Fred H. (Rusty) Gage - US grants

psychophysiology; nervous system transplantation; brain cell; aging; embryo /fetus cell /tissue; tissue donors; chordate locomotion; learning; neuropharmacology; innervation; neurochemistry; sensorimotor system; neurotransmitter metabolism; ethology; stereotaxic techniques; immunofluorescence technique; mature animal; histochemistry /cytochemistry;

Transplantation of embryonic neural tissue to the central nervous system of brain damaged adult host rats has been demonstrated to induce anatomical indices of reinnervation, biochemical indices of neurotransmitter restoration and behavioral indices of functional recovery. Aged rats exhibit specific anatomical, biochemical and behavioral deficits in systems that have been previously demonstrated by this laboratory to respond favorably to transplantation of embryonic neural tissue in the adult host rat. We propose to determine 1) if the aged rat will act as a receptive host of transplanted embryonic neural tissue, and 2) whether the transplants will reverse the behavioral deficits measured. Further experiments will determine 1) the optimal parameters of transplantation, 2) whether transplantation at an earlier date will protect the development of aged related deficits and, 3) whether multiple grafts are more effective than single grafts. Subsequent experiments will test 1) effectiveness of embryonic nerve cells in other brain areas, 2) combined effect of drug treatments and transplants and 3) the effectiveness of cross-species transplantation. We will use histochemical methods to determine the extent of anatomical innervation, biochemical markers as indices of neurotransmitter restoration, and behavioral tests as indices of functional recovery. The overall objective of the research is to introduce the transplantation techniques into the ongoing investigation of aging as a new tool to determine basic mechanisms underlying the aging process.

Subpopulations of rats show a significant decline in learning and memory during the aging process which is not attributable to sensory, motor or motivational deficits. The basal forebrain cholinergic system is also impaired in these behaviorally impaired aged rats. In addition, evidence supports a decline in Nerve Growth Factor (NGF) and NGF receptors and their respective mRNAs in aged animals. Further, high voltage EEG spindles (HVS) in the neocortex which can disrupt cognitive function are increased dramatically with aging and are controlled in part through cholinergic neurons in the basal forebrain. Finally, the inserted form of the amyloid precursor protein (APP-751) which is regulated in part by NGF is abnormally expressed in the aged, behaviorally impaired rats. We wish to determine whether and/or which of these physiological markers of aging are causally linked to the age related learning and memory deficits. To test the generalizability of these results we will repeat the experiments in several rat strains, and will expand the learning test repertoire. We have shown that chronic infusions of the NGF can have a positive influence of retention in the learning task of the aged impaired animals, and that grafts of fetal basal forebrain tissue to the brains of the aged impaired animals have an ameliorative effect on acquisition in the same learning task. In the present set of experiments we propose to test the influence of NGF infusions, intracerebral rafts of fetal basal forebrain and a combination of NGF and intracerebral grafting on the physiological parameters described above in an attempt to determine whether any of these parameters are influenced by the treatments which have ameliorative influences on aspects of aged related behavioral impairments. These experiments are designed to test experimental therapeutic protocols in behaviorally impaired aged animals, and to determine the functional relationship between cholinergic activity, NGF function, and expression of the APP.

Neuronal grafting into the CNS has recently been suggested as a potential approach to CNS therapy through the selective replacement of cells lost as a function of disease or damage. Independently, studies aimed at direct genetic therapy in model systems have recently begun to suggest conceptually new approaches to the treatment of several kinds of human genetic diseases, especially those caused by single gene deficiencies. We have recently suggested that a combination of these two approaches, namely the grafting into the CNS. We have presented evidence for the feasibility of this approach, including a description of some current techniques for mammalian cell gene transfer and CNS grafting. In this application we propose to test the phenotypic effects of implanting cells expressing on of 2 vitally transduced genes - the E. coli B-galactosidase (ladZ gene product) to optimize conditions for grafting, and mouse Nerve Growth Factor (NGF) to establish a phenotypic effect in young animals with brain damage, or aged animals with morphological and cognitive deficits.

Transplantation of embryonic neural tissue to the central nervous system of brain damaged adult host rats has been demonstrated to induce anatomical indices of reinnervation, biochemical indices of neurotransmitter restoration and behavioral indices of functional recovery. Aged rats exhibit specific anatomical, biochemical and behavioral deficits in systems that have been previously demonstrated by this laboratory to respond favorably to transplantation of embryonic neural tissue in the adult host rat. We propose to determine 1) if the aged rat will act as a receptive host of transplanted embryonic neural tissue, and 2) whether the transplants will reverse the behavioral deficits measured. Further experiments will determine 1) the optimal parameters of transplantation, 2) whether transplantation at an earlier date will protect the development of aged related deficits and, 3) whether multiple grafts are more effective than single grafts. Subsequent experiments will test 1) effectiveness of embryonic nerve cells in other brain areas, 2) combined effect of drug treatments and transplants and 3) the effectiveness of cross-species transplantation. We will use histochemical methods to determine the extent of anatomical innervation, biochemical markers as indices of neurotransmitter restoration, and behavioral tests as indices of functional recovery. The overall objective of the research is to introduce the transplantation techniques into the ongoing investigation of aging as a new tool to determine basic mechanisms underlying the aging process.

DESCRIPTION (provided by applicant): The aim of this Program Project is to develop reliable, safe, and rational gene transfer procedures to repair or replace neurotransmitter function in relevant animal models of Alzheimer's Disease (AD). Project 1 focuses on the uses of lentivirus to replace age related decline of the cholinergic system in aged rats by over expression of the CHAT gene regionally; protecting against age related cholinergic cell loss; and testing the therapeutic effects of these genes in the mouse by over expressing a human mutated form of APP. Project 4 will use lentivirus to over express Cre recombinase in transgenic mice with the LOX sites surrounding the CHAT, NGF and P75 genes respectively, and will cross mice with homologous knockout of p75 and NGF gene, with mice over expressing a human mutation of the APP gene, to directly determine the interaction between these related genes. Project 5 will test the hypothesis that development of Alzheimer's disease is due to multiple deficits or "stresses" which lead to pathology. Project 3 will optimize in vivo vectors for gene therapy in primates by comparing AAV and lentivirus for amount, duration and safety of gene expression. In Project 6 recombinant lentiviral vectors expressing mutant PS1 or APP in developing transgenic models of amyloid deposition in the brain, will examine the interactions of these genes in vivo. The Vector Core, will support high recombinant vectors to all projects. The Projects/Cores in this PPG have been chosen for their relevance in addressing these problems and securing a rational approach to gene therapy in AD. The interdependency of resources and skills between Projects/Cores, as well as our past success with this PPG, support the argument that the Program Project is the most efficient and cost-effective way to address these issues.

Parkinson's disease (PD) is a neurodegenerative disorders characterized by a progressive loss of dopaminergic neurons of substantia nigra pars compacta with motor dysfunctions. The etiology of PD is yet unknown, and there is no cure available. Although various pharmacotherapies are modestly successful, late complications of the therapies limit the ultimate utility. We propose the use of genetically modified primary skin fibroblasts that produce catecholamines or neurotrophic factors as donor cells for grafting in rat models of PD. The primary dermal fibroblasts can be biopsied easily and grown well in culture. These primary fibroblast can be genetically modified by retroviral vectors with high efficiency. Tyrosine hydroxylase, aromatic L-amino acid decarboxylase, basic fibroblast growth factor, and brain derived neurotrophic factor will be introduced by this method. The transduced fibroblasts will be examined in vitro to characterize the expression of these transgenes and their biological functions. These cells will then be grafted in the brain to assess the survival, host reactions, and transgene expression in vivo. Once fully characterized, these cells will be studied for functional effects in rat models of PD. The catecholamine-producing cells will be tested for their ability to ameliorate the apomorphine-induced rotational behavior in rats with unilateral lesions of the substantia nigra. The neurotrophic factor- producing fibroblasts will be studied for their ability to promote sprouting and survival of the remaining dopaminergic neurons, and their effect on behavioral recovery. In the future, combinations of both neurotransmitter replacement and neurotrophic factor delivery strategies will also be studied.

The aim of this Program Project is to develop reliable and rational procedures to repair or replace neurotransmitter function in aged, cognitively impaired animals, with the idea that the tools and strategies developed and tested will be relevant to Alzheimer's disease (AD). Our underlying premise is that transfer to the brain of genes that will either protect against age-related neuronal cell death, repair neuronal cell damage, or replace missing neurotransmitter function will delay age-related functional decline, or reverse some of the deficits associated with age-related decline. The therapeutic potential of gene transfer as a for neurological disease is promising, yet substantial technical and theoretical problems remain to be solved before this technology can be seriously considered for clinical application. The Project and Cores in this Program Project have been designed for their relevance in addressing these problems and securing a rational approach to gene therapy in AD. Specifically, Project 1 uses established rat models of cognitive dysfunction to test whether and which gene transfer methods and genes result in long-term functional recovery. Projects 4 and 5 will establish transgenic models of age-related cognitive dysfunction to permit a better understanding of the role of specific neuronal systems in age- related cognitive decline and provide more exacting models to test gene therapy strategies. Project 3 uses primate models as a preclinical test of the results from Project 1, 4, and 5. The information obtained in Project 3 will then be used by Projects, 1, 4, and 5 to better design or modify existing experimental designs to meet the goals of clinically relevant gene therapy. The Cores are designed to support all aspects of the Program Project: 9001 will provide cells, plasmids and vectors; 9002 will assist with surgery, behavioral testing and histology; and 9003 will maintain ES cell lines and assist in the production, generation and maintenance of transgenic animals. The interdependency of resources and skills between Projects and Cores, as well as our past success with a Program Project, supports the use of the Program Project form at as the most efficient and cost effective way to develop and refine gene therapy methods for cognitive dysfunction.

confocal scanning microscopy; biomedical facility; autoradiography; neuronal guidance; immunocytochemistry; intracellular; tissue /cell culture; histopathology;

nervous system transplantation; nervous system regeneration; neurotrophic factors; hippocampus; disease /disorder model; tenascin; cadherins; neuronal guidance; synaptogenesis; fibroblasts; brain injury; fibroblast growth factor; collagen; neuroanatomy; confocal scanning microscopy; laboratory rat; experimental brain lesion; immunofluorescence technique; monoclonal antibody; immunocytochemistry;

The overall aim of the Core - 9002 (Systems Neuroscience) is to provide the instrumental base for the neurobiological components of the individual projects and to develop novel methods. The use of different Alzheimer~s disease (AD)-related animal models and the development and testing of numerous treatment strategies in different species (multimodel approach) is the major focus of this core. Aged, cognitively impaired rats, rats with transections of the fimbria-fornic or the perforant pathway, and mice with specific gene defects for nerve growth factor (NGF), choline acetyltransferase (ChAT) and glutam ate receptor-2 (GluR-2) will function as experimental tools for testing different ex vivo and in vivo gene transfer interventions. 9002 will provide standardized surgical (lesioning techniques, ex vivo and in vivo gene transfer), behavioral (sensorimotor function, general activity, acoustic startle, passive avoidance, water maze, five-choice serial visual discrimination, spatial delayed nonmatch-to-sample), biochemical (ChAT activity, acetylcholine, monoamines and their metobolies, amino acids) and morphological (histochemistry, immunohistochemistry, immunofluorenscence, in situ hybridization, stereology, electron microscopy) methods. This multimethodical approach supports the individual projects and, by standardizing the various methods in multiple AD-related animal models, it will enable this program to make direct comparisons between results from each project. Finally, this standardized multimodel and multimethodical strategy will help to identify potential treatments in age-dependent cognitive dysfunction.

In Project 1 we aim to therapeutically deliver trophic factors or neurotransmitter enzymes to protect against age-related dysfunctions found in aged rats or transgenic mouse models of Alzheimer's disease. The degeneration of cholinergic neurons is one of the most consistent findings in AD. We propose to use viral vectors to investigate therapeutic approaches of direct gene delivery to animal models of AD primarily focused on lentiviral vectors. Three complementary strategies will be developed to reverse the morphological and behavior deficits associated with aging in normal aged rats and a transgenic mouse model of AD. The first strategy is to restore function using ChAT to replace or supplement depleted acetylcholine in aged rats. The second strategy places the emphasis on neuroprotection using the NGF gene and product to protect ChAT neurons from age related death, and enhance the function of the remaining neurons. A progression within these two strategies from constitutive to regulatable expression will be made as we improve our lentiviral vectors. The third strategy focuses on the interplay between amyloid precursor protein (APP), and the cholinergic system. Lentiviral factors delivering either NGF or ChAT will be injected into the hippocampus of transgenic animals over expressing APP to elucidate whether the cholinergic system regulates amyloid processing, alters plaque formation, or may improve behavioral performance. Functional and anatomical effects of direct gene delivery of choline acetyltransferase (ChAT) and nerve growth factor (NGF) to the aged brain have not been evaluated. Using lentiviral vectors, we can deliver these molecules to discrete populations of cells within the central nervous system (CNS), measuring the effects of direct (ChAT), or indirect (NGF) cholinergic enhancement on cognition, neuronal and synaptic changes, and amyloid processing.

DESCRIPTION: (from the applicant's abstract) Most neurons of the adult central nervous system are terminally differentiated, exist through the life of the organism and, when they die, are not replaced. However, evidence exists that small populations of neurons continue to be born in the adult ventricular zone and hippocampus. Given adult hippocampal granule cells continue to be born through the life of the rodent and FGF-2 can induce hippocampal neuroblast proliferation in vitro , we designed a set of experiments to determine if adult hippocampal cells could be isolated and expanded in vitro to an adequate number for transplantation back into the adult hippocampus. Our results show that cells of the adult nervous system can be isolated and characterized, expanded indefinitely in vitro , genetically labeled in vitro , and transplanted back into the adult central nervous system where they can survive and differentiate in a target-specific manner into neurons. In this grant we plan to extend these observations by conducting the following experiments. 1) We will isolate individual cells of the expanded population of the progenitor cells and grow them as clones to determine the pluripotentiality of the progeny of each of the clones. We will define conditions that influence the fate of the progenitor cells toward glial or neuronal lineage. 2) We will determine if, and to what extent, adult progenitors survive engraftment to the developing and adult brain. We will then determine whether the grafted cells will differentiate and which phenotypic markers they express at different time points following implantation. Finally in this aim we will determine exogenous and endogenous factors that influence the extent of cell survival and the fate of cells following grafting. 3) We will implant the cells into the damaged CNS to determine if they can participate in the repair process, or replace missing cells. Independently of our success in the preceding component of this aim we will genetically engineer the progenitor cells to make and secrete the product, then implant the cells into a relevant animal model. Finally in this aim we will inject FGF-2 chronically in the adult brain to induce proliferation and differentiation of the endogenous progenitor cells.

cell transplantation; nervous system transplantation; neurons; spinal cord injury; brain injury; method development; biological models; mature animal;

The overall aim of this project is to characterize factors that act on neuronal stem or progenitor cells in the adult and aging brain and influence or regulate endogenous adult neurogenesis. Knowledge about the biology of these processes will be relevant for future therapeutic approaches to Alzheimer's Disease (AD) and other neurodegenerative disorders, in that manipulation of neuronal stem or progenitor cells in situ night allow neuronal replacement and cellular and structural regeneration. The underlying rationale is that identification of regulatory mechanisms under physiological conditions will provide the necessary tools to manipulate neuronal stem or progenitor cells for therapeutic goals. In this project we propose to investigate molecular events and constraints that regulate neurogenesis in the adult and aged rodent and primate dentate molecular events and constraints that regulate neurogenesis in the adult and aged rodent and primary dentate gyrus. Glutamatergic fibers project to the dentate gyrus and damage to these fibers has been reported to influence neurogenesis. We will therefore also investigate in transgenic animals the role of glutamate receptors in neurogenic regulation. The specific objectives of this project are: 1) Determine if and to what extent hippocampal neurogenesis occurs in adult primates. 2) Determine the effects of long-term environmental stimulation on neurogenesis in the aging brain of mice. 3) Determine the role of learning and physical activity as key factors in environmental stimulation of adult hippocampal neurogenesis. 4) Characterize the role of glutamatergic system n the regulation of neurogenesis in the adult and aging hippocampus.

The observation that endogenous stem cells exist in the CNS of adult mammals has grown into an exciting field of study. The capacity of these cells for repair is a current and very active topic of debate. However, a training conference on this topic has not yet been conducted. By assembling scientists and trainees into a workshop to discuss the manipulation and recruitment of endogenous stem cells, we hope to generate new ideas and alternatives to cell transplantation for many CNS disorders. This August, a Route28 Summits in Neurobiology workshop will be held on the therapeutic potential of endogenous neural stem cells in the adult CNS. This workshop is designed to give students, postdoctoral and clinical fellows exposure to the thoughts and works of the most accomplished researchers in neurobiology. Participants in the workshop will hear talks from eighteen well-known leaders in the fields of CNS trauma, neural degeneration and stem cell biology. This workshop is unique in that it focuses on the direct interaction between students and prominent researchers. Formal talks will alternate with candid and critical discussions led by each speaker. To facilitate interaction, students must integrate the information into mini-grant proposals that address repair issues in the diseased or traumatized CNS. Throughout the course of the meeting, attendees will query faculty members to test theories and conjectures in the context of the Summit topic. Trainee participants will work in groups of five to solve one of three problems presented on the first evening of the meeting. On the 4th day, the groups will have the opportunity to re-address the speakers on any topic and will then finalize and present the strategies they have developed. A panel of senior scientists will score the merit of each proposal based on the creativity, scientific merit and feasibility of the proposed projects. An expanded synopsis of the three award winning proposals, accompanied by critiques from the attending speakers, will be published in a joint Summit Summary. The meeting format highlights the strengths of scientific collaboration in an environment modeled after the granting process. This workshop has two unique goals in mind, to encourage the exchange of ideas between senior faculty and student participants, and to promote rewarding collaborative interactions that may grow into future scientific endeavors.

DESCRIPTION (provided by applicant): Parkinson's disease is a neurodegenerative disorder characterized by a progressive loss of dopaminergic neurons of the substantia nigra pars compacta (SN). The etiology is largely unknown and there is no cure available. Although current pharmacotherapies are modestly successful in ameliorating motor deficits, late complications of the therapies limit their long-term utility. The paucity of adult CNS plasticity is a further limit to structural repair of the striatonigral system. However, there is recent evidence that some areas of the adult brain retain the ability of continued generation of new cells including neurons and that this process contributes to repair following neuronal injury. Our results show that continued cell generation occurs in the adult SN. In addition, we have evidence that the resident proliferating cells in the adult SN have the potential to give rise to neurons. We propose that continued cell generation in the adult SN can be exploited for future endogenous dopaminergic cell replacement therapies in Parkinson's disease. In this grant we will examine cell genesis in the adult SN under three conditions in vivo: cell genesis in the intact adult SN; cell genesis in the adult SN in behavioral paradigms that stimulate neurogenesis in other CNS regions; cell genesis in the SN in an experimental model for Parkinson's disease. These in vivo studies will be complemented by a set of experiments in which we will: investigate the characteristics of proliferating cells derived from the adult SN in vitro; define conditions which will enhance dopaminergic cell generation from these cells. The suggested experiments will provide the groundwork for possible future strategies in which we will try to manipulate cell genesis in the adult SN in order to restore the function of the injured adult SN.

Cells proliferate in the inner granular zone of the dentate gyrus of the adult hippocampus of all mammals. It is hypothesized that some of the dividing cells are stem cells because they can give rise to neurons and glia in the dentate gyrus. Additionally, we have demonstrated that cells from the adult hippocampus can be isolated in vitro and induced to proliferate indefinitely. Under specific conditions the cells can be transplanted back into the adult brain where they can differentiate into neurons and glia. We demonstrated previously that there is a progressive decrease in the number of proliferative cells in the dentate gyrus of adult rodents with aging. This decrease in neurogenesis in the aged dentate gyrus can be partially reversed by environmental stimulation. We do not know whether newly born cells in the aged brain can become neurons that are anatomically and functionally similar to the new neurons in the young adult brain. Further, we do not know if environmental stimulation like voluntary exercise will affect only the proliferation rate and survival of the newly born neurons, or whether it will also affect the anatomical or physiological properties of the differentiated cells. To determine the answer to these questions in vivo we developed new methods that allow us to measure anatomical and physiological properties of individual, newly born cells in acute brain slices. Further, we developed methods to isolate adult stem cells in vitro and measure their functional and electrophysiological properties in vitro. Finally, using transplantation methods established in this lab we can directly compare the effects of the age of the host hippocampus on survival and function of grafted aged versus young adult stem cell populations propagated in vitro.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] Therapeutic advances utilizing gene therapy vectors have recently shown great promise in a number of pre-clinical studies of neurological disorders. The central nervous system, however, presents unique challenges for the development of successful gene therapy approaches and advancement of this therapeutic modality to the clinic. To accelerate gene therapy applications into the clinic, several issues remain to be further examined and resolved. One major issue that has raised significant concern in gene therapy is long-term safety. A second issue that requires further research is the development of efficient delivery strategies to target diseased regions of the brain. Thirdly, effective methods for both long-term and controlled expression of therapeutic transgenes need to be developed. Assessment of the efficacy and safety of proposed methodological advances would require pre-clinical testing in animal models of neurodegenerative disorders. We have recently discovered that adeno-associated vectors (AAV) have the unique property of retrograde transport within the nervous system and we have utilized this finding to efficiently deliver therapeutic transgenes to distinct regions of the brain. Furthermore, we have recently engineered a novel regulatable AAV vector system that allows control of transgene expression. The novelty of the system allows genes to be rapidly turned on with one ligand and rapidly turned off by a second, different ligand. In this project, we propose to develop efficient, targeted gene delivery to the nervous system and test the recrulatable AAV vector system for in vitro and in vivo applications. We will assess the ability of this system to efficiently deliver therapeutic transgenes that will prevent or slow cell death occurring in neurodegenerative diseases, and utilize this new methodology to determine therapeutic windows for intervening in disease progression.

DESCRIPTION (provided by applicant) Neurogenesis in the adult mammalian brain is restricted to the subventricular zone and the dentate gyrus of the hippocampal formation. These regions contain mitotically active cells that are, in fact, neural progenitor cells (NPCs) with the capability of producing both neuronal and glial cells through adulthood. NPCs are also present in non-neurogenic areas of the adult brain including the neocortex and substantia nigra (SN), and they can give rise to neurons when differentiated under permissive conditions. At present, the physiological role of neurogenesis in the adult brain it is unclear, and the mechanisms underlying this spatial restriction are not yet understood. This proposal will test the overall hypothesis that NPCs from the SN have neurogenic potential, but the SN is a restrictive or non-supportive environment for neuronal differentiation and/or survival. The main goal of this proposal is to investigate whether NPCs from the adult SN can generate functional neurons when exposed to permissive environments both in vitro and in vivo. The Specific Aims cover the characterization of the electrophysiological properties (generation of action potentials and formation of functional synapses) of NPCs cultured in defined media, and co-cultured with astrocytes (known to induce neuronal differentiation) or with primary striatal neurons, the physiological target of dopaminergic neurons from the SN. Furthermore, nigral NPCs will be grafted into the dentate to investigate if they can generate functional neurons in a neurogenic area in vivo. To determine whether there are intrinsic differences between NPCs isolated from neurogenic and non-neurogenic regions, functional properties of differentiated NPCs derived from the SN will be compared with those of NPCs isolated from the dentate gyrus. These experiments will establish whether SN-derived NPCs have the potential to differentiate and integrate in the existing circuits, how their function is influenced by the environment, and whether they can be functionally distinguished from DG-derived neurons. This comparison is relevant to understand the basis for the absence of neurogenesis in the non-permissive environments of the brain.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. There are two primary goals of this project. The first is to determine the role of glial processes in the generationof synapses onto newborn neurons in the adult dentate gyrus. The second is to clarify the organization of neurogenic niches in the subgranular layer (SGL) and their relationship to potential supporting networks, such as the vasculature. This work will require the use of EELS microscopy and large-scale high-resolution mapping technologies being developed at NCMIR.

DESCRIPTION (provided by applicant): New neurons are continuously generated from neural stem cells in two discrete regions of the adult mammalian brain: the subventricular zone of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus. We have previously shown that adult hippocampal neurogenesis is regulated by interaction of neural stem cells with the local astrocyte population. Adult hippocampal astrocytes provide an environment that instructs neural stem cells to adopt a neuronal fate. Both diffusible and membrane-bound factors derived from hippocampal astrocytes contribute to these effects; however, the identity of these factors and the respective signaling pathways remain unknown. Our preliminary results indicate that hippocampal astrocytes express members of the Wnt family of proteins, including Wnt3, and that Wnt3 enhances the neuronal differentiation of adult neural stem cells. Conversely, blocking of hippocampal astrocyte-derived Wnts decreases the neuronal differentiation of neural stem cells in astrocyte/neural stem cell co-cultures. Furthermore, perturbation of Wnt signaling in vivo decreases the rate of neurogenesis in the adult hippocampus, suggesting that Wnt signaling plays a central role in adult hippocampal neurogenesis. In this proposal we will investigate the role of hippocampal astrocyte-derived Wnts and Wnt signaling in hippocampal neurogenesis in vitro and in vivo. Moreover, we will begin to investigate the functional consequences of perturbed Wnt signaling on hippocampal neurogenesis and hippocampal function. Finally, we will investigate how manipulation of Wnt signaling in other regions of the adult CNS influences the behavior of endogenous NSCs in non-neurogenic regions. These studies will not only lead to a better understanding of the molecular signals controlling the behavior of adult NSCs but will also provide a first insight into the role of Wnt-dependent neurogenesis in hippocampal function. The studies may also ultimately help in the design of therapeutic approaches in CNS disorders that aim at the recruitment of endogenous NSCs for replacement of dying neurons.

DESCRIPTION (provided by applicant): The discovery that restricted regions of the adult mammalian brain exhibit ongoing neurogenesis promises considerable therapeutic potential, however, there is a current lack of understanding regarding the cellular and molecular mechanisms that control neural stein cell proliferation and fate specification in vivo. Neuronal differentiation is in part controlled by the transcription factor NRSF, which restricts expression of neuron specific genes to neuronal cells. By binding to a ~21 base-pair consensus DNA sequence (NRSE) within their regulatory regions and via recruitment of silencers, NRSF represses these genes. As neural stem cells (NSCs) undergo differentiation into mature neurons, repression is relieved and expression of neuron-specific genes is activated. Recently, a double-stranded RNA (dsRNA) of the same sequence and approximate length as the NRSE was found to mediate this switch. This RNA is necessary and sufficient to specify the neuronal fate of multipotent adult NSCs in vitro and may therefore function as an intrinsic inducer of neuronal differentiation in vivo. Moreover, this finding represents the first example of a novel class of regulatory RNAs, termed small modulatory RNAs (smRNA), whose biogenesis and mode of action is distinct from previously characterized RNAs. The overall goal of this work is to elucidate how NRSE smRNA is expressed and to understand the molecular mechanism by which this RNA regulates adult neurogenesis in vivo. This project is in response to the PAS-04-130 on Collaborative Research in Stem Cell Biology and reflects the collaboration of several scientific groups to address this novel discovery. Specific Aim 1 will focus on how the NRSE smRNA is regulated in NSCs and Specific Aim 2 will examine the functional consequences of regulating this smRNA in vivo. These are aims directly related to skills and experience in the Gage lab. Specific Aim 3 focuses on the structural relationships between the NRSE smRNA and NRSF, and with its DNA binding sequence. The Riek lab has skills and expertise in addressing these structural biology questions. Specific Aim 4 continues the search for new smRNAs by taking advantage of the expertise in ribozyme technology developed by Kuwabara in Japan and applied at the Salk for this project.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] Amyotrophic lateral sclerosis (ALS) is a lethal neuromuscular disease that is associated with the degeneration of spinal and brainstem motor neurons (MNs), leading to atrophy of limb, axial and respiratory muscles. The cause of ALS is unknown, and there is no effective therapy. Factors that generally act to protect neurons from cell death, including neurotrophic factors (NTFs), are obvious candidates for therapeutic evaluation in ALS. While the chronic delivery of large molecules directly to the CNS has proven to be a major obstacle for therapy of CNS diseases, we recently discovered that recombinant adeno-associated viral vectors (AAV) expressing therapeutic genes can be retrogradely transported efficiently from muscle to MNs of the spinal cord. We utilized this novel transport system to deliver glial cell line-derived neurotrophic factor (GDNF) and insulin-like growth factor-1 (IGF-1) to spinal MNs to test their effects in an animal model of ALS. We report that IGF-1 prolongs life and delays disease progression, even when delivered at the time of overt disease symptoms in an ALS animal model. In addition, IGF-1 significantly slows the initiation of the apoptotic process in this disease model. Clinical trials are currently being designed to test this potential therapy in patients with ALS. This proposal seeks to translate the basic science discoveries we have made into a clinical study of AAV-IGF-1 in subjects with ALS. We aim to complete further efficacy studies as well safety and toxicity studies required for the submission of an Investigational New Drug Application (IND) for a clinical study of AAV-IGF-1 in adults with ALS. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): LINE-1 (L1) retrotransposons are active elements in the genome capable of mobilization in neuronal precursor cells, resulting in a mosaic brain. Upon mobilization, L1 insertions can alter gene expression, resulting in a genetic heterogenic population of neurons. However, the physiological consequences of somatic L1 retrotransposition are unknown. Our preliminary results showed that L1 retrotransposition levels are increased in a MeCP2 knockout mouse model, indicating that MeCP2 is important for L1 repression during neuronal differentiation in a methylation-independent fashion. Based on this, we postulate that the phenotypic heterogeneity in Rett Syndrome (RTT) could be, at least in part, due to variable neuronal patterns of L1 activity in the brain. The main objective of this proposal is to characterize the molecular complex that represses L1s in undifferentiated neural stem cells and the switch mechanism that unleashes L1 expression during neuronal differentiation. Specific aims are designed to obtain results with cellular models (both rodents and humans) and to attenuate L1 retrotransposition in MeCP2 knockout mice. A battery of behavioral tests will reveal if L1 attenuation could rescue, at least partially, some of neurological defects associated with this mouse model. [unreadable] PUBLIC HEALTH RELEVANCE: The goal of this proposal is to characterize the role of MeCP2 in the control of retrotransposition in nerve cells. For that purpose, we will use several rodent and human cells to monitor the activity of L1 retrotransposons in presence and absence of MeCP2, and the consequences of an attenuated L1 activity to a mouse model of Rett syndrome. The validation of this hypothesis will certainly open new potential possibilities for therapeutic interventions for RTT and other neurological diseases. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): LINE-1 (L1) retrotransposons are active elements in the genome capable of mobilization in neuronal precursor cells, resulting in a mosaic in the brain. Upon mobilization, L1 insertions can alter gene expression, resulting in a genetic heterogenic population of neurons. The first aim of this proposal is to identify genomic loci of L1s in the human brain as compared to somatic tissues from the same individual in order to map and characterize de novo insertions in the brain. The second aim is to attenuate L1 retrotransposition in a mouse model and thereby investigate the functional significance of L1 in behavior. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to characterize the activity and effects of mobile genetic elements in the germ and neural genomes. We will determine where in the genome the mobile elements insert and separately determine the functional consequences of the mobile elements on behavior of individuals. The outcome of this study will increase our understanding of human diversity and behavior and may contain insights into the genesis of multiple neurological diseases.

DESCRIPTION (provided by applicant): The dentate gyrus is one of two regions in the adult brain that continuously incorporates new neurons throughout the lifetime of mammals. These new neurons, which add to the principal excitatory neuron population in the dentate gyrus, pass through several different morphological and functional states prior to fully integrating into the network. Previous behavioral and computational studies have suggested that this gradual incorporation of new neurons has the potential to have a substantial impact on cognition. While theoretical studies have demonstrated that these neurons may contribute novel forms of information into memories, including temporal information, there is little direct biological evidence showing what the effects of neurogenesis are on animal behavior. This proposal will address the function of adult neurogenesis from several different and important perspectives. First, a novel genetic mouse model that provides temporal selectivity will be used to knockout neurogenesis. This mouse line will allow examination of the functional role of adult-born neurons of specific ages. Such temporal specificity is important given recent theoretical arguments for age-dependent functions for maturing neurons. Second, the long-term specialization of adult-born neurons will be examined by labeling different populations of neurons which will be exposed to different experiences during their maturation. According to both theoretical modeling of adult neurogenesis and preliminary observations, this study should demonstrate that adult-born neurons will preferentially respond to environments that they experience shortly after they are born. Finally, this proposal will investigate the effects of dopamine - a behaviorally-regulated neurotransmitter - on the function of the dentate gyrus involving both existing and adult-born neurons. Preliminary data suggests that dopamine, which has been associated with both rewarding and aversive stimuli, has effects on both immature and mature dentate gyrus neurons. This proposal will explore both the extent of and the mechanisms underlying these effects. Adult neurogenesis has been shown to be associated with several neurological conditions, including depression, aging, and schizophrenia. The studies described in this proposal will serve to help elucidate mechanisms by which neurogenesis affects cognition and memory. Determining the functional impact of new neurons in the adult brain is an important initial step for understanding how adult neurogenesis is involved in these pathological conditions. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to investigate the function of adult neurogenesis in the hippocampus of the adult brain. We will determine how newly born neurons affect the ability for the brain to form new memories during behavior and what type of information these new neurons will encode later in life. The findings of this study will increase our understanding of the adult neurogenesis process which has been implicated in neurological conditions such as aging, depression, and schizophrenia.

DESCRIPTION (provided by applicant): Contrary to the conventional wisdom that the majority of healthy cells in an individual have identical genomes, endogenous L1 retrotransposons are now known to 'jump'during neurogenesis and change neuronal genomes. The diversity and prevalence of unique genomes is unknown, but these are essential data for understanding how mobile element-mediated genetic diversity affects neural circuits. Genetic diversity in a population cannot be measured using typical bulk analysis of a million or so cells;therefore, we propose to develop single cell methods to analyze retrotransposition in individual neuronal genomes. To understand how the diversity and prevalence (i.e. the mosaic composition) of de novo mobile element insertions alters neuron function, we propose three experiments. In one experiment, we will examine the mosaic composition of specific neural circuits in behavioral outliers. A second experiment will test the expectation that new mobile element insertions differentially alter the transcriptome of individual neurons. In a third experiment, we will generate mouse lines with little or no retrotransposition to determine if mobile element insertions are both necessary and sufficient for specific aspects of neuron function. Taken together, the application of single cell genomic approaches to understand neuronal diversity promises to challenge basic assumptions about the genetics of behavior and the origin of human neurodevelopmental disorders. PUBLIC HEALTH RELEVANCE: Our overall objective is to develop single-cell approaches to measure mobile element-mediated genetic diversity in neural circuits. These tools promise to reveal new factors that bring about discordant behavioral phenotypes in monozygotic twins and contribute to polygenic neuropsychiatric diseases such as schizophrenia and autism.

DESCRIPTION (provided by applicant): Addiction represents a complex genetic psychiatric disorder that likely involves a number of different genes. Addiction to cocaine and other psychostimulants, which are among the most addictive of substances, is also the most heritable. Recently, the ability to reprogram fully differentiated tissues, such as skin, into human induced pluripotent stem cells (hiPSCs) has made it possible to study a multigenic psychiatric disease, such as drug addiction. Cell-based human models for addiction will be created by directly reprogramming skin fibroblasts from methamphetamine-addicted patients and controls into hiPSCs (RFA-DA-11-012). Differentiating these hiPSCs into dopaminergic (DA) neurons will enable the identification of specific genetic and functional changes associated with hiPSC neurons treated with psychostimulants. There are three primary Aims for this R21 proposal. In Aim 1, fibroblasts from 'control'humans will be reprogrammed into hiPSCs and then differentiated into DA neurons. These DA neurons will be transcriptionally profiled in untreated, methamphetamine-treated and methamphetamine-withdrawal conditions to identify the gene pathways affected by methamphetamine treatment and withdrawal. In Aim 2, an electrophysiological characterization of DA neurons differentiated from control hiPSC lines will be completed, followed by studies examining the effect of exposure to methamphetamine treatment and withdrawal. In Aim 3, hiPSCs will be developed from humans addicted to psychostimulants and then examined for differences in properties of these patient-derived neurons and their response to methamphetamine treatment and withdrawal. The hypothesis that hiPSC DA neurons from patients addicted to methamphetamine respond uniquely to methamphetamine treatment in a manner distinct from controls will be tested. Preliminary data are provided that show reprogramming of adult cells into hiPSCs, differentiation of hiPSCs into DA neurons, and basic electrophysiological properties. Studies of patient-derived hiPSC neurons have the potential to significantly advance our understanding of a complex multigenic disorder like drug addiction. The proposed studies have the potential to reveal unique functional differences in DA neurons derived from addicted and control patients, as well as their response to methamphetamine. Results from the proposed studies will likely lead to both the development of improved animal models of addiction and provide an innovative approach for examining the effect of new drug therapies for the treatment of addiction. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to create human cell-based models for addiction by reprogramming skin samples from methamphetamine-addicted patients'human induced pluripotent stem (hiPS) cells. By differentiating these disease-specific hiPSCs into dopaminergic neurons, we will identify specific gene expression and electrophysiological changes associated with methamphetamine treatment and withdrawal in vitro. Ultimately, we hope that one day it will be possible to treat the disrupted molecular pathways in neurons that lead to addiction.

The overarching goal of Project II is to identify the defining characteristics of disease neurons in Williams syndrome (WS), and to address the interplay between gene expression and the WS social phenotype. Genetic reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells or iPSCs) by over-expression of specific genes has been accomplished using mouse and human cells. The resulting iPSCs are isogenic to the donor individual, i.e., they carry a similar genetic background, and thus are attractive not only for future therapeutic purposes, with lower risk of immune rejection, but also for the understanding of complex diseases with heritable and sporadic conditions. Thus, the use of human iPSCs as a biological tool to understand complex disorders, such as WS, may provide additional insights into the disease pathology, and new compounds that ameliorate disease progression. Wth an unparalleled history of research on WS as a compelling model for understanding the linkages from molecular genetics to neurobiology to higher cognition and social functions, WS provides a privileged setting for the proposed studies. The remarkable potential of iPSCs has sparked profuse interest and excitement in researchers studying individuals with a variety of neurodevelopment disorders, because of their potential to reveal avenues for intervention. WS offers an excellent model due to a well-defined genetic basis and a robust social phenotype. To this end, the Specific Aims are: (1) to derive iPSCs from individuals with WS as well as typical controls;(2) to analyze the gene expression profile of neural cells derived from controls and WS iPSCs;and (3) to test cross-level hypotheses generated during the course of the Program Project, e.g., the role of specific genes in the atypical WS social behavior, neuronal phenotype, and brain anatomy. The pluripotent stem cells will be driven to differentiate in neurons for a comprehensive transcriptional analysis and future mechanistic explorations based on our hypotheses. The ultimate aim of this research is to discover potential biomarkers and specific therapeutic targets. Elucidating the links between genes and social-affective behavior in WS may provide fundamental insight into the genetic mechanisms and neural circuits responsible for human social behavior

The Neuroscience Center Behavior Core will establish a centralized resource for the analysis of animal behavior. The objectives of this core resource will be to provide uniform behavioral analysis of rodents employing a variety of behavioral platforms appropriate for the research needs of Salk Institute Neuroscience Center faculty and to provide expertise for the interpretation of results. Behavioral studies are an important component of modern neuroscience research. For example, careful analysis of changes in rat behavior in the radial water maze showed that the ability to learn new tasks decreases with age. These changes were eventually found to be reflected at the cellular level, in particular, in the dentate gyrus of the hippocampus where aging is associated with a reduction in the number of axons in the medial perforant path, as well as the density of synaptic contacts impinging on granule cells. Subsequently this reduction in the input to the hippocampus was found accompanied by a decrease in synaptic plasticity, longterm potentiation has a higher threshold and lasts for a shorter time in aged animals, and there is an agedependent decrease in NMDA receptor-mediated responses - all discoveries stemming from the initial analysis of animal behavior. Several ofthe NINDS-funded investigators at the Salk Institute rely on behavioral testing of rodents as a critical component of their projects and most ofthe others would like to incorporate behavioral testing into their NINDS-funded projects. Although we have considerable amount of appropriate equipment for a broad range of behavioral testing of rodents at The Salk Institute, it is presently distributed at multiple sites across the Institute and is maintained, operated and controlled by investigators in individual labs. This situation either precludes the use of behavioral testing or significantly limits it, as investigators that wish to use it in their projects either have to have funding to purchase the equipment and appropriate space to set it up, and further, become trained with its use, and / or collaborate with investigators that have the equipment and experience for the appropriate tests. Many negative factors though are associated with each of these alternatives and are often sufficient to discourage investigators from integrating behavioral testing into their project. The proposed Behavior Core will consolidate at a single convenient location within the Salk Institute the equipment and expertise necessary to perform a wide range of behavioral testing of rodents (primarily mice, but rats can also be accommodated) relevant to the NINDS-funded research projects. The goal ofthe Behavior Core is to provide uniform and comprehensive service for all NINDS-funded investigators at the Salk Institute. This core will eliminate duplication of resources and provide critical expertise across a broad range of analytical tests needed for the proper performance of behavioral studies. The faculty management and technical staffing ofthe Behavior Core will make behavioral testing accessible to all NINDS funded investigators at the Institute and allow them to properiy integrate it into their projects as warranted, regardless of their background training and expertise. This arrangement will also conserve resources, and in several ways serve as a resource multiplier in addressing the issues under study with NINDS funding. This conservation, as well as the communal influence ofthe properly sized user group of NINDS funded investigators, will make potential resources more readily available to invest in the coming years in additional equipment for behavioral testing to keep abreast of technical and issue-related advances that will benefit the NINDS-funded projects.

DESCRIPTION (provided by applicant) Alzheimer's disease (AD) is a neurodegenerative disease characterized by the deposition of amyloid plaque in the extracellular space, formation of neurofibrillary tangles in neurons, and extensive neuronal loss. Growing evidence has shown that increased beta amyloid (A¿) peptide accumulation, aggregation, and deposition in the brain are central to the pathogenesis of AD (1). Recently, the advent of methodologies to reprogram fully differentiated tissues, such as skin, into human-induced pluripotent stem cells (hiPSCs) has made it possible to study AD in a human physiological context. Cell-based human models for AD will be created by directly reprogramming skin fibroblasts from AD patients and controls into hiPSCs. This proposal will focus on reprogrammming skin fibroblasts from sporadic AD patients containing two copies of the ¿4 allele of the apolipoprotein-E (APOE) gene and control subjects containing two copies of the APOE ¿3 allele. Previously established hiPSC lines derived from familial AD patients with mutations in the amyloid precusor proteins (APP) and preselinin (PS) genes will be obtained and differentiated into neuronal cells, including cholinergic neurons. There are three primary aims for this R21 proposal. In Aim 1, fibroblasts from AD patients carrying the APOE ¿4 allele and controls carrying the APOE ¿3allele will be reprogrammed into hiPSCs and then differentiated into neurons. Several parameters associated with AD development such as A¿ expression level, A¿ fibrilgenesis, neuroinflammation, synapse formation and function will be analyzed at different timepoints after neuronal differentiation. Thi will illustrate whether hiPSC-derived neurons can recapitulate the disease progression of AD in vitro. In Aim 2, A¿ 1-42 peptide will be used to treat the hiPSC-derived neurons at low concentrations resembling in vivo A¿ concentration and at high concentrations that induce neuronal toxicity. Various indicators for neuroinflammation, neuronal degeneration and cell death will be measured to examine whether hiPSCs-derived neurons carrying the APOE ¿4 allele react differently to A¿ treatments compared to those carrying the APOE ¿3 allele. In Aim 3, gene expression profiles of hiPSC-derived neurons will be generated using gene expression microarrays. Genes with altered expression in familial AD patients or patients with the APOE ¿4 allele will be cross-referenced with a list of genes modulating A¿ neurotoxicty that we have generated through a genome-wide shRNA library screen. Overlapping genes of interest will be further studied using lentiviral overexpression or shRNA-mediated repression in hiPSC-derived neurons. Studies of hiPSC-derived neurons from AD patients carrying the APOE ¿4 allele have the potential to significantly advance our understanding of the complex mechanisms underlying APOE ¿4-dependent increase of the risk for AD. Results from the proposed studies will likely lead to development of hiPSC-derived neurons-based assays for screening genes or compounds that lead to potential new drug therapies for the treatment of AD. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to create human cell-based models for Alzheimer's disease (AD) by reprogramming skin samples from sporadic AD patients with two copies of the APOE ¿4 allele into human induced pluripotent stem cells (hiPSCs). By differentiating these disease-specific hiPSCs into neurons, we will investigate the underlying mechanisms by which the apoE4 isoform mediates increased risk for AD. Ultimately, we aim to develop genetic or chemical treatments that disrupt molecular pathways that lead to AD.

? DESCRIPTION (provided by applicant): Schizophrenia (SCZD) is a debilitating and typically incurable neuropsychiatric disease that affects 1% of the human population. Disease symptoms, which include hallucinations, paranoia, and impaired cognition, are thought to arise from impairments in neuronal connectivity and plasticity, but etiology of these defects remains unclear. Multiple lines of evidence suggest a strong genetic component to SCZD. Thus, identifying genetic variants associated with SCZD may provide critical tools for understanding and treating the disease. Indeed, recent genome wide association studies have identified >100 loci that are associated with SCZD, but these genetic variants account for only a small percentage of disease incidence. One potential explanation for this unsatisfying result is that SCZD risk alleles are not inherited through the germline, but instead arise through somatic mutations within neurons of affected individuals. Perhaps it is the propensity for somatic mosaicism that is inherited in patients with SCZD. It is now clear that somatic mosaicism of DNA sequence is much more common than previously thought (i.e., all cells within an individual do not contain the same genome), and that this phenomenon is particularly prevalent in the brain. These genomic differences may contribute to the diversity of neuronal function. However, dysregulation of processes that generate or control somatic mosaicism may lead to disease-related genomic instability. Our hypothesis, therefore, is that somatic mosaicism in neurons or their progenitors are a major contributor to SCZD pathogenesis. Aim 1 will use single-cell genomic sequencing techniques to identify somatic copy number variants (CNVs) in neuronal and non-neuronal cell types from patients with SCZD or neurotypic controls. These analyses will focus on the frontal cortex and hippocampus, two brain regions associated with SCZD pathogenesis. Results will determine whether somatic CNVs are overrepresented in SCZD brains, and whether SCZD risk alleles are disproportionately affected by these CNVs. Aim 2 will characterize somatic retrotransposon insertions within these same cell types, asking whether the frequency or location of retrotransposition events is altered in neurons from patients with SCZD compared with controls. A total of 8000 neurons will be analyzed in Aims 1 and 2, making this the most comprehensive analysis of neuronal somatic mosaicism to date. In Aim 3, genomic variants most overrepresented in patients with SCZD (identified in Aims 1 and 2) will be engineered into hESCs for functional validation tests. It has been shown that cultured neurons derived from patients with SCZD exhibit reduced levels of connectivity and have underdeveloped neurites compared with controls. Similar analyses will be performed using isogenic and mosaic cultures of neurons derived from engineered hESCs. Results from these studies will determine whether the level, pattern, or type of somatic mosaicism is altered in SCZD neurons, and potentially identify genes and gene networks most affected by these changes. Identifying causal disease factors will provide new therapeutic targets and move us closer to finding a cure for this devastating disease.

Project Summary For neural networks to be refined and shaped by experience, the expression of new genes in response to activity is critical. Neural activity triggers the expression of immediate early genes (IEGs) such as arc, fos, and egr1 within minutes, but many IEGs are transcription factors that in turn trigger subsequent waves of transcription. These later waves of transcription are necessary for the consolidation of stable, persistent memories, yet identity and function of the genes in these later waves remains unknown. Further, it is unknown if all activated neurons in a circuit are identically modulated, or if cell-specific transcriptional changes can drive emerging functional differences. The extent of this heterogeneity at the level of the individual neuron has been completely unexplored. Only now, with recent advances in single-cell sequencing technologies, is it possible to track gene expression changes in individual activated neurons. This project will track activity-related gene changes in single neurons of the hippocampal dentate gyrus, a region critical for learning and memory, providing for the first time a transcriptional `signature' of the activity in individual neurons. This will be achieved by examining dentate granule cells in three aims; First, the long-term waves of transcription will be characterized during the full time span during which transcription and translation inhibitors have been demonstrated to impair memory. Second, the impact of each transcriptional wave will be explored in terms of changes in cell excitability and the probability of activation to a subsequent event either in vivo or in vitro. Finally, the dentate gyrus is one of the few brain regions that incorporates new neurons during adulthood in mammals. Immature dentate granule cells are highly similar to their mature counterparts, except that they possess distinct electrophysiological properties which help to control function across the entire dentate gyrus. Therefore activity-related transcription of this unique population of immature cells will be characterized, as well as their functional and transcriptional impact on mature cells. Findings from this project will reveal novel links between gene expression and function in individual neurons, identifying novel targets for the maintenance of memory in healthy humans and in restoring memory function in aging or disease states.

PROJECT ABSTRACT Alzheimer?s disease (AD) is conventionally characterized by specific neuropathological features, including the appearance of extracellular amyloid deposits and the accumulation of intracellular neurofibrillary tangles. While several gene mutations are clearly associated with early onset Alzheimer?s disease, the large number of individuals exhibiting delayed onset, aging-associated AD, are likely to harbor many alterations in linked modifier genes that predispose to AD susceptibility. Genetic and genome wide association studies (GWAS) have identified numerous genes and risk alleles that indicate both cell autonomous and non-cell autonomous mechanisms contributing to loss of neurons and cognitive decline. In this regard, the majority of risk variants identified by GWAS reside in non-coding regions of the genome, implying that they act in part to alter gene expression. This proposal responds to the RFA indicating a particular need for approaches designed to delineate the transcriptional and cellular consequences of combinations of SNPs in the risk alleles by generating new cell line reagents to help unravel the question of the causative SNPs and their target genes in specific neurons derived from iPS cells of AD individuals. There are two features of sporadic AD that require molecular explanation- the potential role of aging in AD susceptibility, and the striking gender disparity, with the incidence of AD being exaggerated in females. These issues can only now be addressed based on new technologies and the availability of patient-derived samples. Our proposed research plan takes advantage of the invaluable samples stored at the brain bank of the Shiley-Marcos Alzheimer's Disease Research Center (ADRC) at UCSD, and the iPSC-derived neurons (Salk). This approach will interrogate the effects of different genetic variants with other risk factors (e.g. age, sex), and assess their effects on cell type-specific enhancer landscapes. By merging these data, we can begin to identify the potential causative SNPs that result in altered function of cell-type specific enhancers. We propose using a high throughput 4C screening approach (UMI-4C), and Hi-ChIP, to identify the most likely causative, enhancer-associated SNPs for functionally-implicated coding target genes. Exploiting the power of contemporary gene editing approaches in control or patient-derived iPS cells to specific neuronal cell types, and to astroglia, we can assess the transcriptional phenotypes and functional behaviors of neurons harboring different combinations of risk alleles, both in the isolated cell lines alone and in combination with coculture experiments with astroglia and microglia, as effects of these SNPs may be manifest only with astroglial:neuronal interactions. Together these studies will use powerful contemporary global genomic approaches to determine the coding transcriptional targets of several of the most significant SNPs in enhancers, and the link to roles of estrogen receptor in the gender disparity for AD.

SUMMARY Pathogenesis of A l z h e i m e r ? s d i s e a s e ( AD) is highly age-related and prevalence increases exponentially after the age of 60. While at the age of 70 around 5% of all people are affected, more than one third over the age of 85 are afflicted with AD. In addition to the dramatic increase seen in patients suffering from age-related neurodegenerative disorders in industrialized countries, a substantial increase in AD patients in the developing world is also observed in tandem with their aging populations. Currently, there are no treatments available for AD that could slow, halt or reverse the progression of the disease. This growing problem can only be mitigated if we can gain a more complete understanding of AD pathogenesis, which obviously demands a solid understanding of human biological aging. This project proposed by Dr. Gage and colleagues will, for the first time, challenge the importance of cellular aging in a human model for the disease. The Gage lab has recently shown that direct conversion of human fibroblasts into induced neurons (iNs) preserves signatures of cell aging, allowing the detection of cellular pathologies relevant to human aging. By contrast, induced pluripotent stem cell (iPSC) reprogramming erases age-dependent differences; iPSC-derived neurons thus resemble rejuvenated cells. To better understand the impact of neuronal cell aging on the pathology of sporadic AD, the first aim of this group is to generate both phenotypically old and rejuvenated neurons from a large set of clinically well-characterized AD patients and matched controls. Following an unbiased transcriptome approach, they will analyze for AD-specific gene expression profiles and work to understand which of the AD-specific gene expression signatures and related mechanisms are age-dependent and which are age-independent. Dysregulation of nucleo-cytoplasmic transport and the import receptor RanBP17 are currently emerging as major topics in aging and neurodegenerative disease research. In their second aim, Dr. Gage's team will work to identify the binding partners and exact functions of the yet understudied protein RanBP17. In a third aim, they will harness their recently established reporter system to measure nucleo- cytoplasmic compartmentalization in young and old AD neurons and probe for nuclear transport-based mediators of age-dependent AD pathology using live cell imaging approaches. Age-dependent accumulation of DNA damage contributes to genetic diversity among our cells, a process known as somatic mosaicism. As recent evidence suggests that AD only needs a small seed from which the pathology can spread throughout the brain, somatic mosaicism might play an important role in the development of sporadic AD. In a fourth aim, the Gage team will use simultaneous DNA and RNA sequencing of single post-mortem neurons and iNs from the same patients and ask to what extent DNA copy number variations can turn a neuron into a potential `AD seed' cell. 

Contact PD/PI: ROSENFELD, MICHAEL G PROJECT ABSTRACT Alzheimer?s disease (AD) is conventionally characterized by specific neuropathological features, including the appearance of extracellular amyloid deposits and the accumulation of intracellular neurofibrillary tangles. While several gene mutations are clearly associated with early onset Alzheimer?s disease, the large number of individuals exhibiting delayed onset, aging-associated AD, are likely to harbor many alterations in linked modifier genes that predispose to AD susceptibility. Genetic and genome wide association studies (GWAS) have identified numerous genes and risk alleles that indicate both cell autonomous and non-cell autonomous mechanisms contributing to loss of neurons and cognitive decline. In this regard, the majority of risk variants identified by GWAS reside in non-coding regions of the genome, implying that they act in part to alter gene expression. This proposal responds to the RFA indicating a particular need for approaches designed to delineate the transcriptional and cellular consequences of combinations of SNPs in the risk alleles by generating new cell line reagents to help unravel the question of the causative SNPs and their target genes in specific neurons derived from iPS cells of AD individuals. There are two features of sporadic AD that require molecular explanation- the potential role of aging in AD susceptibility, and the striking gender disparity, with the incidence of AD being exaggerated in females. These issues can only now be addressed based on new technologies and the availability of patient-derived samples. Our proposed research plan takes advantage of the invaluable samples stored at the brain bank of the Shiley-Marcos Alzheimer's Disease Research Center (ADRC) at UCSD, and the iPSC-derived neurons (Salk). This approach will interrogate the effects of different genetic variants with other risk factors (e.g. age, sex), and assess their effects on cell type-specific enhancer landscapes. By merging these data, we can begin to identify the potential causative SNPs that result in altered function of cell-type specific enhancers. We propose using a high throughput 4C screening approach (UMI-4C), and Hi-ChIP, to identify the most likely causative, enhancer-associated SNPs for functionally-implicated coding target genes. Exploiting the power of contemporary gene editing approaches in control or patient-derived iPS cells to specific neuronal cell types, and to astroglia, we can assess the transcriptional phenotypes and functional behaviors of neurons harboring different combinations of risk alleles, both in the isolated cell lines alone and in combination with coculture experiments with astroglia and microglia, as effects of these SNPs may be manifest only with astroglial:neuronal interactions. Together these studies will use powerful contemporary global genomic approaches to determine the coding transcriptional targets of several of the most significant SNPs in enhancers, and the link to roles of estrogen receptor in the gender disparity for AD. Project Summary/Abstract Page 7

PROJECT SUMMARY ? Human Cell Models of Aging Core The SD-NSC Human Cell Models of Aging Core (Human Cell Core) is focused on creating new cellular models of aging from human fibroblasts, including fibroblasts from a unique cohort representing the full breadth of adult chronological age and annotated for physical and functional measures of biological age. Genetic model organisms have long been important for aging research, but there are limitations to the extent to which animal models faithfully represent the molecular and cellular process of human aging. The overarching goal of this Core is to create powerful new human cell-based models to enable a wide range of studies into the molecular and cellular heterogeneities of human aging. Approaches for generating these new human models of aging draw largely from recent advances in creating induced cell types via the direct conversion of skin cell samples. Pioneering work from the Human Cell Core co-Lead, Dr. Gage, has shown that aging characteristics are maintained in these induced cell types. Pairing this approach with equally new approaches for creating 3D organoid cell cultures representative of different organ and tissue microenvironments, enables the creation of unprecedented human cell-based models of tissue aging. These models will enable robust analyses of cellular heterogeneities in human aging processes. As part of this effort, the Core will recruit human subjects ranging in age from 20?70+ years, thereby representing the full breadth of the healthy adult human age span. These participants will be extensively phenotyped for key clinical and physiological features of biological aging, and blood and skin samples will be collected. Skin samples will be used to derive primary dermal fibroblasts, from which both reprogrammed stem cells and directly induced cell types will be created and banked for sharing. The Human Cell Core will develop protocols for the direct inducement of different cell types and for the creation of organoids, including pancreas, kidney, liver, heart, smooth muscle, and vasculature. To promote the uptake of these new human cell models throughout the basic biology of aging research community, the Core will offer workshops and training sessions in conjunction with the Research Development Core. The protocols, specific reagents, and cells will be distributed to NSC affiliates directly from the Human Cell Core, and more broadly through open resources and cell repositories like the NIA Aging Cell Repository at the Coriell Institute and Addgene. Subject phenotype data will be shared along with the cell lines. Importantly, the Human Cell Core is designed to be integrated with the other Research Resource Cores of the SD-NSC, as human cell models (with subject-specific data) can be fed directly into the Heterogeneity of Aging Core for systems-level analysis of aging processes (multi-omic and advanced imaging). These complex datasets can then be fed into the Integrative Models of Aging Core for the application of new and existing integrative analyses. The Human Cell Core, as a stand-alone resource, and as an integrated component of the SD-NSC Core pipeline, will create substantial new opportunities to investigate the influences of cellular heterogeneity in human aging processes.