Lee A. Cunningham - US grants

The long term objective of the proposed research is to develop the experimental and therapeutic use of genetically engineered primary astrocytes as vehicles for neurotrophic factor delivery to the brain. The specific aims of this proposal are to 1) systematically evaluate the level, stability, and autocrine effects of neurotrophic factor expression by primary astrocytes that are genetically engineered to express two related neurotrophins, beta-nerve growth factor (beta-NGF) and brain- derived neurotrophic factor (BDNF) and 2) evaluate the effects of these transgenic astrocytes on the development, plasticity and regeneration of nigral dopaminergic neurons utilizing a rodent model of Parkinson's disease in which the nigrostriatal pathway is unilaterally lesioned with 6-hydroxydopamine. For the proposed studies, constitutive expression of recombinant beta-NGF or BDNF will be conferred to primary type I rat astrocytes in culture using replication defective retroviral vectors, which allow the efficient and unidirectional transfer of cDNA sequences. The level and stability of recombinant beta-NGF and BDNF expression in culture and following transplantation into the adult rat striatum will be assessed at the messenger RNA level by quantitative RNase protection assays using riboprobes specific for beta-NGF or BDNF mRNA. Expression at the protein level will be assessed using an enzyme-linked immunoassay and a rat retinal ganglion cell bioassay for beta-NGF and BDNF proteins, respectively. Autocrine effects of genomic integration and/or expression of the beta-NGF and BDNF transgenes in astrocytes will be assessed by comparing the expression of proteins that are synthesized by normal versus transgenic astrocytes utilizing high resolution two-dimensional gel electrophoresis. Once characterized, the genetically engineered astrocytes will be tested for their neurotrophic effects on developing dopaminergic neurons in vitro and in vivo. The hypothesis to be tested is that BDNF-producing astrocytes selectively influence the development, plasticity and regeneration of mesencephalic dopaminergic neurons. To this end, the effects of BDNF- and NGF-producing astrocytes on the survival and neuronal differentiation of embryonic dopaminergic neurons will be compared in co-culture and following transplantation into the dopamine-denervated striatum of the unilateral 6-hydroxydopamine lesioned rat. In addition, the ability of BDNF-producing astrocytes to promote regenerative sprouting by adult dopaminergic neurons will be evaluated using a partial lesion model of Parkinson's disease in which a portion of nigrostriatal dopamine neurons remains intact. Analysis of neuronal growth, regeneration and function will include histological, biochemical and behavioral indices. The proposed research is designed to assess the utility as well as the limitations of using transgenic astrocytes to deliver recombinant neurotrophic factors to the brain, and to begin to assess the therapeutic potential of transgenic astrocytes for treating neurodegenerative disease.

DESCRIPTION: (Verbatim from the Applicant's Abstract) Glial cell line derived neurotrophic factor (GDNF) is a recently identified peptide shown to elicit marked restorative/protective effects in both rodent and primate models of Parkinson's disease. Although the therapeutic effects implicate its clinical use, since GDNF does not cross the BBB the problem of in vivo delivery still remains an issue. Using an ex vivo gene therapy approach, the applicant has shown that astrocytes transduced with a replication defective retroviral vector express recombinant GDNF in culture and following intracerebral transplantation. Preliminary studies described in the proposal demonstrate that GDNF producing astrocytes provide nearly complete protection of both nigral dopaminergic perikarya and their associated striatal fibers, when implanted near the substantia nigra several days prior to a 6-OHDA lesion in the mouse. The proposed studies are designed to further characterize the expression of recombinant GDNF by grafted astrocytes and to investigate potential in vivo mechanisms by which continuous exposure to low amounts of GDNF confers decreased susceptibility of adult nigrostriatal neurons to 6-OHDA induced toxicity. In addition, experiments are proposed to determine whether GDNF delivered via transgenic astrocytes restores function to atrophic neurons previously damaged by 6-OHDA. Using a murine model of PD, the proposed studies are designed to address the following Specific aims 1) to characterize the long-term expression of GDNF by transgenic astrocytes following intracerebral transplantation 2) to investigate the potential role of the glutathione system and phosphatidylinositol 3 kinase (PI3K) regulated signaling events in GDNF mediated neuroprotection in vivo using transgenic astrocytes and 3) to determine whether GDNF producing astrocytes restore dopaminergic function to damaged nigrostriatal neurons following the onset of degeneration induced by 6-OHDA. These aims will be addressed using a combination of molecular, tissue culture, behavioral and neurochemical methods, in a mouse 6-OHDA model of Parkinson's disease.

Bone marrow-derived stem cells incorporate into multiple tissue types in adult mammals, where they give rise to cells of multiple lineages that cross classic embryological trilaminar boundaries [9,11,17,21,31,40,41]. This recent discovery has opened the possibility that bone marrow transplantation could be clinically useful to treat a broad spectrum of pathologies. Recently, marrow-derived progenitor cells have been shown to migrate and incorporate into the brain to give rise to cells of neuroectodermal lineage, (astrocytes and neurons), in addition to microglia [4,7,25]. Importantly, marrow-derived progenitors appear to display preferential homing to regions of CNS gliosis and degeneration [8], consistent with studies in peripheral tissues demonstrating enhanced engraftment of multi-potential marrow-derived stem cells into sites of injury [21,31] [Jackson, 2001 #360]. The focus of the current proposal is to explore the migration and differentiation of marrow-derived stem cells into the brain in a rodent model of Parkinson's disease, the MPTP-treated mouse. Questions to be addressed in this proposal include: Do marrow-derived progenitors display selective homing to the damaged nigra and striatum in response to MPTP-induced degeneration? If so, do they give rise to cells of neuroectodermal lineage? What is their contribution to the gliosis that accompanies nigrostriatal degeneration? To track marrow-derived progenitors within the CNS, we will utilize bone marrow from transgenic mice that express an enhanced green fluorescent protein (GFP) under the beta-actin promoter. The migration and differentiation of marrow-derived progenitors will be studied in chimeric mice whose endogenous hematopoietic systems have been completely reconstituted with GFP-expressing cells prior to MPTP- treatment, and in mice that receive acute intravascular injections of GFP+ expressing cells prior to MPTP-treatment, and in mice that receive acute intravascular injections of GFP+ expressing marrow following the onset of MPTP-induced degeneration. The immediate goal of the proposed studies is to delineate the relationship between marrow-derived progenitors and the brain in a rodent model of Parkinson's disease. If successful, these studies may provide the basis for future work to development new non-invasive gene therapies for Parkinson's disease, using accessible, renewable and autologous bone marrow stem cells.

DESCRIPTION (provided by applicant): Metalloproteinase activity at the cell surface can strongly influence cell sensitivity to extrinsic death vs. survival signals in a variety of cell types, yet the convergence of metalloproteinases and receptor signaling in the nervous system remains largely unexplored. TIMP-3 (tissue inhibitor of metalloproteinase-3) is a unique natural metalloproteinase inhibitor, in that it is a potent inhibitor of all known metalloproteinase sheddases, and has been shown to play a role in the regulation of receptor-mediated cell death in various non-neuronal cell types. TIMP-3 plays a pro-apoptotic role in many cell types through its ability to inhibit sheddases that target death receptors and their ligands. Our interest in the role of metalloproteinases in CNS damage following stroke led us to investigate the expression of TIMP-3 and the metalloproteinase sheddase, MMP-3, in a rodent model of focal cerebral ischemia. We found that while TIMP-3 and MMP-3 are expressed at low to non-detectable levels in the adult brain, their expression becomes markedly upregulated following ischemia, particularly in cortical neurons undergoing delayed apoptotic death. Using a tissue culture approach, we found that TIMP-3 and MMP-3 are constitutively expressed by embryonic cortical neurons in culture and modulate neuronal sensitivity to receptor-mediated apoptosis induced by the chemotherapeutic drug, doxorubicin (Dox). Metalloproteinase inhibition by TIMP-3 was found to be necessary for Dox-induced apoptosis, whereas addition of exogenous active MMP-3 markedly attenuated apoptosis and blunted death receptor-ligand interactions at the cell surface. These observations strongly implicate a physiologic role for TIMP-3 and MMP-3 in the regulation of receptor-mediated death in the nervous system. While metalloproteinase activity has previously been implicated in vascular damage following cerebral ischemia, the ability of metalloproteinases and their inhibitors to influence neuronal vulnerability to ischemic stress has not been studied. In the proposed studies, experiments are designed to establish a role for metalloproteinase activity in the regulation of receptor-mediated neuronal death following cerebral ischemia, and to explore underlying mechanisms, utilizing both in vitro and in vivo models of ischemic injury, coupled with pharmacological and gene deletion approaches.

DESCRIPTION (provided by applicant): The denate gyrus of the hippocampus is an area where new neurons continue to be generated throughout adult life. Although the role of adult hippocampal neurogenesis is still debated, accumulating evidence suggests that it is involved in the acquisition of certain forms of hippocampal-dependent learning. Based on those findings, we have begun to explore the relationship between learning deficits and neurogenesis in adult offspring of prenatal ethanol exposed rodents. For these studies, we have utilized a mouse model of fetal alcohol exposure (FAE) that is based on free-choice and represents a moderate alcohol access model of FAE. Adult offspring of FAE mice display learning deficits similar to that of the FAE rat, but the FAE mouse model is not confounded by potential diet and pair feeding effects. Preliminary data presented in this proposal demonstrate that FAE does not impair basal neurogenesis under standard housing conditions, but abolishes the neurogenic response to enriched environment. Studies outlined in this exploratory R21 proposal are designed to determine a) whether FAE also impairs the neurogenic response to hippocampal-dependent learning, and b) whether mitigation of the neurogenic response to behavioral challenge is due to intrinsic defects of FAE progenitors and/or due to microenvironmental changes within the neurogenic niche of the adult SGZ. Our preliminary studies are among the first to demonstrate that prenatal exposures can result in persistent defects in adult hippocampal neurogenesis. Since learning disabilities are among the most prevalent and pervasive fetal alcohol-related defects in children, further studies to understand the basis of these deficits in animal models of prenatal ethanol exposure seems warranted.

SPECIFIC AIMS The production of new neurons within the adult dentate gyrus represents a novel form of hippocampal plasticity associated with certain forms of hippocampal-dependent learning. Our recent studies suggest that the production of new granule neurons within the adult dentate gyrus is impaired in mice exposed to moderate ethanol throughout gestation. While several studies have demonstrated robust effects of acute ethanol on neurogenesis in the adult hippocampus and in isolated stem cells in culture (Crews and Nixon, 2003;He et al., 2005;Nixon and Crews, 2004), our studies were the first to demonstrate that fetal alcohol exposure (FAE) results in persistent deficits in adult hippocampal neurogenesis in mice (Choi et al., 2005). Interestingly, these neurogenic deficits only become apparent when the adult FAE mouse is behaviorally challenged. For example, when exposed for several weeks to enriched living conditions, control mice display a 2-fold increase in hippocampal neurogenesis, whereas FAE mice respond with no increase in the neurogenic response. Because we found no difference in the size of the progenitor pool in the dentate subgranular zone, nor any change in the rate of progenitor proliferation, our studies suggest impaired survival and integration of new neurons within the adult dentate under conditions of enriched environment. Currently, our studies are focused on understanding the mechanistic underpinnings of this impaired neurogenic response in FAE mice. Our overarching hypothesis is that the impaired neurogenic response to enriched environment is due to an intrinsic defect in the adult neural stem cells themselves and/or due to microenvironmental changes that persist within the neurogenic niche. To test this hypothesis, we propose the following Specific Aims: Specific Aim 1: Does FAE result in intrinsic defects in adult neural stem cells? To address this question, we will compare the self-renewal and differentiation properties of neural stem cells isolated from adult FAE vs. control mice in vitro. Specific Aim 2: Does FAE result in microenvironmental perturbations that persist within the neurogenic niche of the adult hippocampus? To address this question we will compare the vascular density and microglial properties within the adult subgranular zone/dentate hilus region in FAE vs. control mice, since these components regulate, in part, the neurogenic response to enriched environment.

DESCRIPTION (provided by applicant): The overall goal of this proposal is to elucidate the mechanisms by which prenatal exposure to moderate doses of alcohol results in long-lasting impairment of enrichment-mediated adult hippocampal neurogenesis, and to link this deficit with impairment of hippocampal-dependent learning in our mouse model of fetal alcohol spectrum disorder (FASD). FASD mice display impaired cognitive ability in hippocampal-dependent learning tasks and show signs of depressive disorder. FASD mice also display persistent deficits in adult hippocampal neurogenesis, which become apparent when the mice are behaviorally challenged by exposure to enriched environment. In this proposal, experiments are designed to determine whether the mechanisms underlying the FASD phenotype include impaired neuronal differentiation and maturation of neural stem cells in adult hippocampus, and diminished incorporation of adult-generated dentate granule cells into spatial memory networks. To test this, we will utilize a novel conditional and inducible nestin- CreERT2:YFP transgenic mouse which allows non-invasive labeling of large numbers of adult neural stem cells and their progeny. FASD and Sacc (control) mice will be generated in the nestin-CreERT2:YFP strain and utilized to address the following Specific Aims. Specific Aim 1 To determine whether prenatal ethanol exposure impairs neuronal differentiation and maturation of adult hippocampal neural stem cells. The nestin-CreERT2:YFP strain will be used to fate map and characterize neuronal differentiation of neural stem cells in adult hippocampus of FASD and Sacc (control) mice in vivo (SA1.1) and following isolation of adult NSCs in vitro (SA1.2). Specific Aim 2: To determine whether prenatal exposure to alcohol impairs synaptic maturation and plasticity of adult-born dentate granule cells (DGCs). Tamoxifen will be used to birthdate and YFP label newborn DGCs for electrophysiological recordings in adult hippocampal slice preparations from FASD and Sacc nestin-CreERT2:YFP mice. Using this approach, we will test the hypotheses that prenatal alcohol exposure disrupts the maturation of GABA- and glutamate-mediated currents (SA2.1) and attenuates LTP plasticity in adult born DGCs (SA2.2). Specific Aim 3: To determine whether prenatal ethanol exposure impairs the ability of newly- generated DGCs to preferentially incorporate into spatial memory networks. We will determine whether preferential activation of newborn neurons is attenuated in adult hippocampus of FASD mice during recall of a spatial memory task, as assessed by immediate early gene (c-fos) expression nestin-CreERT2:YFP mice.

Research Component 3 will investigate glycogen synthase kinase-3 (GSK-3) as a potential therapeutic target in FASD. Studies outlined in this proposal will test the hypothesis that inhibition of GSK-3 activity reverses deficits in adult hippocampal neurogenesis and associated learning behaviors in a mouse model of moderate fetal alcohol spectrum disorder (FASD). Although originally discovered as a key metabolic regulator, GSK-3 has experienced resurgence in research interest due to its ability to regulate multiple signaling processes in the developing and adult CNS. Furthermore, GSK-3 inhibition has been shown to have therapeutic effects in a wide array of neurological and neurodevelopmental disorders, including developmental alcohol toxicity. GSK-3 inhibition has been shown to exert therapeutic benefits in preclinical rodent models of stroke, Alzheimer's disease, bipolar disorder, and schizophrenia. The current proposal is based, in part, on published studies performed in collaboration with Dr. Allan's laboratory demonstrating that GSK-3 inhibition restores adult hippocampal neurogenesis and associated learning behaviors in a genetic mouse model of fragile x syndrome. Based on these findings, we hypothesize that GSK-3 inhibition also restores learning and neurogenic defects in our mouse model of moderate FASD. We will test this hypothesis using a combination of pharmacological and genetic approaches. Specifically, we propose to determine whether GSK-3P expression patterns are altered in adult hippocampus of FASD mice (Specific Aim 1), whether pharmacological inhibition of GSK-3 activity restores neurogenesis and improves functional plasticity of newborn granule neurons (Specific Aim 2) and whether inducible and selective gene deletion of GSK-3P in adult hippocampal progenitors improves neurogenesis and learning (Specific Aim 3). If successful, these studies will broaden our understanding of both FASD and GSK-3 mechanisms in adult neurogenesis, and could lead to identification of a novel therapeutic target for improving hippocampal function and reversing behavioral deficits in clinical FASD.

SUMMARY The Preclinical Core (PC) will support the goals of the COBRE Center for Brain Recovery and Repair by establishing a much-needed centralized resource of expertise, training and instrumentation for high quality automated behavioral and structural analyses, broadly applicable across a range of preclinical models of brain and behavioral illnesses. Currently, there is a breadth of investigator expertise in preclinical models of neurological disease across multiple departments at the University of New Mexico Health Sciences Center, but complete lack of a centralized facility that can provide the expertise, training and instrumentation necessary for automated behavioral assessment and structural analyses. In support of the COBRE Center mission, establishment of the PC will accelerate the trajectory of Junior COBRE PIs toward independent NIH-R01 level funding, by providing the know-how, training, instrumentation and proficiency necessary for high throughput preclinical studies of brain recovery and repair. The PC will also foster multidisciplinary interactions between established and young UNM investigators in the discovery of novel therapies for brain recovery and repair using preclinical models that will enhance the overall level of excellence in this vital area of neuroscience research. The following aims will be addressed: Specific Aim 1: Create a centralized resource that provides expertise, training and instrumentation for automated behavioral assessment and structural analyses, applicable across a broad range of preclinical models of brain and behavioral disorders. Specific Aim 2: Support the research objectives of Junior COBRE PIs and cultivate a new cohort of COBRE investigators using preclinical models within the Center for Brain Recovery and Repair. Specific Aim 3: Develop a PC user base of established, funded investigators for continued growth and sustainability of the core facility. Achieving these aims will be essential to the long-term viability of the Center for Brain Recovery and Repair and will enhance multidisciplinary collaborations at UNM, across New Mexico and other IDeA states.

PROJECT SUMMARY/ABSTRACT The overall goal of this proposal is to establish the relevance of adult hippocampal neurogenesis as a potential therapeutic target for fetal alcohol spectrum disorder (FASD), utilizing a well-characterized mouse model of prenatal alcohol exposure (PAE). The significance for adult hippocampal neurogenesis as a potential therapeutic target in fetal alcohol spectrum disorder (FASD) is based on preclinical rodent models that demonstrate long- lasting deficits in neurogenesis following developmental alcohol exposure, the critical role of neurogenesis in many hippocampal-dependent behaviors that are also disrupted in clinical FASD, and recent evidence that neurogenesis continues throughout life in the human hippocampus. Using voluntary drinking paradigms to model PAE in mice, our laboratory previously demonstrated marked impairment of the neurogenic response to enriched environment (EE) that is associated with disruption of neurochemical, morphological and electrophysiological indices of EE-mediated network activity. Experiments outlined in this proposal are designed to test the overall hypothesis that impaired EE-mediated neurogenesis is relevant for deficits in behavior and hippocampal network mechanisms in PAE, and can be restored using genetic and/or pharmacological therapeutic approaches. This hypothesis will be tested by addressing the following specific aims. Specific Aim 1: To determine whether impaired EE-mediated neurogenesis is directly correlated with impaired pattern discrimination learning in PAE mice. We will utilize two complex neurogenesis-dependent cognitive tasks in which pattern discrimination/separation is tested in both contextual and spatial domains. Behavioral performance will be correlated with impaired neurogenesis in PAE-EE mice, and with immediate early gene expression as a readout of network activation. Specific Aim 2: To determine whether impaired EE-mediated neurogenesis in PAE mice leads to compensatory remodeling of afferent synaptic input to aDGCs. We will utilize neuroanatomical approaches including rabies-based retrograde tracing to determine whether dendritic complexity and/or the distribution of monosynaptic afferent inputs to aDGCs are selectively altered in PAE-EE mice. Specific Aim 3: To determine whether sensitivity to EE-mediated neurogenesis in PAE mice is restored by genetic and/or pharmacological intervention. We will utilize a genetic gain-of-function approach to augment the survival of aDGCs in an attempt to restore EE-mediated neurogenesis in PAE mice. In addition, we will test whether the neurogenic antidepressant, fluoxetine (FLX), restores EE-mediated neurogenesis and behavior in PAE. If so, causal relationships between neurogenesis and FLX-mediated behavioral improvement will be tested by selective, genetic silencing of aDGCs.

PROJECT SUMMARY The Preclinical Core supports the mission of the Center for Brain Recovery and Repair by providing well- validated measures of both structural and functional alterations for investigators utilizing preclinical models of neurological disorders. The Core is a centralized resource providing intellectual guidance, technical expertise, training and instrumentation for high quality automated behavioral and structural analyses, broadly applicable across a range of preclinical models of brain and behavioral illnesses. Through cultivation of new research programs and collaborative interactions between young investigators and established experts, the Core has played an essential role in elevating the excellence of brain injury and repair research at UNM. Overall, the goal of the Core is to leverage our success in Phase I to catapult preclinical research capacity to the critical mass of funded investigators required to support the Center?s mission of self-sustainability beyond IDeA-level funding. We will continue to provide the resources and expertise required to support interdisciplinary cutting-edge research projects of Project Leads in neurophysiological mechanisms of brain recovery and repair, and to expand our user base of new and established investigators through project development and strategic investment in state-of-the-art instrumentation, approaches and methodologies. Via the cultivation of innovative new research programs and increased collaborative interactions between junior investigators and established scientists the Core will contribute to the sustainability of the Center for Brain Recovery and Repair as a nationally recognized leader for translational research in brain recovery. Since being established in 2015, the Preclinical Core has helped advance the field by leading in the development of translational EEG as a platform for assessing homology of neural activity during behaviors across species. Phase II builds on these successes with the combination of physiological and behavioral approaches, and adds higher throughput anatomical assessments to speed studies utilizing sophisticated confocal imaging. Continuing from Phase I, the Core will promote the Center mission of accelerating the trajectory of Project Leads toward independent NIH-R01 level funding for Junior Investigators or establishing themselves in the field of brain injury for more senior Investigators. The Preclinical Core will continue to foster multidisciplinary interactions between clinical and preclinical investigators at UNM and across the Mountain West. We will also provide a platform for the discovery of novel therapeutic avenues for brain recovery and repair using preclinical models that will enhance the overall level of excellence in this vital area of neuroscience research.