Michel Baudry - US grants

The long-term goals of our research are to understand the mechanisms governing growth and degenerative processes in the central nervous system. We previously advanced the hypothesis that disturbances in the regulation of mitochondrial calcium transport at the level of the rate-limiting enzyme of mitochondrial metabolism, pyruvate dehydrogenase, is a key event in the triggering of degenerative processes which follow denervation and possibly in various pathological conditions. Our previous studies have provided various means of testing this hypothesis. As in the past, the proposed experiments will take advantage of our knowledge of the various biochemical and morphological alterations (degeneration and sprouting) which follow deafferentation of the hippocampus from its main input, the entorhinal cortex. Our specific aims are (1) to identify the factors which regulate pyruvate dehydrogenase activity and mitochondrial calcium transport in intact and denervated hippocampus of rats of different ages (in particular we propose to determine the role of pyruvate carboxylase and to test the involvement of polyamines and various neuroactive peptides in the regulation of mitochondrial metabolism), (2) to study the effects of chronic insulin treatment on the morphological alterations induced in the rat hippocampus by entorhinal cortex lesions, and (3) to determine the effects of entorhinal cortex lesion on biochemical and morphological alterations in hippocampus of rats of different ages. These studies should significantly add to our understanding of the mechanisms by which presynaptic afferents participate in the regulation of intracellular calcium concentration in the target cells and possibly provide tools to manipulate growth and degenerative processes which might ultimately be applicable in a wide range of pathological conditions.

DESCRIPTION (Investigator's Abstract): The long-term goals of our research are to understand the mechanisms underlying growth and degeneration in the adult central nervous. Our general hypothesis is that the balance between growth and degenerative processes taking place under physiological or pathological conditions is critical to determine the changes in nervous- tissues occurring during normal conditions and as a result of insults or injuries. Our previous work has identified a number of mechanisms that are closely linked with growth and degenerative processes. In particular, all the conditions leading to neuronal pathology trigger the induction of several genes, including the gene for ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis. Polyamines have been linked with growth processes as well as with degenerative processes through their interactions with DNA, protein synthesis: calcium signaling and more recently with NA receptors. Several key questions have been raised by recent findings obtained during the tenure of our proposal: (i) What are the changes in polyamine levels in different cellular compartments following various forms of insults or injuries in adult and developing animals? (ii)What are the causes for the marked dissociations between seizure activity, ODC induction and neuronal pathology taking place during the postnatal period and exhibiting a major shift after the third postnatal week? (iii) Are the-between polyamines, NMDA receptors, calcium, DNA and protein synthesis important aspects of the polyamine functions in the CNS? The aims of the present proposal are to provide answers to these questions. Specifically we propose toe: (1) Determine the changes in polyamine levels and activity and expression of enzymes regulating polyamine levels in susceptible neuronal populations following excitotoxic lesions at different ages; (2) Evaluate the effect of inhibitors of the enzymes regulating polyamine levels (alone or in combination) on neuronal pathology elicited by excitotoxic agents; (3) Study the mechanisms involved in the dissociation between seizure activity, ODC induction, and neuronal pathology during the postnatal period; and (4) Study the effects of polyamines on NMDA receptor mediated responses in acute hippocampal slices. These studies should provide not only a better understanding of the cascade of events which follows neuronal injuries, but could also provide new ways to better control the degenerative processes occurring in a number of neurodegenerative diseases, while stimulating regenerative and growth processes.

One of the most dramatic symptoms associated with Alzheimer's disease (AD) and, to a lesser extent, with aging, is a gradual impairment in learning and memory. This impairment is in part due to the presence of plaques and tangles in several brain structures, including the cerebral cortex and the hippocampus, as well as to the degeneration of selectively vulnerable neuronal populations. Although the mechanisms involved in the neuropathologies associated with aging and AD are not known, it is widely accepted that alterations in glutamatergic transmission might play critical roles in both learning impairment and neuronal death. This view has been supported by numerous studies indicating the participation of glutamate receptors in both learning and memory and in neuronal degeneration. Our previous work has revealed the existence of several mechanisms regulating the properties of flutamate receptors and has documented the roe of these mechanisms in learning and memory and in neuronal degeneration in adult rat brain. The proposed studies are intended to expand these studies to the aging rat brain and to brain from AD patients and to test several hypotheses relating changes in glutamate receptors and /or in mechanisms regulating their characteristics to changes in synaptic plasticity and excitotoxicity in aged rats. Possible alterations in glutamate receptor properties and regulation in AD brain will also be evaluated. The following specific aims will be pursued: (1) to determine the changes in glutamate receptor properties and regulation in aging rat brain and human AD, (2) to study potential changes in mechanisms regulating glutamatergic transmission in aging rat brain and human AD, (3) to evaluate potential changes in the mechanisms linking glutamate receptors and neuronal degeneration in aging rat brain, and (4) in view of the regulation of the NMDA receptor by polyamines, to study potential correlation between CSF and blood polyamine levels and disease severity in AD patients. The research will use a variety of molecular and biochemical techniques to study the properties in young and old rats as well as in brain samples from AD patients. It will also use in vitro and in vivo models of synaptic plasticity to study mechanisms regulating glutamate receptors, glutamatergic transmission, and the excitotoxic properties of glutamate in young and old rats. These studies will provide important information concerning the mechanisms involved in age-related modifications of glutamatergic transmission and identify critical steps that could account for the learning deficits and neuronal damage observed with aging and AD. They might therefore suggest new avenues for designing optimal strategies to alleviate the learning deficits and to prevent the neuronal damage associated with these states.

technology /technique development; psychology; nervous system; biomedical resource; bioengineering /biomedical engineering; model design /development; behavioral /social science research tag;

This project addresses the cellular mechanisms underlying the dual actions of estrogen in women with Alzheimer's Disease, i.e., the improvement of the cognitive deficits and the reduced incidence of AD in women with estrogen replacement therapy. These effects of estrogen suggest that estrogen or some equine estrogen constituent acts as a cognitive enhancer and/oras a neuroprotective agent. These hypothesis will be tested in in vitro models of synaptic plasticity and neurodegeneration. Long-term potentiation (LTP) is widely considered to represent a cellular mechanism for information storage and compounds modulating LTP have been found to modulate learning and memory processes. LTP induction and expression are critically dependent on 2 classes of inotropic receptors, the NMDA and the AMPA receptors, respectively. Specific Aims 1 and 2 will therefore be directed at determining the effects of acute and chronic estrogen treatment of hippocampal slices maintained in culture for several; weeks on the characteristics of AMPA and NMDA receptors and on the properties of LTP. Treatment of cultured hippocampal slices with excitotoxin or beta- amyloid peptide (betaAP) produces selective neuronal damage which exhibits several features of neuronal death occurring in AD. Specific Aims 3 and 4 will therefore evaluate the neuroprotective effects of estrogen in these models. Effects of estrogen in these in vitro systems will be compared with its effects in cultures of dissociated neurons as well as in in vivo models of synaptic plasticity and neurodegeneration.

EIA-0130883-Theodore W. Berger-University of Southern California-Title: Neurobiological Nonlinear Dynamics for Biomimetic Signal Processing-Title-The fundamental goal of the proposed research is to derived a new generation of temporal and spatio-temporal pattern recognition systems based on the nonlinear dynamics, network architecture, and synaptic plasticity properties of the hippocampus, a cortical brain system responsible for the formation of new pattern recognition memories. From a neurobiological perspective, the proposed experimental/modeling work promises to generate (1) first-characterizations of high-order nonlinearities of cortical brain tissue, i.e., predictive models of the input/output transformations in spatio-temporal activity performed by individual hippocampal neurons, and to (2) investigate the increasingly likely possibility of dynamic neural "learning rules", i.e., requisite conditions for the induction of synaptic plasticity that depend on the past history of activity. In addition, the proposed research will investigate (3) the role of known hippocampal network topology in neurobiological signal processing and hierarchical feature extraction. From a theoretical/computational perspective, the proposed work is designed to (4) develop novel methodologies essential for characterizing nonlinearities of neurobiological systems, as well as to (5) further expand a newly developed paradigm for biologically realistic neural system modeling (the "dynamic synapse neural network architecture") that has already demonstrated a heretofore unmatched capability for identifying optimal feature sets for temporally and spatio-temporally coded information.

Several lines of evidence indicate that fast excitatory transmission mediated by AMPA receptors regulates expression of BDNF. As predicted from this, increasing the size of excitatory postsynaptic currents with positive modulators of AMPA receptors (ampakines) increases BDNF expression both in cultured brain slices and in vivo. These results raise the possibility of using AMPA receptor modulators to produce sustained increases in the brain's concentration of a critical trophic factor. However, preliminary studies using a novel cultured slice technique for 'chronic' recording of synaptic responses led to the discovery that ampakines depress excitatory responses after about 24-36 hours. Follow on experiments showed that the depression is accompanied by a marked reduction in the number of AMPA receptors in membrane fractions. Separate work in the laboratory of Dr. Gall uncovered a parallel effect with regard to BDNF induction: prolonged exposure causes cultured slices to become refractory to ampakines. The proposed work has five objectives relating to these findings. Specific Aim One will define the time course for the onset of, and recovery from, EPSP depression. This information will be compared to the temporal parameters of the BDNF refractory period (see Project One). Specific Aim Two will test the hypothesis that down-regulation of AMPA receptors is responsible for EPSP depression. Number and affinity of the membrane receptors will be measured first; subsequent work will assay for changes in levels of receptor subunits and turnover. Specific Aim Three addresses additional mechanistic questions relating to EPSP depression: What are the triggering events? Are signaling pathways linking AMPA receptor activation to AMPA receptor regulation and BDNF expression disturbed? (Also see Project One). Specific Aim Four investigates the functional consequences of AMPA receptor down-regulation. Long term potentiation, long term depression, and seizure susceptibility will be tested prior to and after the onset of the effect. Studies on how LTP is affected by ampakine-induced elevation of BDNF in the absence of depression are described in Project Three. Specific Aim Five will test for the occurrence of AMPA receptor down-regulation and signaling in vivo. In all, the results of the proposed study are expected to yield new and fundamental information concerning AMPA receptor regulation. They are also expected to be critical in the development of an AMPA receptor based strategy for up-regulating a critical trophic factor.

[unreadable] DESCRIPTION (provided by applicant): Parkinson' s disease is a neurodegenerative disease that specifically affects dopaminergic neurons in the substantia nigra. Although several hypotheses have been proposed to account for the specificity of the neurodegenerative features of the disease, the exact cause of the disease remains to be elucidated. Significant advances in our understanding of the possible causes of the disease were provided by the serendipitous discovery that a neurotoxin, 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP), elicits a pattern of neurodegenerative features in humans and experimental animals identical to that seen in patients with Parkinson' s disease. A potential target to prevent neurodegeneration in Parkinson' s disease is the calcium-dependent protease calpain. Calpain levels are elevated in post-mortem substantia nigra of patients with Parkinson' s disease, MPP+ neurotoxicity in granule cell cultures is associated with calpain activation and blocked by calpain inhibitors, and calpain has been implicated in several neurodegenerative diseases. We have recently obtained a series of novel and potent calpain inhibitors and have demonstrated their potency in preventing NMDA-induced calpain activation in cultured hippocampal slices. The current proposal is aimed at testing the hypothesis that calpain activation plays a critical role in animal models of PD and that calpain inhibitors are neuroprotective in these models. We will first determine the potency and efficacy of calpain inhibitors to prevent MPTP toxicity in cultured slices from rat mesencephalon. We will then use structure activity relationship in conjunction with additional assays to identify the best inhibitors to be tested in in vivo models. Finally, we will test the hypothesis that calpain is activated and that calpain inhibitors are neuroprotective against MPTP-mediated neurotoxicity and behavioral impairments in vivo in C57BI/6 mice, and against rotenone-mediated neurotoxicity in rats. Conversion of the pro-apoptotic factor Bid to its active, truncated form tBid will be tested as part of the mechanisms by which calpain activation induces cell death. These studies will test the hypothesis that calpain inhibitors might prevent neurodegeneration not only in Parkinson' s disease but also in a variety of conditions resulting from exposure to environmental toxins. Finally, because calpain has also been implicated in the mechanisms underlying Amyotrophic Lateral Sclerosis (ALS), our proposal could lead to significant advances in the treatment of this neurodegenerative disease as well. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Neonatal hypoxic ischemia (H/I) is largely responsible for neonatal encephalopathy, which occurs in 2-4 out of 1000 births per year in the US. While the resulting mortality has decreased, there has been a corresponding increased rate of children developing serious neurological disorders, including motor and learning disabilities, cerebral palsy and seizures. Although the mechanisms underlying H/I-induced neuronal damage are not completely understood, it has repeatedly been proposed that excitotoxicity due to over-excitation of glutamate receptors plays a critical role. Identification of downstream events from glutamate receptor activation has been the object of intense investigation, and the calcium dependent protease calpain has been implicated in ischemia-induced neuronal damage. Metabotropic glutamate receptors have recently emerged as new players in ischemic neuronal death, although their roles remain controversial. Studies have focused on one of the metabotropic glutamate receptors, mGluR1, as this receptor activates two signaling cascades, one leading to neurodegeneration through the stimulation of phospholipase C, the synthesis of inositol-3-phosphate and the release of calcium from internal stores and another one linked to neuroprotection through the PI3K-Akt pathway. We recently found that NMDA receptor activation results in calpain-mediated truncation of the C- terminal domain of mGluR11, one of the isoforms of mGluR1. As a result of this truncation, mGluR11 loses its neuroprotective signaling, although it maintains a normal calcium signaling function. In other words, truncated mGluR11 becomes an exclusively "neurodegenerative receptors", suggesting the existence of a positive feedback loop linking NMDA receptors, calpain activation and mGluR1 in excitotoxicity and neurodegeneration. Furthermore, we found that a small peptide consisting in a sequence of amino acids surrounding the cutting site linked to the HIV tat-peptide was neuroprotective against excitotoxicity both in vitro and in vivo. This R21 application is therefore directed at obtaining in vivo preclinical data to develop the tat-mGluR1 peptide or a modified form of this peptide for therapeutic use against neonatal H/I-induced neuronal damage. We will therefore test the potential neuroprotective effects of the tat-mGluR1 peptide in an in vitro and an in vivo model of neonatal H/I (Specific Aim #1). We will design and test the neuroprotective effects of analogs of tat-mGluR1 (Specific Aim #2). Positive results will then encourage the clinical development of this approach for the treatment of neonatal hypoxia/ischemia. PUBLIC HEALTH RELEVANCE: The rationale for the proposed studies is derived from recent studies in the PI's laboratory that have identified a new mechanism for excitotoxic neuronal damage as well as a promising neuroprotective peptide. This new mechanism involves the existence of a previously unknown positive feedback loop linking several molecular processes postulated to participate in glutamate-mediated neuronal death. The proposed studies will evaluate the neuroprotective effects of the peptide in an in vitro and an in vivo model of neonatal hypoxic/ischemic neuronal damage. If the peptide shows neuroprotective effects when applied after the hypoxic/ischemic insults, these studies will provide the in vivo proof-of-concept needed to pursue the clinical development of this approach not only for neonatal hypoxia/ischemia-induced neuronal damage, but also for neuronal damage occurring in other forms of neurodegenerative diseases.

DESCRIPTION (provided by applicant): The hippocampal formation is critically involved for the long-term storage of various forms of information, and it is widely believed that the phenomenon of long-term potentiation (LTP) of synaptic transmission is a molecular/cellular mechanism participating in memory formation. Progress in our understanding of LTP has led to the discovery of multiple processes interacting in complex ways that are critically important for different steps of memory formation. Although several high level models of hippocampal function have been developed, they do not incorporate detailed molecular information of the type necessary to understand the contribution of individual molecular events to the overall network function of the hippocampus. It is therefore our goals to develop new technological tools based on mathematical modeling and computer simulation of the molecular processes taking place in realistic biological networks to reach such an understanding. We believe that this approach will not only provide an intimate understanding of the contribution of specific molecular events to overall network function and synaptic plasticity, but also facilitate the design of better and safer therapeutic approaches for learning and memory impairments. Scientists at the University of Southern California have had a long- standing collaboration to understand the molecular and cellular mechanisms of LTP and to develop models to translate basic research into real-life applications. In collaboration with Rhenovia Pharma, we have initiated the development of an integrated platform that incorporates some of the elements of field CA1 of hippocampus. The proposed bioengineering research partnership between 2 research teams at the University of Southern California and Rhenovia will further develop this platform, validate the outputs of the simulation by in vitro experimentation in hippocampal slices and test the possible use of the platform to identify molecules or combination of molecules that could result in facilitation of LTP induction. In particular, we propose to incorporate cholinergic modulation of CA1 network function in order to better understand the links between theta rhythm synchronization of neuronal firing and LTP formation, as well as various types of metabotropic glutamate receptors in order to explore the roles of these receptors in synaptic transmission and synaptic plasticity processes. Finally, integrating various GABA receptors will provide a unique tool to better understand the effects of a large number of drugs currently used to treat a wide range of diseases from epilepsy to Alzheimer's disease PUBLIC HEALTH RELEVANCE: Learning and memory impairments are important aspects of numerous neurological and neuropsychiatric diseases. Identifying new pharmacological treatments for cognitive impairment is both urgent and difficult in view of the complexity of the mechanisms involved in memory formation. The proposed work is directed at developing bioinformatics tools to facilitate this process by providing a better understanding of the molecular and cellular events participating in memory formation as well as a platform for testing the efficacy of drugs or combination of drugs for improving memory formation.

? DESCRIPTION (provided by applicant): Angelman Syndrome is rare but severe neurodevelopmental disorder characterized by intellectual disability, motor impairment, and happy demeanors. Genetic cause of the disease is the deletion/abnormal expression of maternal chromosome 15q11-q13, which includes the UBE3A gene that codes for the E6-associated protein (E6-AP). A mouse model (AS mice) of the human disease has been generated by deletion of maternal Ube3a; these mice exhibit impaired long-term potentiation (LTP) of synaptic transmission in hippocampus, learning of various hippocampus-dependent tasks, and motor functions. Preliminary results indicated that abnormal calpain-2 activity might contribute to synaptic and cognitive impairment in AS mice, as treatment with a calpain-2 inhibitor restored normal LTP in slices prepared from AS mice. The rationale for the preliminary study was based on our recent discovery that calpain-2 activation during LTP consolidation functions as a molecular brake that limits the magnitude of long-term potentiation in hippocampus. A major component of the molecular brake is calpain-2-mediated degradation of the tumor suppressor PTEN, and AS mice have enhanced calpain-2 expression and decreased PTEN levels in hippocampus. These results suggest that calpain-2 might be a good target to restore normal learning in the Angelman Syndrome. While we have identified the dipeptide ketoamide, Z-Leu-Abu-CONH-CH2-C6H3(3,5-(OMe)2) as a relatively selective calpain-2 inhibitor at low concentrations, at higher concentrations it also inhibits calpain-1, which is necessary for LTP induction. As PTEN is selectively cleaved by calpain-2 and not calpain-1, we posit that identifying PTEN properties underlying its selectivity for calpain-2 will provide criticl information to develop calpain-2 selective inhibitors, which will not inhibit calpain-1. The proposed studies are directed at using detailed molecular dynamics simulation to identify critical features in PTEN that account for calpain-2 selectivity. We will then use a multi-level virtual screening method to design new and selective calpain- 2 inhibitors. These inhibitors will be tested first on in vitro assays for calpain-1 and -2 and for interactions with other cysteine proteases and then for their effects on LTP and in learning in the AS mice. Successful design and testing of a selective calpain-2 inhibitor will then lead to a potential U01 submission for using such inhibitors not only for AS treatment but also for other indications with learning and memory impairment.

This proposal is directed at purchasing a confocal microscope to be used by several teams of interdisciplinary scientists for research and education at Western University of Health Sciences (WesternU), and to be shared with various neighboring Institutions (Cal State Fullerton University, Cal Poly Pomona, and the Claremont Colleges). Specifically, this project will not only gradually replace the current and outdated confocal microscope but will add the following research capabilities: live imaging with less phototoxicity, spectral imaging, faster imaging, allowing for accurate FRET analysis, and high-resolution 3D imaging. The requested equipment will be located in a Core Facility dedicated to imaging technologies, and will be accessible to qualified researchers and students. This instrument will also be used to train postdoctoral fellows, graduate students and undergraduate students from the neighboring Institutions in imaging techniques. Finally, it will also be used in a partnering program with a neighboring High School, Garey High School, directed at enhancing their STEM programs. Overall, the project is significant in that it will provide state-of-the-art research capability to both WesternU and to the region. It will also provide valuable research training and experience for student users as well, and facilitate collaborative research between WesternU and neighboring institutions.

The microscope will be used by at least 10 primary research teams of WesternU, and will be used for several research projects: Analysis of protein-protein interactions by several investigators investigating signaling pathways involved in regulation of protein synthesis, receptor trafficking, and synaptic function and plasticity; Analysis of calcium imaging in various neuronal populations in response to a variety of extracellular signals; 3D reconstruction of neuronal dendrites under different physiological or pathological conditions; Analysis of neurogenesis and synaptogenesis.

UBE3A, an E3 ligase in the ubiquitin-proteasomal system, plays important roles in brain development and function. Optimal CNS UBE3A expression is crucial since its deficiency results in Angelman syndrome (AS), while its over- expression increases the risk for autism. Yet, the precise function of UBE3A in the CNS remains largely unknown. In AS mouse models, Ube3a deficiency leads to deficits in motor function, learning and memory, and social interactions. We have recently reported that Ube3a deficiency leads to increased activation of mTORC1 but decreased activation of mTORC2, the two core complexes in the mechanistic target of rapamycin (mTOR) signaling pathway. However, how UBE3A deficiency results in mTORC1 over-activation remains unknown. Emerging evidence indicates that the presence of amino acids is essential for full mTORC1 activation. Amino acid-induced mTORC1 activation depends on the recruitment of mTORC1 to late endosomal/lysosomal membranes, a process involving the formation of heterodimers of a RagA or RagB, with a RagC or RagD through binding to the Ragulator. The anchoring of Ragulator to lysosomal membrane is through one of its components, p18. Myristoylation and palmitoylation in the N-terminal domain of p18 are critical for its lysosomal localization. Our preliminary results revealed that p18 levels were increased as a result of Ube3a deficiency in COS1 cells and in AS mouse brain. We further showed that p18 could be ubiquitinated by Ube3a in COS1 cells. These findings led us to propose the central hypothesis that, under normal conditions, lysosomal localized p18 levels are controlled by Ube3a-mediated ubiquitination, which targets p18 for proteasomal degradation. A correlate of this hypothesis is that lack of Ube3a-mediated p18 ubiquitination and degradation in AS mice results in imbalanced mTORC1/mTORC2 signaling and abnormal spine morphology and synaptic plasticity. We will first identify the ubiquitination sites in p18 and determine whether N-terminal acylation affects Ube3a- mediated p18 ubiquitination. We will then determine whether Ube3a-mediated p18 regulation plays important roles in synaptogenesis, synaptic plasticity, and experience-dependent remodeling of synapses using a multidisciplinary approach. Since both UBE3A deficiency and overexpression are linked to neurodevelopmental disorders, understanding UBE3A-mediated regulation of p18 and of mTOR signaling and its role in synaptogenesis and synaptic plasticity should shed light on basic neurobiological mechanisms, as well as on several neurological and neuropsychiatric diseases.