William C. Mobley - US grants

Neuronal survival, differentiation, and maturation are influenced by soluble trophic agents. The importance of trophic factors has been best demonstrated in the case of nerve growth factor (NGF) and its responsive peripheral neurons. Sequestration of endogenous NGF by appropriately timed injection of NGF antibodies destroys sympathetic and sensory neurons. Identification and characterization of central nervous system (CNS) trophic agents has been hampered by the tiny amounts present in nervous and other tissues. It appears that this problem may be circumvented in the case of basal forebrain cholinergic neurons. NGF injected into the cerebral ventricle of neonatal rats produces dramatic and selective increases in the activity of choline acetyltransferase (ChAT), the neurotransmitter synthetic enzyme for cholinergic neurons. It has been postulated that NGF is the endogenous trophic factor for these cells. In this proposal, experiments will be conducted to directly test this hypothesis. First, it will be proven that NGF (and not an impurity) is responsible for the effect on ChAT activity. NGF will be purified by high-performance, reverse-phase liquid chromatography. To determine the nature and pattern of its activities, the effect of NGF administration on several neurochemical markers for cholinergic and other neurons will be examined. These studies will record the response of septohippocampal cholinergic neurons before and during the period of synapse formation. The physiological role of endogenous NGF will be examined during the same developmental stages by intracerebroventricular injections of affinity-purified NGF antibodies. These studies will indicate whether cholinergic neurochemical markers are selectively depressed by the presence of these antibodies. If so, this will indicate that NGF does function as an endogenous trophic factor for basal forebrain cholinergic neurons. It is expected that the proposed studies will lead to an improved understanding of the growth and development of these neurons. It is hoped that they will provide insights regarding the role that trophic factors generally play in the developing CNS. Furthermore, they may indicate the potential consequences of altered synthesis or release of trophic factors and suggest means for evaluating the contribution of such disturbances to neurological disease.

Neurotrophic factors have an important role in supporting the survival, differentiation and function of neurons. By examining the actions of a specific neurotrophic factor, it should be possible to define essential features of the trophic relationship. This perspective may allow one to modulate neuronal survival and differentiation and, perhaps, to intervene in neurological disorders which threaten neuronal viability and function. Nerve growth factor (NGF) may be a trophic factor for cholinergic neurons of the basal forebrain. Death of these cells in Alzheimer's disease is thought to have an important role in memory loss. In this proposal we will further define the site and mechanism of NGF actions in the rat central nervous system (CNS) and will determine whether NGF supports the viability and differentiation of basal forebrain cholinergic neurons. NGF mediated induction of gene expression for the beta-amyloid precursor protein (APP) and the prion protein (PrP) will allow us to localize NGF-responsive neurons via in situ hybridization histochemistry. Radiolabeled probes will be applied to tissue sections to localize APP and PrP mRNA in individual neurons. We expect to see a response in forebrain cholinergic neurons; other populations may also respond. The mechanism of NGF actions on APP and PrP will be examined at the level of gene transcription and mRNA stability. These studies will examine NGF responses in basal forebrain in vivo and in pheochromocytoma (PC 1 2) cells in vitro. Agonists and antagonist NGF peptides will be prepared. In vitro assays will be used to characterize the potency and efficacy of peptides on neurite outgrowth and survival; their site of action will be examined in ligand-binding and affinity crosslinking studies. Potent, stable antagonists will be administered in vivo to attempt to block the actions of endogenous NGF. We expect to see inhibition of cholinergic neurochemical differentiation and decreased viability. NGF agonist peptides may stimulate cholinergic function. The proposed studies are expected to define key features of NGF actions in the CNS. They may suggest approaches for elucidating the actions of other CNS neurotrophic factors. Such studies may lead to new insights regarding the role of NGF and other trophic factors in neurodevelopmental and neurodegenerative diseases. The ability to deliver biostable neurotrophic agonists or antagonists may provide a means for treating such disorders.

Neuron cell death features prominently in the development of the nervous system and is a key pathological component in neurodegenerative disorders such as Alzheimer's disease (AD). Currently, there is no effective treatment for AD. One view of pathogenesis suggests that neuronal death in development and disease are due to activation of cell death genes and that controlling expression of these genes will make it possible to prevent degeneration. Target-derived neurotrophic factors prevent neuronal death in development; they also act to prevent degeneration induced by lesions of mature neurons. They may prevent cell death in AD. It is our hypothesis that nerve growth factor (NGF) will prevent selective, spontaneous degeneration of basal forebrain cholinergic neurons (BFCN) in a recently developed animal model of AD. The model is based on the finding that all Down syndrome (DS) patients develop AD pathology, including degeneration of BFCN. Mouse trisomy (TS) 16 is an animal model of DS. We have found that TS16 basal forebrain transplants demonstrate atrophy of BFCN after several months in vivo. NGF, or a vehicle control, will be infused into the cerebral ventricle of adult female mice carrying transplanted basal forebrain neurons from TS 16 and control (diploid) fetuses. The number and size of TS16 cholinergic neurons will be compared to controls, and the incidence of extracellular amyloid and neurofibrillary tangles will be assessed. These studies will determine whether or not NGF treatment prevents atrophy and death of BFCN and will give evidence for or against NGF mediated toxicity. If NGF is shown to be both effective and safe, these studies would give strong support to NGF trials in AD patients. The NGF gene family members brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) may also act on BFCN; they will be tested for activity on lesioned BFCN and, when appropriate, in the transplant model. To pursue the goal of neurotrophic drug design, we will attempt to define NGF domains important for receptor binding and activation. The strategy to be used is based on the fact that NGF, BDNF and N.T-3 activate distinct populations of neurons. Structural differences between them must lead to activation of distinct receptors. Chimeras in which variant domains from BDNF or NT-3 replace those of NGF will be tested for activity and binding. When activity is changed to that for the factor whose domains were introduced, we will have identified structural regions important for NGF receptor activation. The proposed studies will markedly advance studies of neurotrophic therapy by extending observations to an animal model of the BFCN degeneration. NGF and neurotrophic factors related to NGF may be shown effective and safe in preventing cholinergic cell death. Finally, our studies will pave the way for future neurotrophic design.

Neurobiology is in a period of explosive growth and change. The development of new techniques, the application of advances in genetics and immunology, the influx of talented scientists from other areas of biomedical science, and the increasing conviction that the time is ripe for major advances in neurobiology, have led to a period of unprecedented growth. Rapid progress will undoubtedly continue through the next decade and will bring major payoffs. Progress in neurobiological research will lead not only to greater understanding of the brain, but will lay the foundation for new modes of diagnosis, treatment and management of neurological and psychiatric disease. Child Neurology deals with illnesses that are among the most debilitating and expensive, and yet this discipline is among the least touched by the modern revolution in biology. Individuals trained in both clinical and basic neuroscience would provide the most effective bridge between disciplines, and could translate basic neuroscience advances into new approaches for understanding and treating neurological diseases in children. This application proposes to develop such individuals through the creation of a research training program for Child Neurology residents at UCSF. The program would integrate clinical and research activities of selected residents in a coherent plan for career development. An effective training program must provide broad and rigorous exposure to ideas and techniques that transcend the bounds of traditional disciplines. We propose to achieve this goal by developing for each trainee a program that incorporates rich experiences in both basic and clinical neuroscience. Clinical training activities will be carried out in the Division of Child Neurology where trainees will become experienced, thoughtful clinicians able to bring critical thinking to bear on clinical problems. Basic science training will consist of both didactic and research activities to be carried out over a minimum of two years. Research training will be carried out in the laboratory of mentors who are established neuroscientists, representing a variety of disciplines, who are committed to trainee development. Formal and informal didactic activities will be carried out under the umbrella of the UCSF Neuroscience Program. The PI and his Advisory Committee will be responsible for candidate recruitment and trainee selection, monitoring of trainee progress, and support of the trainee's transition to an independent research career. A successful training program will result in the production of a number of highly talented Child Neurologist- neuroscientists who pursue productive investigative careers that impact significantly on neurological disease in children.

The broad long-term goal is to elucidate the mechanisms responsible for the dysfunction and loss of neurons in degenerative neurological disorders. In Alzheimer's disease (AD) and Down syndrome (DS), age- related degeneration of basal forebrain cholinergic neurons (BFCNs) contributes significantly to dementia. Defining the molecular events leading to BFCN degeneration would significantly advance our understanding of pathogenesis. In studies on the Ts65Dn mouse, a genetic model for DS, we documented age-related degeneration of BFCNs. Nerve growth factor (NGF) is essential to the function of normal BFCNs and recent findings suggest that failed NGF signaling contributes to their degeneration of Ts65Dn. We will test the hypothesis: that a failure in NGF signaling is responsible for the degeneration (i.e. progressive dysfunction) of BFCNs in the Ts65Dn mouse. The proposed Specific Aims are: 1) To characterize the functional status of BFCNs and document age-related dysfunction of these neurons in Ts65Dn mice. Using unbiased stereology, biochemical and behavioral studies we will document the time of onset of BFCN dysfunction in Ts65Dn mice. 2) To determine if NGF signaling is defective in BFCNs in Ts65Dn and, if so, to determine whether the abnormality predate degeneration of BFCNs. Abnormal NGF retrograde transport will be used to document the existence of a signaling defect and biochemical studies will define its nature and time of onset. 3) To determine whether disrupting NGF signaling reproduces the cholinergic abnormalities seen in Ts65Dn. If the NGF signaling defect demonstrated in Aim 2 is sufficient to produce the degeneration of BFCNs in Ts65Dn, animals defective for NGF signaling should show the same degenerative events. We will examine mice treated with NGF antibodies to sequester endogenous NGF, mice in which one copy of the NGF gene has been disrupted, and mice in which both copies of the gene for the NGF TrkA receptor has been knocked-out. 4) To define NGF actions on degenerating BFCNs in Ts65Dn. Current data suggest that NGF may be capable of reversing other aspects of BFCN degeneration. In this Aim, we will determine whether NGF reverses the degenerative phenotypes defined in Aim 1. 5) To determine whether BFCN degeneration is found in other genetic models of Ds and AD and, if so, whether failed NGF signaling is present. We will examine BFCNs in several models of AD. If BFCN atrophy is detected, we will determine whether there is an abnormality of NGF signaling. We will also examine Ts65Dn animals expressing either human Apo E3 or E4 to determine whether, as expected, BFCN degeneration is worse in ApoE4 expressing mice. Evidence that abnormal NGF signaling is responsible for BFCN degeneration in models of AD and DS will give important new insights into pathogenesis and may suggest novel approaches to prevent or reverse dementia in these patients.

Nerve growth factor (NGF) is a target-derived factor and interacts with its receptor, TrkA, on the axon tip, to exert its effects on neuronal differentiation and survival. To date, little is known on how NGF signalling in axons is communicated to the cell body. Recent evidence suggests that neurons exploit small vesicles that contain activated NGF/TrkA complexes, to convey the NGF signal from the distant axon to the nucleus, where changes in geneexpression are induced. Aim of the project is to examine occurrence and identity of proteins thatare associated with activated NGF/TrkA in these endosomes. To this end, protein maps will be generated of vesicles from NGF-treated versus control cells, using 2-D gel electrophoresis, and mass spectrometry will be used to identify differential gel spots.

Our studies aim to define the genetic and molecular bases for the degeneration of specific neuronal populations in the brains of elderly Down syndrome (DS) patients. The principal focus of this application is the degeneration of basal forebrain cholinergic neurons (BFCNs), whose age-related loss contributes significantly to dementia in both Alzheimer's disease (AD) and DS patients. Defining the pathogenesis of BFCN degeneration may lead to treatments that reverse or prevent dementia. In studies leading to this application, we examined the Ts65Dn mouse, a genetic model for DS in which the mouse chromosome 16 (MMU16) homolog to the DS critical region is found in three copies. We documented behavioral, morphological, and biochemical phenotypes shared by Ts65Dn mice and S patients. Importantly, these mice showed age- related degeneration of BFCNs. Our recent findings suggest that a failure in NGF signaling contributes to BFCN degeneration. Moreover, we showed that infusion of exogenous NGF reversed the degenerative changes. We propose to test the hypothesis: that a failure in NGF signaling is responsible for the degeneration (i.e. progressive dysfunction) of BFCNs in the Ts65Dn mouse. We have powerful new tools to aid us in testing the hypothesis, two new partial trisomic mice, Ms1Ts65 and Ts1Cje. We will document abnormalities in NGF signaling, BFCN function and cholinergically-influenced cognitive behaviors in Ts65Dn and ask if they co-segregate in the new trisomies. Co-segregation will suggest that increased dosage of the same gene(s) responsible for all three phenotypes. This will support the hypothesis, as will evidence that NGF treatment reverse BFCN degeneration and improves cholinergically- influenced behaviors. We propose the following Specific Aims: 1) To characterize NGF signaling in Ts65Dn, and to map abnormalities to either Ts1Cje or Ms1Ts65. We will define the NGF signaling defect, determine the molecular basis for the abnormality, and map it genetically. 2) To characterize the functional status of BFCNs in Ts65Dn in Ts65Dn, and to map age-related cholinergic degeneration to either Ts1Cje or Ms1Ts65. We will further characterize BFCN dysfunction in Ts65Dn mice and define which aspects of BFCN synaptic and perikaryal function map to Ts1Cje and Ms1Ts65. 3) To characterize cholinergically-influenced cognitive behaviors in Ts65Dn, and to map age-related abnormalities to either Ts1Cje or Ms1Ts65. We will characterize cognitive changes in aging Ts65Dn mice, focusing on those influenced by BFCNs. Abnormalities will be mapped in studies on Ts1Cje and Ms1Ts65. 4) To attempt to reverse with NGF treatment the dysfunction of BFCNs and abnormal cholinergically-influenced behaviors in Ts65Dn, Ts1Cje and Ms1Ts65. If the hypothesis is correct, we will see that NGF treatment reverse many, and perhaps all, the BFCN and cholinergically-influenced behavioral abnormalities that map with NGF signaling abnormalities. We believe that the proposed studies will provide important new perspectives on the neurobiology of DS. Moreover, the long-lived and productive collaboration of the Mobley and Epstein laboratories, and the availability of uniquely important animal resources will allow considerable progress in defining important DS phenotype-genotype relationships.

Nerve growth factor (NGF) is a target-derived factor and interacts with its receptor, TrkA, on the axon tip, to exert its effects on neuronal differentiation and survival. To date, little is known on how NGF signalling in axons is communicated to the cell body. Recent evidence suggests that neurons exploit small vesicles that contain activated NGF/TrkA complexes, to convey the NGF signal from the distant axon to the nucleus, where changes in gene expression are induced. Aim of the project is to examine occurrence and identity of proteins that are associated with activated NGF/TrkA in these endosomes. To this end, protein maps will be generated of vesicles from NGF-treated versus control cells, using 2-D gel electrophoresis, and mass spectrometry will be used to identify differential gel spots.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Nerve growth factor (NGF) and other neurotrophic factors (NTFs) are produced and released in target tissues to activate specific receptors on the distal axons of innervating neurons. The signals thus produced are retrogradely transported to the cell bodies to regulate cytosolic and nuclear events important for survival and differentiation. An important unresolved issue is the mechanism(s) by which signals generated by NGF in axon terminals are transmitted. In the last funding period, we tested the signaling endosome hypothesis which states that the NGF signal is retrogradely transported through endosomes that contain NGF bound to its activated TrkA receptor in complex with associated signaling proteins. We gathered support for the "signaling endosome" hypothesis, and isolated such organelles, but many questions remain as to the structure and function of signaling endosomes. The hypothesis tested in this application is that signaling endosomes are an important source of retrogradely transmitted NGF signals. In the proposed Aims, we will examine the signaling pathways that use endosomes to signal, characterize the proteins that participate, examine signaling from endosomes in vitro and in vivo, and test the role that endosome-derived signals play in maintaining mature DRG neurons, cells that respond robustly to NGF but that are independent of NGF for survival. In Aim 1, examining intact cells and endosome-containing fractions, we will define signaling pathways that employ endosomes. Immunostaining and confocal microscopy will be used as will biochemical studies of signaling proteins. In Aim 2, using compartmented cultures, we will test what signaling pathways use endosomes to retrogradely transport NGF signals from axon terminals to cell bodies. We will define the cell biological steps required to make and traffic signaling endosomes. Endocytosis of NGF will be inhibited using bead-bound NGF. Other steps critical to endocytosis and trafficking events will be inhibited using Lentivirus to express dominant negative isoforms of proteins or to induce RNAi to suppress synthesis of endogenous proteins. In Aim 3, we will explore the cellular events induced by retrogradely transported signaling endosomes at the level of downstream signaling events, gene expression and cellular morphology. Finally, in Aim 4, we will examine the biochemical properties of signaling endosomes isolated by FACS sorting or by magnetic immuno-adsorption and will test signaling from these endosomes in vitro and in vivo. The proposed studies are expected to provide new insights into the cell biology of NGF actions and may help to elucidate a role for failed signaling through endosomes in the pathogenesis of Alzheimer's disease and Down syndrome. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We are entering an era of unprecedented opportunities for understanding and treating disorders of the developing nervous system, which exact immense suffering and cost. Recent advances in genetics, molecular and cellular biology, systems and behavioral neuroscience, and bioinformatics have provided us with powerful new tools and concepts that can be used to both discover the cause of childhood neurological disorders and to create effective therapies. There is no paucity of good ideas and tools with which to work tangibly and with success toward alleviating the burden of neurological disorders in children. But there are important roadblocks to success: the low priority given to disorders of the developing nervous system by the neuroscience research community; the low priority given to developmental disorders by industry; and the disconnection between neuroscience discovery and the delivery of new treatments. Moving forward an agenda that successfully addresses developmental disorders requires that we create a talented cadre of investigators whose training prepares them to take on leadership roles in translational developmental neuroscience. We propose a training program that combines excellence in clinical Child Neurology and in basic or clinical science research with the experience needed to understand how new insights are translated into advances in care. Organized by a dedicated faculty, supported by Stanford University School of Medicine, and engaging a talented group of mentors, the program incorporates: 1) excellence in clinical Child Neurology, 2) mentored basic or clinical research in developmental neuroscience, 3) experience in the methods of design and conduct of clinical trials, 4) training in translational research and 5) exposure to the concepts and methods used to define and develop new therapies. The program in "Translational Developmental Neuroscience" will produce child neurologist-neuroscientists that can successfully translate developmental neuroscience insights into new treatments.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) features the dysfunction and loss of basal forebrain cholinergic neurons (BFCNs) whose degeneration contributes to cognitive difficulties. The long term goal of this project is to define the cellular and molecular basis for the degeneration of BFCNs. One clue is that the hallmarks of AD, including BFCN degeneration, are present in elderly people with Down syndrome (DS) (i.e. trisomy 21), many of whom also show progressive cognitive decline. To link increased expression of one or more pf the genes on chromosome 21 to BFCN degeneration examined the Ts65Dn mouse, a genetic model for DS. We showed that degeneration of BFCNs is linked to failed retrograde axonal transport of nerve growth factor (NGF). In recent studies, we showed that failed NGF transport and degeneration of BFCNs are caused by increased expression of the gene for the amyloid precursor protein (APR), present in three copies in these mice. The defect in transport was recapitulated in mice transgenic either for wild type human APR or for a mutant APR that causes AD. Preliminary data suggest that increased APR C-terminal fragments (CTFs) within endosomes disrupts NGF transport. Our hypothesis is that in DS an increase in full length APR, and/or its transmembrane C-terminal fragments (CTFs), within endosomes acts to inhibit retrograde transport of NGF and NGF-TrkA signaling leading to neuronal dysfunction and degeneration. Using Ts65Dn and transgenic APR mice we will: 1) characterize further the defects in axonal structure and function that result from increased expression of APR;2) determine whether or not increased expression of APR decreases NGF- TrkA signaling in the axons and cell bodies of BFCNs and to define the cellular compartment involved;3) show whether or not failed NGF-TrkA signaling is responsible for BFCN degeneration and abnormal hippocampal learning;and 4) define in vitro the mechanism by which APR overexpression acts. Using a culture system that allows for precise tracking of NGF transport, and building upon preliminary studies showing that Ts65Dn DRG neurons also show a marked deficit in NGF transport, we will determine which APR isoforms are responsible for disrupted transport and signaling and discern the mechanism(s) employed. These studies are an important first step in clarifying the pathogenesis of BFCN neurodegeneration in the setting of increased APR expression and may motivate novel treatment strategies.

DESCRIPTION (provided by applicant): We ask for partial support for the 2009 Neurotrophic Factors Gordon Research Conference. The conference will be held in June of 2009, from June 21st through 26th, at Salve Regina University in Newport, Rhode Island. This conference, held every other year, is viewed as the most important meeting for conveying the most significant discoveries in the neurotrophic factor field. It attracts virtually all of the leading scientists studying neuronal growth factors, their mechanisms of action and their roles in development, plasticity and diseases of the nervous system. Moreover, in recent years it has been attended by a significant number of clinicians and scientists in the related fields of developmental neuroscience, and neurological and mental health disorders and by industry representatives whose companies engage in research in neurological and mental health disorders. Over the years, many of the major breakthroughs in the field have been showcased at this meeting. The Conference has played a catalytic role in building the neuroscience community by bringing together both young and established investigators with diverse interests in neurotrophic factors and with expertise in molecular biology, cell biology, signal transduction, synaptic transmission, development, behavior and disease. The goals of the 2009 Conference are: 1. To communicate and disseminate new findings about known and novel functions of neurotrophic factors, including the impact of such factors in the biology of a variety of different types of stem cells and their potential therapeutic uses. 2. To explore recent advances regarding the synthesis, secretion, signaling mechanisms of neurotrophic factors and their actions in development, plasticity, and the maintenance of neuronal circuits in the young, aged, and diseased nervous system. 3. To disseminate knowledge about the emerging methodologies that are for the first time making it possible to examine in "real-time" the trafficking, signaling and actions of neurotrophic factors. 4. To increase knowledge within the community of the new tools now available that allow scientists to more effectively pursue neurotrophic factor actions in mental and neurological disorders. 5. To increase collaboration between research groups using molecular, genetic, and cell biological approaches with neurologists as well as industry scientists interested in the use of neurotrophic factors for therapeutic purposes. 6. To provide a venue for discussion of new ideas and to facilitate the exchange of reagents and unpublished information. 7. To provide an atmosphere for students, postdoctoral fellows and new faculty to meet and interact with senior scientists studying growth factor biology. 8. To highlight the contributions to our field being made by underrepresented individuals, especially women and minorities. The Conference will enhance communication between scientists working in diverse disciplines to increase their ability to understand neurotrophic factors and to deduce how exploiting their actions may allow us to enhance the lives of individuals with disorders of the nervous system. PUBLIC HEALTH RELEVANCE: The public health relevance of the Conference is its ability to accelerate the transmission of knowledge from fundamental neuroscience to the benefit of patients that suffer with a variety of disorders - Alzheimer's disease, Down syndrome, Parkinson's disease, Huntington's disease, depression, obesity, epilepsy, stroke, and peripheral neuropathies, including those that complicate diabetes and other chronic medical disorders. As such, it fills an important gap in creating and sustaining research with enormous potential benefit to the health and well being of America and the rest of the world. Indeed, we expect this conference to catalyze many new collaborations that address questions important to understanding and treating neurological and mental disorders.

DESCRIPTION (provided by applicant): Trisomy 21, Down syndrome (DS), affects approximately 400,000 people in the U.S., causing cognitive disability, which includes the neuropathology of Alzheimer's disease and late-life dementia. Based on the prevailing gene dosage effect hypothesis, a cognitively relevant phenotype in DS is caused by the triplication of one or more human chromosome (HSA) 21 genes. Our preliminary observations from the mouse-based studies suggest that these causative genes are indeed present and the search for them is both possible and productive. The long-term objective of this project is to identify these causative genes by using mouse-based genetic analysis, which is built upon the recent successes of our team: (1) We have developed two optimal reference mouse models for DS using efficient Cre/loxP-mediated chromosome engineering: Dp(16)1Yu/+, which is trisomic for the entire 22.9-Mb HSA21 syntenic region on mouse chromosome (MMU) 16, and Dp(10)1Yu/+;Dp(16)1Yu/+;Dp(17)1Yu/+, which is trisomic for all three HSA21 syntenic regions on MMU10, MMU16 and MMU17. (2) We have narrowed down the genomic region associated with the cognitive disability of DS to the smallest segment in the mouse genome: the Cbr1-Fam3b chromosomal segment containing 30 HSA21 gene orthologs. The triplication of this segment in mice causes abnormalities in cognitive behaviors, synaptic structures and hippocampal long-term potentiation, a major cellular mechanism that underlies learning and memory. To achieve our objective, we propose, in Specific Aim 1 of this application, to characterize the most important cognitively relevant phenotypes of the optimal reference mouse models for DS. To establish the basic phenotypic parameters to facilitate the genetic dissection, we will characterize the synaptic structures and plasticity in the hippocampus as well as cognitive behaviors of Dp(16)1Yu/+ and Dp(10)1Yu/+;Dp(16)1Yu/+;Dp(17)1Yu/+ mice. We will also measure the size and number of neurons in the hippocampal circuits of Dp(16)1/+ mice at the different ages to ascertain the neurodegenerative phenotype. In Specific Aim 2, we will analyze the Cbr1-Famb3b segment to identify a minimal genomic region for the DS- associated synaptic and cognitive phenotypes. We will generate new mouse mutants carrying nested duplications and deletions within the Cbr1-Fam3b segment by chromosome engineering and, by using these mutants, we will employ a subtractive/additive strategy in which synaptic and cognitive phenotypes are linked to progressively smaller genomic segments until a minimal critical region is defined. This effort will lay the groundwork to identify a causative gene(s) located within the minimal critical region(s) for these phenotypes, which will set the stage for the unraveling of the molecular mechanism of DS-associated cognitive disability as well as provide the conclusive support for the aforementioned hypothesis. Therefore, we expect, through these studies, to considerably accelerate progress in understanding and treating cognitive disability in DS. PUBLIC HEALTH RELEVANCE: Cognitive dysfunction affects essentially all children and adults with trisomy 21, Down syndrome (DS);with no effective treatments available, fully 400,000 people in the U.S. experience developmental delays in mental function as children and progressive decline of cognitive skills associated with the neuropathology of Alzheimer's disease during aging. Innovative approaches to unraveling the underlying mechanisms and to developing effective therapies are urgently needed. We propose to use chromosome engineering to create new mouse mutants to define linkages between cognitively relevant phenotypes of DS and minimal critical genomic regions, with the ultimate goal of identifying the causative genes, an accomplishment that would greatly accelerate progress toward understanding and treating cognitive dysfunction in DS.

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. Axons are the "highways" of cargo transport and communication between neurons. It is important to understand the axonal sub-structural changes in neurodegenerating neurons'axons vs. healthy axons. These axonal 3D sub-structural changes can be annotated using cryo-ET acquired data.

DESCRIPTION (provided by applicant): The ability to test hypotheses in a variety of neuroscience fields has exploded due to the new ability to monitor and manipulate key cellular events in living animals and other models of disease and neuronal function in a high throughput fashion using modern cell biological approaches. Our previous award provided the foundation for a world-class Neuroscience Core, in support of NINDS-funded aims of UCSD Neuroscientists. With this application, we seek to incorporate high-throughput and high-sensitivity approaches in our established Neuroscience Imaging Core through purchase and active management of new equipment for our Major Users. Furthermore, we aim to increase the number NINDS-funded investigators participating in this initiative from 8 to 28 labs, to reflect ou outstanding cellular neuroscience community and number of qualifying projects. The University of California, San Diego Neurosciences Core has grown to be a centerpiece for research within the neuroscience community. Its existence has resulted in remarkable yields in productivity, expanded scientific scope and ability to test hypotheses using cutting edge technology and in vivo approaches. In this application, we propose move into exciting new areas through: 1. In vivo brain imaging of live neural tissue with high-sensitivity multiphoton microscopy. 2. High throughput electron microscopic ultrastructural imaging taking advantage of the latest breakthroughs in serial block face scanning electron microscopy (SBFSEM) and emergent probe technologies developed at UCSD. 3. More than tripling the number of Major User NINDS-funded investigators at UCSD. The research that this equipment will support involves neuronal stem cell differentiation, migration, axonal guidance, activity-dependent plasticity, connectivity, injury repair, degenerative disease and developmental biology. The requested systems were chosen because of their high data quality, unsurpassed abilities to document events in the nervous system not previously possible, and their ease of use, which is critical for a multi-user Core. The Neuroscience Core offers on-hand technicians to provide training for each of the services provided. The Core leverages the expertise of the other UCSD efforts including the National Center for Microscopy and Imaging Research (NCMIR), which will provide expertise and staff to meet the proposed goals to support training and research. The flexibility, dynamic range, sensitivity, and processing capabilities that will be provided by these tools is essential fr the next phase of the NINDS-funded work that has important implications for multiple nervous system diseases.

The overall aim of the ADCS is to advance research in the development of interventions that might be useful for treating, delaying, or preventing AD, particularly interventions that might not be developed by industry. In particular, the ADCS has focused on instrument and trial methodology, and the testing of potential therapeutics that might not otherwise be studied by the pharmaceutical industry. For the coming grant cycle, the ADCS will continue its efforts to advance therapeutics through controlled clinical trials, development o novel instruments and trial designs, recruitment efforts (with particular attention to recruitment f minority subjects). The organization will continue its recent emphasis on collaboration and data sharing. Specific Aims: Aim 1: Test interventions to Improve cognition, slow the rate of decline, or delay/prevent the onset of AD. Three of the four projects in this application aim to slow disease progression. Aim 2: Test an intervention to ameliorate behavioral symptoms. We will extend promising early results supporting an adrenergic approach amelioration of behavioral symptoms with a multicenter trial of prazosin. Aim 3: Design new instruments for use in clinical trials. For the present cycle, we have incorporated instrument development into our largest project, the A4 trial. Aim 4: Develop novel and innovative approaches to AD clinical trial design. The A4 trial utilizes a new trial design to test a leading intervention at the earliest feasible stge of disease, preclinical AD. Aim 5: Develop novel and innovative approaches to AD clinical trial analysis. The Biostatistics Core will continue efforts to advance analytical approaches to AD trial design. Work will continue on optimal modeling of longitudinal data, including novel methods to link diverse datasets. Aim 6:.Expand the range of individuals studied in AD studies to include at-risk individuals and those with MCI. The ADCS has focused its methodological research on early-stage trials, and for this cycle, the two largest projects target preclinical AD and mild cognitive impairment. Aim 7: Enhance the recruitment of minority groups into AD studies. For the coming cycle, the ADCS Minority Recruitment Core will expand outreach efforts, and we will require sites to meet minority enrollment targets in our two largest trials.

Project Summary/Abstract Huntington disease (HD) is caused by a mutation in exon 1 of the gene for Huntingtin (HTT). The mutant protein (mHTT), which contains an expanded stretch of glutamine residues (polyQ), is responsible for all HD manifestations, including dysfunction and degeneration of neurons. The overall goal of this Project is the discovery and development of effective treatments for HD. The Project is informed by evidence that mHTT is a client protein of the cytosolic chaperonin TRiC and that TRiC overexpression prevents or rescues mHTT- induced phenotypes in model systems. In particular, we have shown that individual TRiC subunits (CCTs), as well as the soluble apical portion of CCT1 (ApiCCT1), reverse deficits in BDNF trafficking and signaling in BACHD neurons in vitro. Project 2 will decipher whether or not TRiC and TRiC-derived proteins (hereafter, TRiC reagents) act in vitro to prevent dysfunction and degeneration of cortical and striatal neurons from the BACHD model. Our hypothesis is that that increasing the levels of TRiC or TRiC-derived reagents will abrogate and/or reverse mHTT- linked pathogenesis. We propose four Aims to examine in vitro quantitative and temporal features of HD pathogenesis. In Aim 1 we will further define deficits in axonal trafficking and signaling of BDNF in the BACHD model in vitro; we will confirm and further explore the ability of TRiC-derived reagents to prevent or reverse deficits. We will begin with reagents proven effective (CCT3, 5 and Apical CCT1) and test reagents coming from Project 1. In Aim 2, we will define deficits in BACHD neuron phenotypes: synapse formation, synaptic connectivity, gene expression, structure of neuronal somas and processes, mitochondrial function and calcium homeostasis. The timing of and possible progression of deficits will be defined and the impact of TRiC reagents explored. In Aim 3, we will define deficits in proteostasis in BACHD neurons examining clearance of mHTT that may involve protein folding, ER stress, and the UPS and autophagy/lysosome pathways. Whether introduction of TRiC reagents prevent and/or reverse changes will be examined. In Aim 4, we will examine iPSC-differentiated neurons from HD patients to determine whether deficits defined in Aim1-3 are detected and, if so, determine whether they are TRiC reagent-responsive. Project 2 is expected to enhance considerably understanding of HD pathogenesis and to define a novel approach to potential treatment. It is possible that TRiC and TRiC-inspired approaches may elucidate pathogenesis and treatment of other neurodegenerative disorders, including Alzheimer disease, Parkinson disease and ALS.

We aim to prevent Alzheimer disease (AD) in adults with DS (trisomy 21), referred to as AD in DS (AD-DS) by enhancing processing of the amyloid precursor protein (APP) to reduce the levels of the C-terminal 99 residue fragment (C99) and A?42. The therapeutic premise is based on: 1) increased APP gene dose is necessary for AD-DS, a finding replicated in mouse models of DS. By normalizing APP dose in DS models we eliminated: a) age-related degeneration of neurons in locus coeruleus (LCNs) and basal forebrain complex (BFCNs), b) hyper- phosphorylation of Tau, and c) enlargement of early endosomes; 2) increased C99 and A?42 acted via increased activation of Rab5 to induce changes in endosomes resulting in reduced trafficking of neurotrophic signals and BFCN atrophy. The evidence suggests that increased C99 and A?42 cause degeneration via deficits in endosomal trafficking of neurotrophic signals and motivates treatments to reduce C99 and A?42. Because both are substrates for ?-secretase, increasing ?-secretase activity should decrease levels and prevent or mitigate endosomal and degenerative phenotypes. BPN15606, a ?-secretase modulator (GSM), increases ?-secretase activity. In vitro, BPN15606 potently reduced C99 and A?42, reduced Rab5 activation and restored endosome size and trafficking of neurotrophins. In vivo, it reversed endosomal enlargement, improved LCN number, reversed Tau hyper-phosphorylation and enhanced cognition. In mouse models of AD-DS and AD/cerebral amyloidosis we will test the therapeutic hypothesis that BPN15606 will reduce the levels of C99 and A?42 to prevent and/or lessen neurodegeneration. The mechanistic hypothesis tested is that BPN15606 will normalize endosomal structure and function, neurotrophin signaling and trafficking, and improve cognition. Extensive pharm/tox studies qualify BPN15606 for our studies. Specific Aims: 1. To detail the time of onset of neurodegeneration in mouse models of AD-DS and AD/cerebral amyloidosis. Studies of the Dp16 model of AD-DS and Line 41 model of AD will be submitted to unbiased stereological studies of morphology and biochemistry, to quantitatively define onset of degeneration of neurons and synapses (Dp16: LCNs, BFCNs; Line 41: BFCNs, CA3), emergence of p-Tau and amyloid plaques. 2. To examine the effect of BPN15606 treatment before and after onset of neurodegeneration in the AD-DS model. Dp16 and 2N mice will be treated with an effective, safe dose of BPN15606. Guided by quantitative GO/NOGO criteria, the extent to which enhanced APP processing results in lessening of degenerative, endosomal and cognitive phenotypes will be assessed. 3. To examine the effect of BPN15606 treatment before and after onset of neurodegeneration in the AD/cerebral amyloidosis model. The same approach will guide BPN15606 studies in the Line 41 mouse. BPN15606-mediated reductions in degeneration will support the therapeutic hypothesis; normalization of endosomal and cognitive phenotypes will support the mechanistic hypothesis. These studies are intended to inform and guide trials of BPN15606 in AD-DS.

We aim to prevent Alzheimer disease (AD) in DS (trisomy 21) (AD-DS). Using antisense oligonucleotides (ASOs), we will selectively target RNA for the amyloid precursor protein (APP) in mouse models of AD-DS (Dp16) and AD/cerebral amyloidosis (Line 41). The therapeutic premise is based on: 1) increased APP gene dose is necessary for AD-DS. As replicated in models of AD-DS, normalizing APP dose eliminated: a) age-related neurodegeneration in locus coeruleus and the basal forebrain complex, b) hyper- phosphorylation of Tau, and c) enlargement of early endosomes; 2) pointing to a mechanism by which increased APP gene dose acts, increased full-length APP (fl-APP), its 99 residue C-terminal fragment (C99) and A?42 each increased Rab5 activity, thus enlarging early endosomes, disrupting endosomal trafficking of neurotrophic signals, and causing atrophy of BFCNs; 3) therefore, reducing levels of these APP products is a rational approach to preventing or lessening the impact of increased APP gene dose in AD-DS, including effects on endosomes. ASOs have recently been shown to safely and effectively treat CNS disorders. Indeed, FDA approval for ASOs in Spinal Muscular Atrophy motivates trials of ASOs in other CNS diseases. In preliminary studies we showed that intracerebroventricular (ICV) injection of ASOs targeting mouse and human APP (i.e. mAPP-ASOs and hAPP-ASOs) reduced APP mRNA and protein levels. Using mouse models of AD-DS and AD/cerebral amyloidosis we will test the therapeutic hypothesis that APP-ASOs will selectively reduce the levels of APP mRNA and its products to prevent and/or lessen neurodegeneration. The mechanistic hypothesis is that APP-ASOs will normalize endosomal structure and function, neurotrophin signaling and trafficking, and improve cognition. Using defined GO/NOGO criteria as a guide, we will pursue these Specific Aims: 1. To investigate newly designed APP-ASOs in vitro for efficacy and target specificity. Using an existing mAPP-ASO as benchmark, additional mAPP-ASOs will be designed to increase potency for targeting APP mRNA and its products and normalizing endosome size. 2. To establish optimal APP-ASO doses and dose-intervals based on empirically defined in vivo pharmacokinetic (PK) and pharmacodynamic (PD) properties. We will define effective, non-toxic doses and treatment intervals for advancement of mAPP-ASOs and hAPP-ASOs to in vivo studies in Aim 3. In the Dp16 model, we will target a ~33% reduction of mAPP RNA, i.e. to 2N values; in Line 41 mice we will target a 50% reduction. Aim 3. To investigate in vivo APP-ASO efficacy in ameliorating neurodegeneration and normalizing endosomal phenotypes. To test the therapeutic hypothesis, we will ask if APP-ASOs given before degeneration in Dp16 mice and plaque deposition in Line 41 mice prevent these changes. Next, we will ask if degeneration in Dp16 mice can be reversed by APP-ASO treatment. The mechanistic hypothesis will be informed by whether or not APP-ASO reductions in degeneration are correlated with normalization of endosomal phenotypes.