of AD mice, as well as the proteins whose translation is regulated by each eIF2? kinase.

Abstract It is known that protein homeostasis is impaired in Alzheimer?s disease (AD) and frontotemporal dementia (FTD). A multitude of studies have indicated that the proteome is altered throughout the disease process, and ribosomal dysfunction occurs prior to advanced disease states. We have previously shown that de novo protein synthesis is impaired in APP/PS1 mutant mice, which express known human mutations associated with early-onset AD. Mounting evidence suggests changes in cell functioning occur decades prior to the development of symptoms in AD. In addition, the lack of success of clinical drug trials based on the amyloid hypothesis indicates greater understanding of the AD process is necessary to develop novel and effective therapies. To address these issues, we have used novel proteomic technology to label newly synthesized proteins in asymptomatic APP/PS1 mice, as well as symptomatic APP/PS1 mice that exhibit AD-like memory deficits. Our preliminary studies indicate that protein synthesis is dysregulated in these mice, and significant changes in the synthesis of protein components of the ribosome are observed even prior to symptom onset. As these findings may underlie the pathology of this proteopathy, we propose to investigate the impact of this ribosomal dysregulation and test our overall hypothesis that that age-dependent alterations observed in the synthesis of specific ribosomal proteins (RPs) in amyloid and tau model mice alter the translation of selective mRNAs that ultimately result in synaptic and memory impairments. Moreover, as RP synthesis has recently been shown to be dysregulated in the rTg4510 mouse model of AD- and FTD-like tauopathy, we hypothesize that alterations in RP synthesis in response to AD- and FTD-like tau dysregulation could impact ribosomal protein content, and similarly affect translational activity. We first will determine the RP content of functional ribosomes from asymptomatic and symptomatic APP/PS1 mice. Then, we will determine the translational activity of ribosomes from AD model mice. Finally, we will determine RP stoichiometry and translational activity of ribosomes from the K3 and PS19 mouse models of tauopathy and neurodegeneration. The results of these experiments will further our understanding of the neurodegenerative disease process, paving the way for similar investigations in AD and FTD patient cells, and subsequently identifying therapeutic targets for the treatment of AD and FTD.

Project Summary/Abstract Over the last 15 years, several laboratories, including my laboratory, have identified multiple signaling pathways that regulate translation via the translation initiation factors eIF4E and eIF2? during protein synthesis-dependent forms of long-lasting synaptic plasticity and various memory processes in rodents, including the consolidation, reconsolidation, and extinction of auditory and contextual threat memory. These findings have generated much excitement because they demonstrate the complex biochemical regulation of translation during synaptic plasticity and memory. Despite this progress, a number of critical and unresolved questions regarding the requirement for de novo protein synthesis in memory consolidation remain unanswered. We plan to focus on auditory and contextual threat memory to determine the cell types in the amygdala and hippocampus, respectively, that require eIF4E- and eIF2?-dependent translation for memory consolidation, reconsolidation, extinction, and discrimination. We also plan to examine the cell type-specific requirement for de novo translation in memory using more complex types of behavioral paradigms, including Dysregulated translation has been shown by a number of laboratories, including my laboratory, to contribute to synaptic dysfunction and aberrant behaviors in neurodegenerative diseases such as Alzheimer?s disease (AD) and neurodevelopmental disorders such as fragile X syndrome (FXS) and autism spectrum disorder (ASD). However, using molecular approaches to dissect circuit dysfunction in these diseases/disorders has been lacking. Therefore, we plan to examine the role of cell type-specific translational dysregulation in mouse models of AD, FXS, and ASD. Moreover, we will identify the inappropriately translated mRNAs and their newly synthesized protein products using translatomic and de novo proteomic approaches that we developed to identify mRNAs/proteins that are translated/synthesized improperly in mouse models of AD and FXS. These questions will be addressed by utilizing the powerful multidisciplinary combination of new groundbreaking genetically-engineered mice and viruses, electrophysiological recordings, immuno-cytochemistry, innovative methods to measure de novo protein synthesis in vivo, cell-type specific translational profiling, and de novo proteomics. The results of these studies will provide fundamental insights into the molecular events in both excitatory and inhibitory neurons that support consolidation, reconsolidation, and extinction of memory. Moreover, these studies have the potential to provide therapeutic targets for multiple brain disorders that are associated with dysregulated translation.