Lennart Mucke - US grants

The dementia of Alzheimer's disease (AD) is accompanied by accumulation of abnormal proteins in form of amyloid deposits in victims' brains. We hypothesize that these proteins are detrimental to surrounding brain cells and are a major cause of the symptoms seen in AD. To test this hypothesis in the living organism, we propose to express human amyloid components, specifically, different forms of mutated and non-mutated amyloid beta protein precursors (APP695, 751, 770) and the protease inhibitor alpha1- antichymotrypsin (ACT), in the brains of transgenic mice with the help of recombinant DNA constructs. CDNAS encoding these human proteins will be recombined with transgenic vectors (derived from the neuroA-specific enolase (NSE) gene and the glial fibrillary acidic protein (GFAP) gene) for the neuron-specific (NSE) or astrocyte-specific (GFAP) expression of foreign proteins in the brains of transgenic mice. These vectors are chosen because both neurons and astrocytes may function as a source of amyloid proteins in AD. The transgenes will be introduced into the germline of mice by microinjection of fertilized egg cells. Transgenic mice will be analyzed for the expression of the human CDNAS at the RNA and protein levels. The transgenic mouse model will be characterized by detailed neuropathologic and biochemical analysis. A battery of neurobehavioral tests will assess if overproduction of APP and/or ACT in the brains of the transgenic mice induces impairment of memory/learning or other forms of neurologic dysfunction. Behavioral abnormalities will be correlated with neuropathologic findings. If amyloid components are causative factors in the dementia of AD, inhibition of their formation, deposition or action should allow treatment of the disease. To help predict their usefulness in the treatment of AD, protease inhibitors, tachykinin agonists such as substance P, and other agents will be tested in the transgenic models for their ability to inhibit the structural and functional alterations induced by the overexpression of APP/ACT. The molecular mechanisms of drug actions and amyloid formation will be studied in vivo as well as in explant cultures of transgenic nervous tissue. This project will assess in vivo the role the following factors could play in the development of AD: i) different forms of APP, ii) an APP point mutation found in familial AD, iii) neuronal vs. astroglial processing of APP, and iv) protease inhibitors such as ACT. The transgenic models proposed should allow the efficient preclinical assessment of drugs aimed at different manifestations of AD and facilitate the development of new therapeutic and diagnostic strategies.

HIV-1 infection is often associated with damage to the central nervous system (CNS). The precise molecular and cellular processes that cause this damage are still unknown. Increasing evidence suggests that HIV-1-derived proteins, particularly envelope proteins and Nef, may play important causal roles in the development of AIDS dementia. To study their effects on the CNS in vivo, we propose to express proteins of the neuroinvasive HIV-1 isolate YU-2 (including gp160 with and without cleavage site mutation, gp120, Flag'ed gp120, gp41 and Nef) in astrocytes or macrophages/microglia of transgenic mice. This will be accomplished with the help of fusion gene constructs which allow the prolonged, reproducible delivery of selected proteins (devoid of any contaminations) to specific areas/cells of the intact CNS. Regulatory sequences from the glial fibrillary acidic protein gene will be used to express the above HIV-1 proteins in astrocytes and sequences from genes encoding the high-affinity Fc receptor or lysozyme to express gp120 in macrophages. Fusion genes will be microinjected individually into fertilized mouse oocytes. The resulting transgenic mice will be bred and the expresssion of transgenes in their offspring characterized. Mice from transgenic expressor lines are expected to show structural and molecular alterations of the CNS, which will be quantitated with a well established battery of tests, including laser confocal microscopy of in situ hybridized/immunolabeled brain sections, electron microscopy, RNAse protection assays, Western blots, and bioassays. To assess the relevance of these models to human disease, alterations in the CNS of transgenic mice will be compared with those in HIV-1 infected human brain tissue, obtained postmortem. Mechanisms of HIV-1-induced CNS damage will also be analyzed in vitro by exposing neurons from nontransgenic mice to HIV-1 protein-expressing transgenic astrocytes/microglia and by testing which pharmacologic reagents block the resulting neurotoxic effects. Well characterized transgenic models will then be used to assess in vivo the effectiveness of drugs aimed at detrimental HIV-CNS interactions. By characterizing the CNS effects of different HIV-1 proteins, this project will further our understanding of HIV-1 associated neurologic disease and help identify important targets for therapeutic interventions. By providing models for the preclinical assessment of drugs, this study will facilitate the evaluation and development of therapeutic strategies for the treatment and prevention of AIDS dementia.

HIV-1 associated impairments of cognition and motor performance can culminate in dementia and paralysis. Here we propose to use transgenic (tg) mice, engineered to express pathogenetic factors in their central nervous system (CNS), to study interactions between viral and hot proteins that could contribute to the development of HIV associated dementia and motor complex. Lines of singly tg mice expressing gp120 or interleukin-6 (IL-6) will be crossed to generate bigenic mice that co-express gp120 and IL-6 in their CNS. Brains from bigenic and singly tg will be compared in a well established battery of neuropathological and molecular analyses to assess whether co-expression of gp120 and cytokines enhances/alters the brain damage observed when these factors are expressed individually. This scenario is relevant to HIV-1 encephalitis in which inflammatory responses lead to the co-expression of viral products and cytokines. To evaluate the neuropathogenic potential of interactions between gp120 and the human CD4 receptor (hCD4), CNS alterations of gp120 singly tg mice will be compared with those induced in gp120/hCD4 bigenic mice (engineered to express hCD4 on brain macrophages/microglia). While the co-expression of cytokines or hCD4 may enhance gp120 induced neurotoxicity, we have found that neuronal overexpression of the human amyloid precursor protein (hAPP) can effectively prevent gp120-induced brain damage. Different strains of gp120/hAPP bigenic mice will be generated and analyzed to determine whether this neuroprotective effect is due primarily to hAPP-mediated stabilization of the neuronal calcium homeostasis or to other hAPP effects. We also plan to examine the hypothalamic-pituitary axis of gp120 tg Balb/cByJ mice to evaluate whether the dwarfism and low blood glucose level that develop in this strain reflect gp120-induced disturbances of central neuroendocrine circuits. If this postulate is confirmed, this stain/phenotype could provide a useful readout for the assessment of treatments aimed at detrimental HIV-CNS interaction. To further dissect he effects of gp120/cytokine, gp120/hCD4 and gp120/hAPP interactions on neurons at the molecular level, experiments in tg mice will be complemented by analyses of neuronal/glial co-cultures. The above experiments can be expected to advance our understanding of HIV-1 associated neurological disease and to help identify important targets for therapeutic interventions.

HIV-1 infection is often associated with damage to the central nervous system (CNS). The precise molecular and cellular processes that cause this damage are still unknown. Increasing evidence suggests that HIV-1-derived proteins, particularly envelope proteins and Nef, may play important causal roles in the development of AIDS dementia. To study their effects on the CNS in vivo, we propose to express proteins of the neuroinvasive HIV-1 isolate YU-2 (including gp160 with and without cleavage site mutation, gp120, Flag'ed gp120, gp41 and Nef) in astrocytes or macrophages/microglia of transgenic mice. This will be accomplished with the help of fusion gene constructs which allow the prolonged, reproducible delivery of selected proteins (devoid of any contaminations) to specific areas/cells of the intact CNS. Regulatory sequences from the glial fibrillary acidic protein gene will be used to express the above HIV-1 proteins in astrocytes and sequences from genes encoding the high-affinity Fc receptor or lysozyme to express gp120 in macrophages. Fusion genes will be microinjected individually into fertilized mouse oocytes. The resulting transgenic mice will be bred and the expresssion of transgenes in their offspring characterized. Mice from transgenic expressor lines are expected to show structural and molecular alterations of the CNS, which will be quantitated with a well established battery of tests, including laser confocal microscopy of in situ hybridized/immunolabeled brain sections, electron microscopy, RNAse protection assays, Western blots, and bioassays. To assess the relevance of these models to human disease, alterations in the CNS of transgenic mice will be compared with those in HIV-1 infected human brain tissue, obtained postmortem. Mechanisms of HIV-1-induced CNS damage will also be analyzed in vitro by exposing neurons from nontransgenic mice to HIV-1 protein-expressing transgenic astrocytes/microglia and by testing which pharmacologic reagents block the resulting neurotoxic effects. Well characterized transgenic models will then be used to assess in vivo the effectiveness of drugs aimed at detrimental HIV-CNS interactions. By characterizing the CNS effects of different HIV-1 proteins, this project will further our understanding of HIV-1 associated neurologic disease and help identify important targets for therapeutic interventions. By providing models for the preclinical assessment of drugs, this study will facilitate the evaluation and development of therapeutic strategies for the treatment and prevention of AIDS dementia.

HIV-1 associated impairments of cognition and motor performance can culminate in dementia and paralysis. Here we propose to use transgenic (tg) mice, engineered to express pathogenetic factors in their central nervous system (CNS), to study interactions between viral and host proteins that could contribute to the development of HIV associated dementia and motor complex. In collaboration with Dr. Campbell, we will cross singly tg mice expressing gp120, interleukin-6 (IL-6) or interferon-alpha1 (IFN- alpha1) to generate bigenic mice that express gp120 together with either IL-6 or IFN-alpha1. Brains from bigenic and singly tg will be compared in a well established battery of neuropathological and molecular analyses to assess whether co-expression of gp120 and cytokines enhances/alters the brain damage observed when these factors are expressed individually. This scenario is relevant to HIV-1 encephalitis in which inflammatory responses lead to the co-expression of viral products and cytokines. To evaluate the neuropathogenic potential of interactions between gp120 and the human CD4 receptor (hCD4), CNS alterations of gp 120 singly tg mice will be compared with those induced in gp 120/hCD4 bigenic mice (engineered to express hCD4 on brain macrophages/microglia). While the coexpression of cytokines or hCD4 may enhance gp120-induced neurotoxicity, we have found that neuronal overexpression of the human amyloid precursor protein (hAPP) can effectively prevent gp 120-induced brain damage. Different strains of gp 120/hAPP bigenic mice will be generated and analyzed to determine whether this neuroprotective effect is due primarily to hAPP-mediated stabilization of the neuronal calcium homeostasis or to other hAPP effects. We also plan to examine the hypothalamic-pituitary axis of gp 120 tg Balb/cByJ mice to evaluate whether the dwarfism and low blood glucose level that develop in this strain reflect gp 120-induced disturbances of central neuroendocrine circuits. If this postulate is confirmed, this strain/phenotype could provide a useful readout for the assessment of treatments aimed at detrimental HIV-CNS interactions. To determine whether the cerebral expression of detrimental or neuroprotective factors is reflected in alterations of neuronal functions, the mice described above will be analyzed by the Functional Assessment Core in electrophysiological and neurobehavioral paradigms. To further dissect the effects of gp 120/cytokine, gp120/hCD4 and gp 120/hAPP interactions on neurons at the molecular level, experiments in tg mice will be complemented by analyses of neuronal/glial co-cultures. This in vitro analysis will include measurements of intraneuronal calcium levels and electrophysiological responses, as proposed in Dr. Gruol's component. The above experiments are expected to advance our understanding of HIV-1 associated neurological disease and to help identify important targets for therapeutic interventions.

We have established transgenic (tg) models in which cerebral expression of HIV-1 proteins induces glial and neuronal alterations that resemble neuropathologic changes found in humans with HIV-1 associated dementia (1). Here we propose to use these tg models to further characterize the processes that underlie HIV-induced neurotoxicity in vivo and to identify critical targets for therapeutic interventions. Aim #1: Compare GFAP-gp120 tg mice with non-tg controls with respect to: a) neuronal susceptibility to exogenously administered excitotoxic amino acids, b) evidence for alterations in cerebral calcium homeostasis, and c) evidence for neuronal apoptosis. Aim #2: Treat GFAP-gp120 tg mice with drugs aimed at specific neuropathogenic pathways (including meman-tine, other adamantanes, nimodipine, nitroglycerin, and phenylbutylnitrone) and compare the extent of their nervous system damage with that of sham-treated controls. Aim #3: Generate bigenic mice in which brain expression of gp120 is combined with the expression of superoxide dismutase (SOD) or mutated glutamate receptor subunits. Analyze the nervous system of bigenic mice at the structural and molecular level and compare the results with those obtained in the singly tg parental strains. Aim #4: Determine whether the expression of gp120 in the brains of tg mice is associated with functional neuronal/behavioral alterations. If so, assess the responsiveness of these abnormalities to the manipulations outlined in Aims #2 and #3 above. The proposed experiments are expected to help determine the molecular processes underlying gp120 neurotoxicity in vivo. They will also allow the preclinical assessment of therapeutic strategies that could benefit patients with HIV- 1 associated dementia and related disorders.

DESCRIPTION Alzheimer's disease (AD) is characterized clinically by dementia and neuropathologically by amyloid plaques, amyloid angiopathy, dystrophic neurites, NFT's, gliosis, and loss of neuronal subpopulations and synapses. Increasing evidence suggests that the AB peptide derived from APP plays a central role in AD. The goals of this project are to characterize neuronal and glial products that contribute to amyloidogenesis and neurodegeneration and identify cortical AD-related pathogenetic pathways that could be targeted by therapeutic interventions. Mice expressing a PDGF-promoter driven, alternatively spliced hAPP minigene, (PDGF-APP mice) develop several aspects of AD neuropathology. This proposal is to utilize this and related models to study cerebral amyloidogenesis and amyloid-induced neurodegeneration in vivo with the following aims: 1) Determine what factors influence AB production and B-amyloid deposition in vivo.; 2) Determine whether development of neuronal/synaptic degeneration in PDGF-hAPP mice depends on B-amyloid formation; 3) Determine whether B-amyloid in the brain results in aberrant induction of neuronal apoptosis and whether this process involves excitotoxic mechanisms.

DESCRIPTION (provided by the applicant): The accumulation of beta-amyloid peptides (Abeta) in the brain plays a key role in Alzheimer's disease (AD). Inflammatory responses of glial cells may also be involved, but their role appears to be complex, with some responses protecting and others damaging the brain. To harness microglial activities therapeutically, one must first identify the mediators of their beneficial functions. Recent reports suggest that immunization with Abeta can prevent and partially reverse AD-like brain alterations in transgenic mouse models, possibly by activating anti- amyloidogenic functions of microglia. Those studies focused on amyloid plaques and were not designed to dissect the underlying molecular mechanisms. Our studies of human amyloid protein precursor (hAPP) transgenic mice suggest that AD-related synaptic/neuronal dysfunction and degeneration are caused by nondeposited forms of Abeta. Such plaque-independent neuronal deficits were inhibited by human apolipoprotein (apo) E3, but not by apoE4, the best established genetic risk factor for the most common form of AD. Here we will assess whether Abeta vaccination can inhibit plaque-independent neurodegeneration and cognitive impairments, and whether it can do so even in the high-risk environment created by apoE4. Using hAPP and hAPP/apoE transgenic mice, we will determine whether Abeta vaccination prevents, inhibits, or reverses loss of presynaptic terminals and cholinergic neurons (Aim 1) and deficits in spatial and nonspatial learning and memory (Aim 2). Since the processes underlying the therapeutic effects of Abeta vaccination may be reflected in changes in gene expression, we will also use DNA microarrays to compare the gene expression profiles of specific brain regions and plaque-associated microglia in transgenic mice that have or have not been immunized with Abeta (Aim 3). We will then determine whether the genetic or pharmacological manipulation of specific microglial gene products diminishes or augments the therapeutic effect of Abeta vaccination (Aim 4). These studies will significantly expand the preclinical evaluation of Abeta vaccination with respect to AD-related neurodegeneration and cognitive impairment. They will also advance our understanding of the processes that underlie the therapeutic effects of this novel treatment, and thereby facilitate its optimization. Lastly, the proposed project will shed light on the roles of microglia in the pathogenesis of AD-related neuronal deficits. These goals and perspectives are congruent with those of RFA AG-01-003.

Description (Provided by applicant): Neurological diseases that result in the degeneration of synapses culminate in severe cognitive deficits and (behavioral disturbances. These dementing illnesses present increasing medical and socioeconomic problems and raise a wide range of fundamental neuroscientific questions. Alzheimer's disease (AD) is the main cause of age-related dementia and its prevalence is increasing rapidly due to the increasing number of elderly in our population. AD is associated with a prominent degeneration of synapses and with an abnormal accumulation of amyloid beta peptides (AB) in the brain. A-beta is derived from the amyloid protein precursor (APP), which is enriched in synapses. The precise relationship between APP, A-beta, and synaptic loss remains obscure. The current proposal aims to shed light on this relationship. We previously used the platelet-derived growth factor (PDGF) beta chain promoter to express human APP (hAPP) in neurons of transgenic mice. In these studies, we focused on the density of synaptophysin-immunoreactive presynaptic terminals because the loss of these structures correlates well with cognitive decline in AD. High levels of neuronal A-beta production in PDGF-hAPP mice resulted in a decrease of presynaptic terminals and elicited major deficits in synaptic transmission. In Aim 1, we will investigate whether the profile of synaptic protein alterations in PDGF-hAPP mice resembles that in AD brains, affecting some proteins more than others, and we will examine whether these alterations are preceded or followed by changes in pre- or postsynaptic neurons. In Aim 2, we will assess to what extent the development of synaptic, alterations in PDGF-hAPP mice depends on the activity of the protein tyrosine kinase Fyn, ablation of which has been shown to prevent A-beta-induced neurotoxicity in tissue culture. In Aim 3, we will immunize PDGF-hAPP mice, with A-beta to determine whether this approach can induce the clearance of synaptotoxic A-beta species and inhibit synaptic degeneration. In Aim 4, we will investigate the relationship between synaptic alterations and behavioral deficits in PDGF-hAPP mice and determine whether the latter can also be prevented or reversed by A-beta vaccination. Achieving these aims will help assess the potential usefulness of novel therapeutic strategies for this most prevalent neurodegenerative disorder. It will also advance our understanding of the function of synaptic proteins in synapse loss and degeneration, which is the focus of RFA NS-01-002.

DESCRIPTION (provided by applicant): We will address the hypothesis that different neurodegenerative diseases are caused by the accumulation of distinct proteins with pathogenic conformations (proteopathies). These disorders are a complex biomedical, behavioral, and social problem as they are increasing in frequency, cause major disability, and remain largely untreatable. If the ways in which different proteins damage nerve cells overlap, treatments may be developed to prevent and reverse more than one of these conditions. We have assembled five interactive projects and four essential cores to study the mechanisms by which proteins associated with Alzheimer's, Parkinson's, or Huntington's disease impair neuronal function and survival. The program is multidisciplinary and relies on state-of the-art technology, including X-ray crystallography, robotic microscopy, transgenic and gene-targeted mouse models, cellular biology, neuropathology, and behavioral neuroscience. Project 1, "Polyglutamine Conformation and Neurodegeneration," aims to differentiate whether visible aggregates or other conformational states of mutant huntingtin are responsible for Huntington's disease-related neurodegeneration. Project 2, "Protein Structure in Apolipoprotein E4-associated Neurodegeneration," will test whether the Alzheimer's disease-promoting effect of apolipoprotein E4 depends on the conformation and stability of this molecule. Project 3, "Apolipoprotein E in Neurobiology: Cellular Mechanisms," will examine whether apolipoprotein E4 promotes Alzheimer's disease-like pathology through amyloid beta peptide (Abeta)-dependent pathways or via independent mechanisms. Project 4, "Causes and Consequences of alpha-Synuclein Aggregation," will assess whether pathogenic interactions between Abeta and alpha-synuclein could contribute to the development of Parkinson's disease and other Lewy body diseases. Project 5, "Mechanisms of AbetaD-induced Neuronal Deficits," will analyze the molecular cascades that link the formation of neurotoxic Abeta assemblies to Alzheimer's disease-related cognitive decline and test whether these cascades can be modulated by apolipoprotein E isoforms and alpha-synuclein. The Cores (A: Administrative; B: Tissue Culture; C: Animal; D: Neuropathology/Imaging) will provide the common services necessary to accomplish the goals of the program project. Our studies will shed light on diverse neurodegenerative diseases and could provide the knowledge needed to better treat and prevent them.

DESCRIPTION (provided by applicant): We will address the hypothesis that different neurodegenerative diseases are caused by the accumulation of distinct proteins with pathogenic conformations (proteopathies). These disorders are a complex biomedical, behavioral, and social problem as they are increasing in frequency, cause major disability, and remain largely untreatable. If the ways in which different proteins damage nerve cells overlap, treatments may be developed to prevent and reverse more than one of these conditions. We have assembled five interactive projects and four essential cores to study the mechanisms by which proteins associated with Alzheimer's, Parkinson's, or Huntington's disease impair neuronal function and survival. The program is multidisciplinary and relies on state-of the-art technology, including X-ray crystallography, robotic microscopy, transgenic and gene-targeted mouse models, cellular biology, neuropathology, and behavioral neuroscience. Project 1, "Polyglutamine Conformation and Neurodegeneration," aims to differentiate whether visible aggregates or other conformational states of mutant huntingtin are responsible for Huntington's disease-related neurodegeneration. Project 2, "Protein Structure in Apolipoprotein E4-associated Neurodegeneration," will test whether the Alzheimer's disease-promoting effect of apolipoprotein E4 depends on the conformation and stability of this molecule. Project 3, "Apolipoprotein E in Neurobiology: Cellular Mechanisms," will examine whether apolipoprotein E4 promotes Alzheimer's disease-like pathology through amyloid beta peptide (Abeta)-dependent pathways or via independent mechanisms. Project 4, "Causes and Consequences of alpha-Synuclein Aggregation," will assess whether pathogenic interactions between Abeta and alpha-synuclein could contribute to the development of Parkinson's disease and other Lewy body diseases. Project 5, "Mechanisms of AbetaD-induced Neuronal Deficits," will analyze the molecular cascades that link the formation of neurotoxic Abeta assemblies to Alzheimer's disease-related cognitive decline and test whether these cascades can be modulated by apolipoprotein E isoforms and alpha-synuclein. The Cores (A: Administrative; B: Tissue Culture; C: Animal; D: Neuropathology/Imaging) will provide the common services necessary to accomplish the goals of the program project. Our studies will shed light on diverse neurodegenerative diseases and could provide the knowledge needed to better treat and prevent them.

Alzheimer's disease (AD) and non-AD dementias are increasing in frequency, cause major disability, and remain largely untreatable. They may be caused by the accumulation of distinct proteins with pathogenic conformations. Transgenic mice expressing human amyloid precursor proteins (hAPP) accumulate amyloid-beta peptides (Abeta) in their brain and develop pathological and behavioral alterations resembling AD. However, the mechanisms underlying cognitive deficits in AD and in hAPP mice are largely unknown. There also is an urgent need, from both a diagnostic and therapeutic perspective, for the identification of reliable biochemical markers of Abeta-induced cognitive decline. We have pinpointed molecular alterations that accurately reflect and possibly contribute to AD-related cognitive impairments. Deficits in spatial learning and memory in mice expressing familial AD-mutant hAPP correlated most robustly with reductions in the calcium-binding protein calbindin-D28k in granule cells of the dentate gyrus, a brain region critically involved in learning and memory. These reductions were age-dependent and correlated with the relative abundance of Abeta1-42 but not with the amount of Abeta deposited in amyloid plaques. In our preliminary studies, we have identified strong reductions in calbindin levels also in granule cells of humans with AD with the greatest reductions seen in the most severely demented cases. In Specific Aim 1, we will determine whether calbindin reductions in granule cells of the dentate gyrus correlate with specific cognitive deficits (e.g., in spatial learning and memory) and genetic factors (e.g., APOE haplotype) in humans with AD and non-AD dementias. In Specific Aim 2, we will examine in these cases how calbindin reductions in granule cells relate to the presence and severity of other neuropathological alterations and to the accumulation of Abeta and other neurotoxic proteins in different brain regions. In Specific Aim 3, we will analyze hAPP transgenic mice with oligonucleotide arrays and protein arrays to search for additional molecular indicators and mediators of Abeta-dependent behavioral deficits. The clinical relevance and usefulness of the most interesting molecular alterations will then be evaluated in human subjects, either in the context of this ADRC or in independent studies. We predict that this Project will shed light on the pathways leading from the accumulation of Abeta to cognitive decline, ascertain the usefulness of a promising indicator of Abeta-induced neuronal deficits, and pinpoint novel markers and mediators of pathological cognitive decline in aging. The success of this project depends critically on the support of the proposed cores and on the overall environment this ADRC would create at UCSF.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD), the main cause of age-related dementia, is increasing in prevalence due to the increasing number of elderly in our population. AD is associated with prominent impairments of neurons and synapses and with an abnormal accumulation of amyloid-B peptides (AB) in the brain. AB, which is derived from the amyloid precursor protein (APP), appears to play a causal role in AD, although it remains uncertain how it erodes cognitive functions and how this process can be prevented or reversed. The results we obtained during the preceding funding period have begun to shed light on these questions. We determined that human APP (hAPP) transgenic mice producing high levels of human Af5 in the brain have an AD-like pattern of synaptic alterations (previous Aim 1), that the enzyme Fyn is involved in some AB-dependent synaptic alterations (previous Aim 2), that premature mortality and neuronal deficits in hAPP mice can be prevented or delayed by the genetic modulation of Fyn or apolipoprotein E (previous Aim 3), and that the extent of cognitive deficits in hAPP mice is tightly linked to the neuronal depletion of factors that are regulated by excitatory synaptic activity (previous Aim 4). Some of these factors were also depleted in corresponding neuronal populations of humans with AD. These results provide a solid foundation for the current application and underline that we are in a good position to advance this interesting area of research. Here we propose to determine how exactly hAPP/Abeta interacts with Fyn-related signaling cascades to cause neuronal impairments (new Aim 1), whether the pharmacogenetic inhibition of Fyn can prevent and reverse neuronal deficits in hAPP mice (new Aim 2), whether hAPP/Abeta-dependent neuronal alterations are caused by an imbalance of excitatory and inhibitory synaptic activities (new Aim 3), and whether the pharmacological or genetic manipulation of neurotransmitter receptors or Fyn-related signaling pathways can prevent and reverse behavioral deficits in hAPP mice (new Aim 4). This study could reveal how increased levels of AB impair important cognitive functions such as learning and memory. It could also resolve whether these impairments can be prevented or reversed by therapeutic manipulation of synaptic activity and neuronal signaling. Ultimately, our study may provide useful guidance in the development of drugs to maintain and improve memory and other cognitive functions in AD.

We will study the roles of the amyloid precursor protein (APP) and APP-derived amyloid beta peptides (Abeta) in the pathogenesis of Alzheimer's disease (AD), the most frequent proteopathy of the aging central nervous system. We previously overexpressed wildtype human APP (hAPP[WT]) or familial AD-mutant human APP (hAPP[FAD]) in neurons of transgenic mice. Amyloid deposition in these mice depended on absolute and relative levels of the 42-amino acid form of Abeta (Abeta1-42) and on interactions of this fibrillogenic peptide with other molecules. Synaptic deficits correlated with Abeta levels but not with the deposition of Abeta into amyloid plaques, suggesting a plaque-independent neurotoxicity of Abeta. Expression of apolipoprotein (apo) E3, but not of apoE4, prevented or delayed synaptic loss and memory impairments in hAPP[FAD]/apoE doubly transgenic mice. Aged hAPP[FAD] mice with high Abeta1-42 levels also had losses of cholinergic neurons in the basal forebrain. Recently, we found that behavioral deficits in hAPP[FAD] mice were tightly correlated with reductions in the calcium-binding protein, calbindin-D28 K (CB), in the dentate gyrus. In Aim 1, we will determine why neuronal CB levels in the dentate gyrus are strongly diminished in hAPP[FAD] mice but only minimally in hAPP[WT] mice. We will assess the dependence of CB reductions on the levels of deposited and nondeposited Abeta species and evaluate the role of calcium channels in the CB alterations. In Aim 2, we will examine whether CB reduction plays a critical role in the development of Abeta-induced behavioral deficits. We will correlate CB levels with functional neuronal deficits in hAPP mice and examine whether regulatable overexpression of CB modulates these deficits. In Aim 3, we will characterize the cholinergic deficits in hAPP mice and assess their mechanisms and behavioral consequences. We will study the cholinergic basal forebrain system of these mice, evaluate whether they have deficits in the expression, transport, or release of brain-derived neurotrophic factor, and test whether increasing acetylcholine levels improves their behavioral deficits. In Aim 4, we will examine doubly transgenic mice to be generated in other components of this program to determine if and how the development of Abeta-induced neuronal deficits is modulated by apoE and alpha-synuclein. These studies will shed light on the molecular cascades that lead from the pathogenic assembly of Abeta to functional neurological decline. The proposed experiments follow up on a solid body of preliminary data and actively contribute to the cohesiveness of the program. Their success depends on most, if not all, components of the proposed program.

Genetically modified mouse models have been very useful in research on neurodegenerative disorders, and the proposed program will take maximal advantage of this valuable resource. The main goal of the Animal Core is to help the project leaders of this program address their research questions conclusively with the minimal number of animals 1and experiments possible. To achieve this goal, we propose five specific aims. Aim 1: Maintain all animals in a I manner that ensures the reliable selection of experimental and control groups for timely distribution to the different projects. Rodents will be housed in specific-pathogen-free animal care facilities on the San Francisco General Hospital campus of the University of California, San Francisco, and on the La Jolla campus of the University of California at San Diego (UCSD). Receipts of mice and rats, matings, pregnancies, births, weanings, coat colors, ear markings, genotyping results, pedigrees, weights, health problems, deaths, and transfers or shipments of mice will all be recorded in a database that will be accessible to all project leaders and their coworkers via the computer networks of the Gladstone Institutes and the University of California. Aim 2: Breed the different lines of genetically modified mice and determine the genotype of the resulting offspring by PCR. Breedings will include timed matings for the generation of primary neural cell and tissue cultures, and crosses between distinct lines to generate mice with two or more genetic modifications. The core personnel have ample experience in genotyping genetically modified mice and maintaining complex animal databases. Aim 3: Receive mice from the Gladstone microinjection facility and blastocyst core and identify new transgenic founders and knockin mice by PCR and southern blot analysis. Gladstone and UCSD maintain state-of-the-art facilities for the microinjection of transgenes into one-cell mouse embryos and for gene targeting to inactivate or selectively modify endogenous mouse genes. Aim 4: Periodically analyze all lines of mice to confirm that levels and patterns of (trans)gene expression have remained stable. Cerebral levels of transgene expression will be tested by quantitative fluorogenic RT-PCR. The distribution of transgene expression will be characterized in collaboration with the Neuropathology/Imaging Core. We will also collaborate closely with the Neuropathology/Imaging Core and veterinary staff to optimize our protocols for the anesthesia and perfusion of animals and for the removal, dissection, and proper storage of neural tissues. Aim 5: Ship mice to Dr. Masliah at UCSD and to investigators at other institutions and advise them on the genotyping and husbandry of the mice. We have distributed our animal models to many institutions and will continue to make our models available to the scientific community.

[unreadable] DESCRIPTION (provided by applicant): This application represents the first competing renewal of our program project, which uses an interdisciplinary team approach to address the unifying hypothesis that most, if not all, aging-related neurodegenerative disorders are caused by the intracellular or extracellular accumulation of specific proteins that have assumed pathogenic conformational states (proteinopathies). The resulting neurodegenerative disorders, which include Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD) and other Lewy body diseases (LED), remain largely untreatable and represent a complex biomedical, behavioral and social problem. Medical breakthroughs are urgently needed in this area, and the surest way to such breakthroughs is to determine how exactly these diseases result in the dysfunction and degeneration of nerve cells. Our program addresses this important need by bringing together investigators with diverse areas of expertise, widely overlapping interests in proteinopathies, and an established track record of fruitful collaborative interactions. Our approach takes advantage of a great number of valuable resources and technologies, including robotic microscopy, molecular imaging, genetically engineered mouse models, RNAi mediated gene silencing, and cell type-specific expression of mechanistically informative viral constructs. Using these and other strategies, we will study the processes by which diverse proteins impair neuronal function and survival and compare our results to determine whether there are common mechanisms of neurodegeneration. We will also study the susceptibility of different neuronal populations to common versus disease-specific pathogenic processes to elucidate why these diseases so selectively attack specific neuronal populations. Project 1, "Mechanisms of Cell-Specific Huntingtin-lnduced Neurodegeneration" aims to elucidate cell autonomous and cell non-autonomous mechanisms that contribute to the susceptibility of striatal neurons to mutant huntingtin. Project 2, "Microglial Kynurenine Pathway and Selective Neuronal Vulnerability," will test if genetic or pharmacological inhibition of the microglial kynurenine pathway is protective in mouse models of AD and HD. Project 3, "Apolipoprotein E in Alzheimer's Disease: Cellular Mechanisms," will study the regulation of apolipoprotein E expression in neurons and explore Apin dependent roles of different apolipoprotein E isoforms in the pathogenesis of AD. Project 4, "Causes and Consequences of a-Synuclein Aggregation," will assess in combined models of AD and PD if interactions between a-synuclein and A[unreadable] lead to neurodegeneration of specific neuronal populations through activation of glutamate receptors and proteases that cleave a-synuclein. Project 5, "Mechanisms of Aft-Induced Neuronal Deficits," will test whether the modulation of specific neuronal or glial molecules can block aberrant neuronal overexcitation and ameliorate behavioral abnormalities in mouse models of AD and other proteinopathies. The Cores (A: Administrative, B: Tissue Culture, C: Animal, D: Microscopy/Neuropathology) will provide the common services necessary to accomplish the goals of the program project. Our studies will shed light on diverse neurodegenerative diseases and could provide the knowledge needed to better treat and prevent them. [unreadable] [unreadable] PRINCIPAL INVESTIGATOR: Dr. Lennart Mucke is a Distinguished Professor at UCSF and Director of the Gladstone Institute of Neurological Diseases (GIND). He is a demonstrated and accomplished leader in the neurodegenerative disease field with broad clinical and basic expertise applicable to all aspects of the PPG. He has effectively coordinated a highly successful first term of the PPG involving a team of investigators at multiple sites, with whom he has interacted productively. Recently, Dr. Mucke has overseen a successful relocation of the Gladstone laboratories of the PPG to a new location on the Mission Bay campus. He is eminently qualified to continue to lead this PPG. [unreadable] [unreadable] REVIEW OF INDIVIDUAL COMPONENTS [unreadable] [unreadable] CORE A - Administrative Core, Dr. Lennart Mucke [unreadable] [unreadable] DESCRIPTION (provided by applicant): Core A will be responsible for the general organization of the program and for promoting productive interactions among all projects and cores and between this program and the scientific community. The core will also promote, monitor, and report on the progress of the program. To achieve these goals, we propose the following specific aims. Aim 1: Define the overall organization of the program and adjust its structure according to research developments and opportunities. The core leader has developed an interactive program in disease-related neuroscience at the applicant institution, and the current proposal emerged directly from these research activities. The proposal also relates closely to the core leader's long-standing and fruitful collaboration with Dr. Masliah at UCSD, whose interests overlap widely with those of the other members of this program. Aim 2: Promote exchange among the projects and cores and monitor their progress toward the goals of the program. Research in this program will be carried out in five projects and four cores. Core A will organize regular meetings of all program personnel to review and discuss progress and adjust experimental strategies as needed. We will continue to encourage and facilitate the development of web pages and databases to promote the exchange of information among program members. Aim 3: Ensure the efficient generation and safe storage of data, the timely preparation of progress reports, and the dissemination of research findings and materials. This will include instruction in safety procedures, backup of electronic files, and assistance with manuscript preparation, travel arrangements, and shipments of materials. Aim 4: Arrange for external reviews of the research activities in the projects and cores and foster the acquisition of new approaches that could promote the progress of the program as a whole. The entire program will be reviewed annually by members of an external advisory board. Additional experts will be invited to provide ad hoc input on specific components. Aim 5: Play an active role in the further development of UCSF's Mission Bay campus and help the project/core leaders take advantage of the many conceptual and technological advances that will emerge at this site. During the preceding funding period, we relocated all three Gladstone Institutes, and with them most of this program, to the Mission Bay campus. By consolidating our laboratories, cores, and animal care facilities into the same research building located within walking distance from many UCSF colleagues with overlapping interests, this move further enhanced the strength and potential of our program. Our new environment will greatly contribute to the success of the research proposed in this renewal application. [unreadable] [unreadable] [unreadable]

Genetically modified mouse models have been very useful in research on neurodegenerative disorders, and the proposed program will take maximal advantage of this valuable resource. The main goal of Core C is to help the project leaders address their research questions conclusively with the minimal number of animals and experiments possible. To achieve this goal, we propose six specific aims. Aim 1: Maintain all animals in a manner that ensures the reliable selection of experimental and control groups for timely distribution to the different projects. Rodents will be housed in specific-pathogen-free animal care facilities in the Gladstone building at UCSF's Mission Bay campus and in the Medical Teaching Facility at UCSD's La Jolla campus. Detailed information on these animals will be recorded in a database that is accessible to all project leaders and their coworkers via the computer networks of the Gladstone Institutes and the University of California. Aim 2: Breed the different lines of genetically modified mice and determine the genotype of the offspring by polymerase chain reaction (PCR). The core personnel have ample experience in genotyping genetically modified mice and maintaining complex animal databases. Aim 3: Receive mice from the Gladstone microinjection facility and blastocyst core and identify new genetically modified lines of mice. Gladstone and UCSD maintain state-of-the-art facilities for transgene microinjection and gene targeting. Aim 4: Periodically analyze all lines of mice to confirm that levels and patterns of (trans)gene expression have remained stable. Cerebral levels of transgene expression will be tested by quantitative fluorogenic PCR. The distribution of transgene expression will be characterized in collaboration with Core D. We will also collaborate closely with Core D and veterinary staff to continually optimize our protocols for the anesthesia and perfusion of animals and for the removal, dissection, and proper storage of neural tissues. Aim 5: Ship mice to Dr. Masliah at UCSD and to investigators at other institutions, and provide the investigators with advice on the genotyping and husbandry of the mice. We have distributed our animal models to many institutions around the world and will continue to make our models available to the scientific community. Aim 6: Maintain a comprehensive mouse colony database to efficiently store and manage detailed information for current and future experiments. We will continue to optimize our database to store mouse colony data in a clear and accessible manner, minimize the effort and time required to enter data, and facilitate the development of individualized modules to best suit the needs of specific investigators and studies.

Within the overarching theme of "Proteinopathies of the Aging Central Nervous System," Project 5 has focused on Alzheimer's disease (AD). The insights we gained during the preceding funding period and the ever increasing threat AD poses to public health have motivated us to maintain this focus in the current proposal. We will also continue to utilize transgenic mice with neuronal expression of human amyloid precursor proteins (hAPP) and amyloid-p (A(3) peptides, because there is substantial evidence for mechanistically informative overlap between these models and the human condition. In our original application, we promised to shed light on the processes by which Ap elicits neuronal deficits. We found that neurons in the dentate gyrus and entorhinal cortex[unreadable]brain regions affected early and severely by AD[unreadable]are particularly vulnerable to the A|3-induced depletion of proteins that are critical for learning and memory. Several molecules were identified that may mediate this process. We also identified strategies to prevent A|3- induced neuronal deficits in hAPP mice. For example, reduction of the tau protein effectively prevented A(3- dependent memory deficits and molecular neuronal alterations. Although the mechanism underlying this striking rescue remains to be fully elucidated, we already know that it does not depend on changes in A(3 levels or deposition. Rather, tau reduction appears to prevent aberrant increases in neuronal network excitability. Our new proposal builds on the most promising findings we obtained during the preceding funding period. In Aim 1, we will examine whether A|3 affects vulnerable neurons directly or indirectly through changes in other regions from which these neurons receive excitatory inputs. In Aim 2, we will determine if the modulation of excitotoxicity-related neuronal or glial molecules can block Ap-induced neuronal overexcitation, eliminate aberrant network activities, and ameliorate behavioral abnormalities in hAPP mice. In Aim 3, we will assess whether tau reduction can prevent neuronal deficits also in mouse models of Parkinson's disease and Huntington's disease. Confirmation of these untested hypotheses should help elucidate the mechanisms that underlie A|3-dependent cognitive deficits and pave the way for the development of better treatments for AD and other neurological disorders. The proposed studies involve collaborative interactions with all other project leaders and depend on support from all four cores. The mechanistic and therapeutic insights we will gain in this project should help answer some of the key questions pursued in the other projects and, thus, will benefit the program as a whole.

DESCRIPTION (provided by applicant): The Gladstone Institute of Neurological Disease (GIND) at he University of California, San Francisco (UCSF) uses an interdisciplinary teann approach to the investigation of neurodegenerative disorders. Alzheimer's disease (AD) is the main cause of aging-related dementia. Because of the increasing longevity of populations around the world, AD is a medical problem of mounting social and economic impact. Indeed, the predicted increase in AD cases could make our health care system collapse in the not-too-distant future. Unfortunately, available treatments provide only partial and temporary symptomatic relief, and no strategy Is available to prevent or reverse AD. To counteract the rapidly rising prevalence of AD in the US, we propose to recruit an investigator who can advance our ability to develop novel approaches to the treatment and prevention of AD. The new investigator will help us expand our efforts to discover and validate novel drug targets for AD;to screen, develop, and evaluate drugs aimed at such targets;and to develop and explore novel bench-to-bedside models. The successful candidate will have a full faculty position as assistant investigator at Gladstone and a joint appointment as assistant professor in UCSF's Department of Neurology with membership in the University's academic senate. S/he will also have appointments in the Neuroscience Graduate Training Program (a branch Of the Program in Biological Sciences (PIBS) Graduate Training Program), the Biomedical Sciences Graduate Training Program, and the Medical Scientist (MD/PhD) Graduate Training Program at UCSF. These appointments provide ready access to talented graduate students. Although faculty with primary appointments at Gladstone are welcome to teach at UCSF, they do not have specific teaching requirements, which maximizes the amount of time they can devote to research. This arrangement will guarantee that the new investigator can devote at least 75% of time to research. As director of the GIND since its inauguration in 1998 and PI of a recently renewed program project grant focusing on proteinopathies of the aging central nervous system, the PI of this proposal has extensive experience in nurturing the careers of junior faculty and in integrating them into interdisciplinary teams tackling major unresolved biomedical problems. The above constellation makes our application fully responsive to this P30 mechanism and should ensure that we will achieve our goals within the proposed timeframe.

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. The early deficits of Alzheimer's disease (AD) likely result from disturbances of synaptic transmission. To understand AD pathogenesis, particularly changes of signal transduction pathways at the synaptic junction, post-synaptic density (PSD) proteins will be prepared from brains of human amyloid precursor protein (hAPP) transgenic mice and nontransgenic controls and used for phosphoproteomics analyses. PSD proteins will be prepared using sucrose density fractionation and then processed and differentially labeled with isotope-coded labeling reagents. The phosphorylated peptides in the sample will be enriched using metal affinity chromatography and both the phospho-enriched and the phospho-depleted portion of the sample will be further fractionated using other liquid chromatography approaches. The differentially labeled, fractionated samples will be analyzed on the ABI QSTAR Elite Q-TOF - LC/MSMS and the relative abundance of the different PSD protein and their phosphorylation levels will be compared between hAPP mice and controls. The whole project will be conducted in a collaborative manner using the phosphoproteomics analysis technical platform developed in the mass spectrometry facility at UCSF.

Within the overarching theme of "New Approaches to Heterogeneity in Dementia," Project 2 has focused on Alzheimer's disease (AD). The insights we gained during the preceding funding period and the ever increasing threat AD poses to public health have motivated us to maintain this focus in the current proposal. We will also continue to utilize genetically engineered mice with neuronal expression of human amyloid precursor proteins (hAPP) and amyloid-p (AP) peptides, because there is substantial evidence for mechanistically informative overlap between these models and the human condition. In our original application, we promised to shed light on the processes by which Ap elicits neuronal deficits. We found that neurons in the dentate gyrus and entorhinal cortex - brain regions affected early and severely by AD - are particularly vulnerable to the Ap-induced depletion of proteins that are critical for learning and memory. Several molecules were identified that may mediate this process. We also identified strategies to prevent Apinduced neuronal deficits in hAPP mice. Our new proposal builds on the most promising findings we obtained during the preceding funding period. Specifically, we discovered that the depletion of calciumdependent proteins and associated memory deficits in hAPP mice are likely caused by spontaneous nonconvulsive epileptiform activity in cortical and hippocampal networks. Memory deficits, depletions of calciumdependent proteins, and abnormal network activity could be prevented in hAPP mice through a genetic manipulation that blocks neuronal overexcitation. Independent lines of evidence suggest that epileptiform activity may also play a pathogenic role in humans with AD. We therefore postulate that aberrant excitatory neuronal activity might play an important causal role in the pathogenesis of Ap-induced cognitive impairments in hAPP mice and in AD. This hypothesis will be tested in three new specific aims. In Aim 1, we will examine whether markers of abnormal neuronal activity are increased in brains of" patients with mild cognitive impairment (MCI), AD, or other dementias. In Aim 2, we will test whether available anti-epileptic drugs can prevent or reverse EEC abnormalities in AD-related mouse models. In Aim 3, we will test whether any of these anti-epileptic drugs can also prevent or reverse cognitive deficits in these models. Confirmation of these untested hypotheses should help elucidate the mechanisms that underlie Ap-dependent cognitive deficits and pave the way for the development of better treatments for AD. Although there is plenty of ' evidence for a potential role of epilepsy in the development of AD, there appear to have been no rigorous clinical trials of anti-epileptic drugs in patients with MCI or early AD. The experiments described in our application could pave the path towards such a clinical trial and provide critical guidance in the selection of the most promising drugs. The proposed ADRC will provide an ideal environment for us to achieve these goals.

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. Cognitive deficits in Alzheimer's disease (AD) and related mouse models correlate closely with synaptic deficits. We recently discovered that tau reduction effectively prevents cognitive and synaptic deficits in human amyloid precursor protein (hAPP) transgenic mice, which have high levels of A[unreadable] in the brain and develop several AD-like abnormalities. Because synaptic functions critically depend on the posttranslational modification (PTM) of synaptic proteins, A[unreadable] may alter this process in a tau-dependent manner. To understand AD pathogenesis and how tau reduction prevents AD-like abnormalities, particularly changes of signal transduction pathways at the synaptic junction, synaptosomal and post-synaptic density (PSD) proteins will be prepared from brains of behaviorally tested hAPP/tau+/+, hAPP/tau[unreadable]/[unreadable], tau+/+ and tau[unreadable]/[unreadable]mice. Protein samples will be prepared using sucrose density fractionation and then processed and differentially labeled with isotope-coded labeling reagents. The phosphorylated peptides in the sample will be enriched using metal affinity chromatography, the O-GlcNAcylated peptides in the sample will be enriched using WGA affinity chromatography, and both the PTM-enriched and the PTM-depleted portions of the sample will be further fractionated using other liquid chromatography approaches. The differentially labeled, fractionated samples will be analyzed on the LTQ Orbitrap platform with ETD capabilities and the relative abundance of the different synaptosomal proteins and their PTMs will be compared between the four genotypes. The whole project will be conducted in a collaborative manner using the phospho- and O-GlcNAc-proteomics analysis technical platform developed in the mass spectrometry facility at UCSF.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. It was recently discovered that reduction of endogenous microtubule associated protein tau prevents the learning impairment usually seen in human amyloid precursor protein (hAPP) overexpressing mice, a mouse model of Alzheimer's disease. This prevention occurs despite a lack of tau pathology in the Alzheimer's mouse model and without a reduction in amyloid plaques in the brain of this mouse, implying that tau is downstream of amyloid beta pathology in this mouse model. This could occur through an active role of tau, via the formation of a toxic tau species, or a more passive role of tau, where removal of tau disrupts a key pathway in amyloid pathology. In order to explore the toxic tau hypothesis, we will prepare tau samples from hAPP and nontransgenic mice to compare the post-translational modifications in the tau samples by mass spectrometry. If a significant difference in post-translational modification of tau is seen, then biochemical methods will be used to explore the role of the modification, or modifications, in the amyloid pathology and behavioral deficits in the Alzheimer's disease mouse model.

DESCRIPTION (provided by applicant): While Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD) are distinct diseases; we suspect that they share certain pathogenic mechanisms, particularly abnormalities in the intracellular distribution of cargoes that regulate synaptic activity. In some forms of FTLD, these abnormalities may result from specific mutations in the microtubule-associated protein tau that imparts gains of adverse function. In AD, they may result from indirect pathogenic interactions between wildtype tau and amyloid-¿ (A¿) peptides, which are widely thought to cause AD. This etiologic difference may account, at least in part, for the fact that these conditions affect different neuronal populations. Identifying common molecular mechanisms in AD and FTLD might help preserve both of these neuronal populations. We hypothesize that impairments of axonal transport by A¿ and wildtype tau in AD and by mutant tau in FTLD require interactions of tau with the tyrosine kinase Fyn. We further hypothesize that A¿ and tau cause abnormal distribution of NMDA receptors (NMDARs) in dendritic spines, at least in part, by increasing the Fyn/ephB2 tyrosine kinase ratio in the postsynaptic density. These pre- and postsynaptic mechanisms are not mutually exclusive. They might result from Fyn-mediated effects on tau, tau-mediated effects on Fyn, or both, and could contribute to A¿- and tau-dependent neurological deficits in dementing disorders. To assess these possibilities, we propose to Aim 1. determine whether interactions between tau and Fyn are required for A¿ and tau to impair synaptic and behavioral functions; Aim 2. determine whether interactions between tau and Fyn are required for A¿ and tau to impair axonal transport and to reduce the ratio of intrasynaptic to extrasynaptic NMDARs; and Aim 3. determine whether ephB2 protects against A¿-induced neuronal dysfunction by increasing the ratio of intrasynaptic to extrasynaptic NMDARs and whether ephB2's protective capacity depends on kinase activity. We hypothesize that human A¿ oligomers and FTLD-mutant tau will no longer impair synaptic and behavioral functions when interactions (binding or phosphorylation) between Fyn and tau are blocked (Aim 1). We further hypothesize that this protective effect involves at least two mechanisms: improvements in the axonal transport of synaptic activity-related proteins (Aim 2a) and increases in the ratio of intrasynaptic to extrasynaptic NMDARs (Aim 2b). We suspect that the latter mechanism also accounts for the ability of ephB2 to protect against A¿-induced neuronal dysfunction (Aim 3). The proposed experiments will test a number of mechanistic hypotheses that have not yet been tested conclusively and address questions to which there currently are no firm answers. The answers we expect to obtain will shed light on the mechanisms that cause neurological decline in AD and FTLD and could help identify novel strategies to prevent and reverse these devastating diseases.

An organism's behavior can be considered the final output of its nervous system. Therefore, careful evaluation of behavior and of the factors that perturb it is essential to any comprehensive effort to understand the function or dysfunction of the nervous system. Many investigators at Gladstone/UCSF have developed complex rodent models to assess fundamental neuroscientific processes or major neurological diseases, but lack the expertise and equipment necessary for the careful behavioral and electrophysiological phenotyping of these valuable models. It would be expensive and inefficient for each of these investigators to hire trained behavioral neuroscientists and to purchase highly specialized equipment whenever they wished to evaluate a transgenic phenotype or assess the effects of a drug, surgical treatment or genetic intervention. The use of a core facility for such experiments is practical and highly in demand. To address this need, we propose to establish a Behavioral Core to achieve the following specific aims: 1) enable and support diverse behavioral studies ranging in focus from basic to disease-oriented neuroscience;2) encourage collaborations among core users around common themes and potentially related findings observed in different projects;3) provide specialized expertise in the phenotypical characterization of mouse models of human neurological disorders;and 4) assist in the design and execution of pharmacological interventions and preclinical drug trials aimed at distinct behavioral domains or abnormalities. The proposed core would support at least 26 NINDS-sponsored qualifying projects, as well as a variety of projects that are sponsored by Other NIH institutes. In addition, the core would help junior scientists to obtain preliminary data for their first NIH grant applications. By promoting interactions among its users, the core would also promote interdisciplinary research in behavioral neuroscience on a broader scale, linking investigations into the molecular, cellular, anatomical, genetic, and neurophysiological substrates of behavior and behavioral disorders. Because in neurology and psychiatry improvements in behavioral alterations often represent the most relevant outcome measures of therapeutic interventions, the core would also play a key role in advancing translational efforts at Gladstone and UCSF.

DESCRIPTION (provided by applicant): Tau, a major microtubule-associated protein (MAP), has been implicated in Alzheimer's disease (AD) and a variety of other neurological conditions collectively referred to as tauopathies. Remarkably, the functions tau fulfills in the adult brain and the exact roles it plays in the pathogenesis of these conditions remain uncertain. The current application aims to address these flagrant knowledge gaps by generating novel mouse models in which the expression of tau can be suppressed in specific brain regions and neural cell types in adult mice. Another important factor involved in AD is the amyloid-[unreadable] (A[unreadable]) peptide, which is derived from the amyloid precursor protein (APP). Crossing human APP (hAPP) transgenic (TG) mice with high levels of A[unreadable] in the brain onto a complete (Tau-/-) or partial (Tau+/-) tau-deficient background prevented most of their A[unreadable]-dependent abnormalities, including impairments in learning and memory, abnormal neural network activity, and various related biochemical and anatomical neuronal alterations. Tau reduction achieved this striking rescue without changing A[unreadable] levels or deposition in the brain. Furthermore, our hAPP/Tau+/+ mice never develop neurofibrillary tangles, and their wildtype murine tau is expressed at physiological levels, has normal solubility, and shows no evidence for the type of abnormal phosphorylation or aggregation seen in AD and mutant-tau TG models. Tau reduction is the most effective strategy to prevent A[unreadable]-induced neuronal deficits we have identified so far, but the safety of this approach in adult animals and the underlying mechanisms remain unknown. Our preliminary studies also revealed that tau reduction makes mice without hAPP/A[unreadable] expression more resistant against chemically induced seizures, suggesting a novel role for tau in the regulation of neuronal/synaptic activity. However, why and how tau reduction increases resistance to epileptic seizures is unknown. The above constellation of preliminary findings and unresolved questions underlines the significance and novelty of the experiments described in the current application. We currently have no effective ways to treat or prevent AD. If tau reduction was as efficacious and safe in preventing cognitive dysfunction in humans with AD as it is in hAPP mice, further exploration of this strategy might lead to a therapeutic breakthrough. In addition, tau reduction might be of benefit in other neurological disorders in which excitotoxicity plays a role. To address these challenges and opportunities, we propose the following specific aims. Aim 1. Decrease tau levels in brains of adult hAPP mice and NTG controls with viral vectors expressing anti-tau shRNA. Aim 2. Generate transgenic mice with regulatable expression of anti-tau shRNA. Aim 3. Analyze the models generated in Aims 1 and 2 behaviorally, anatomically and biochemically. Generation of the proposed mouse models should allow us to directly address the following questions to which there currently are no clear answers: Is tau reduction efficacious and safe when implemented in adulthood? In which brain region and neuronal cell types (e.g., neuronal vs. glial, excitatory vs. inhibitory neurons) does tau have to be reduced in order to prevent A[unreadable]- dependent cognitive decline and to increase resistance against chemically induced seizures? In the long run, the proposed models could help decipher the function(s) of tau in the adult brain, its role in disease-related neuronal dysfunction, and the underlying molecular mechanisms. PUBLIC HEALTH RELEVANCE: Amyloid-[unreadable] (A[unreadable]) is widely thought to cause Alzheimer's disease (AD). We recently discovered that reducing the protein Tau can prevent A[unreadable] from causing memory deficits and related neuronal impairments in AD mouse models. In this proposal, we will assess the efficacy and safety of this novel therapeutic strategy at the preclinical level.

DESCRIPTION (provided by applicant): This grant focuses on Alzheimer's disease (AD), the most frequent neurodegenerative condition, and on the question of how elevation of amyloid-beta (A-beta) levels in the brain contributes to its pathogenesis. Recently, we discovered that exploration of a novel environment causes putative DNA double strand breaks (DSBs) in neurons of wildtype (WT) mice. The DSBs were most abundant in memory centers and were repaired within 24 hours. Transgenic mice with neuronal expression of familial AD-mutant forms of the human amyloid precursor protein (hAPPFAD mice), which simulate several aspects of AD, had increased neuronal DSBs at baseline and more severe and prolonged DSBs after exploration. Treatment with the anti-epileptic drug levetiracetam suppressed aberrant network activity, normalized levels of DSBs and improved synaptic functions as well as learning and memory in hAPPFAD mice. In primary neuronal cultures from WT mice, exposure to human A-beta oligomers increased DSBs, and this effect was prevented by blocking NR2B-containing NMDA-type glutamate receptors. Thus, A-beta may exacerbate and prolong activity-related increases in neuronal DSBs, possibly as a result of synaptic and network dysfunction. By changing the expression of genes involved in cognitive functions and the regulation of neuronal activities, this process could promote a vicious cycle and contribute to the pathogenesis of AD. To test these hypotheses, we will (1) further characterize the nature and causes of neuronal DSBs in WT and hAPP-J20 mice, (2) determine the mechanisms by which levetiracetam counteracts A-beta-induced increases in neuronal DSBs, (3) determine whether neuronal DSBs and the associated histone variant YH2A.X specifically affect learning/memory and related gene expression, and (4) validate key findings in other mouse models and in humans with AD. The most novel aspects of this proposal include the hypotheses that physiological brain activity causes transient neuronal DSBs that DNA integrity in neurons is regulated by the activity of NR2B-containing glutamate receptors, and that pathologically elevated levels of A-beta alter these processes by changing the activities of synapses and neuronal networks. Innovative approaches include the use of chromatin immunoprecipitation and massively parallel DNA sequencing to identify the genes affected and the use of a novel APP knockin mouse that overproduces A¿ but not APP. Investigational anti-A-beta treatments have been associated with serious side effects that were probably unrelated to the reduction of A-beta levels per se. It is therefore desirable to identify alternative or complementary therapeutic strategies to make the brain more resistant to A-beta-induced neuronal dysfunction. Protecting the neuronal genome against A-beta's adverse effects could be particularly critical in this regard.

DESCRIPTION (provided by applicant): Cognitive functions such as learning and memory are of fundamental biological importance and diseases that affect these functions are among the most challenging biomedical problems of our time. We recently obtained evidence indicating that elevating levels of the pleotropic protein klotho enhances cognitive functions in mice and humans and can prevent synaptic and cognitive impairments in a transgenic mouse model simulating key aspects of Alzheimer's disease (AD). Klotho is a class I transmembrane protein whose ectodomain is released by proteolytic cleavage. In the periphery, it is produced predominantly by the kidney. Within the central nervous system (CNS), it is produced predominantly by the choroid plexus, with lower levels of expression seen in specific neuronal populations, particularly in the hippocampus. While diverse lines of evidence suggest that klotho delays aging and aging-related diseases, the functions it fulfills in the CNS and the roles it migh play in neurological disease are largely unknown. In our preliminary studies, increasing klotho production throughout the body of transgenic mice enhanced learning and memory not only in middle-aged and old mice, but also in young mice. Moreover, heterozygous human carriers of a KLOTHO variant (KL-VS) had elevated levels of klotho in the serum and performed better in a battery of cognitive tests than non-carriers. Again, this klotho effect was seen across a wide age range. Global klotho elevation also prevented AD-related synaptic, neural network and cognitive dysfunctions in human amyloid precursor protein (hAPP) transgenic mice. We hypothesize that klotho improves neural functions in both normal and diseased brains through a mechanism that is independent of aging. We also hypothesize that augmenting klotho or its effects can counteract the pathogenic effects of elevated amyloid-? (A?) levels in the brain and may help prevent AD. To test these hypotheses and help unravel the functions of this fascinating protein in the brain, we propose three specific aims. Aim 1 will explore whether increasing the peripheral production of klotho improves synaptic and cognitive functions or whether klotho levels must be elevated within the blood-brain barrier to achieve these beneficial effects. It will also ascertain the relative importance of secreted klotho released from choroid plexus epithelial cells into the cerebrospinal fluid versus full-length klotho expressed in specific neuronal populations. Aim 2 will examine the functional importance of klotho expression in the CNS at different life stages and define the potential pathogenic impact of klotho depletion from the brain, which occurs with aging and is prominent in humans with AD. Aim 3 will explore potential mechanisms by which increased klotho levels enhance synaptic and cognitive functions, with a particular emphasis on glutamate receptors and related signaling pathways. The proposed studies will fill important knowledge gaps and could provide critical guidance for the development of klotho-related therapeutics in independent projects.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, affecting over 5 million people in the U.S. No effective treatments are available to prevent, halt, or reverse the disease. Although aging is the most important nongenetic risk factor for AD, young mice have been used for the vast majority of preclinical studies in AD-related mouse models, mainly for practical and financial reasons. Even at young ages, these models share several pathological features with AD. However, they clearly do not simulate the full complexity of the human condition. We hypothesize that aged mouse models will simulate the human condition to a greater extent than young mouse models and that assessing candidate therapies in aged mouse models will better predict the efficacy of these therapies in later clinical trials. In this UH2/UH3 proposal, we will elucidate the phenotypic impact of natural aging in human amyloid precursor protein (hAPP) transgenic mice from line J20?one of the most extensively used AD-related mouse models. In addition, we will compare the efficacy of promising candidate therapies in young and old mice from this line. While strategies targeting amyloid-? (A?) have justifiably received considerable attention over the past decade, it is still unclear whether they will turn out to be both efficacious and safe in ongoing clinical trials. We reported in well-cited publications that treatment with the anti-epileptic drug levetiracetam and genetic reduction of tau ameliorate synaptic, network and cognitive dysfunction in hAPP-J20 mice, and these findings have been confirmed by other groups in independent mouse models. However, it remains to be determine whether these strategies also have beneficial effects in aging brains that have had longer exposures to pathologically elevated levels of A?. We therefore propose to investigate the efficacy of levetiracetam treatment and genetic reduction of tau in old hAPP-J20 mice.

The increased life expectancy of populations around the world has led to a striking increase in the prevalence of Alzheimer's disease (AD), the most common and costly among neurodegenerative disease. It has become increasingly evident that DNA damage is exacerbated in neurons of AD patients, which may lead to neuronal dysfunction and contribute to disease development and progression. In recent studies, we demonstrated an increase in DNA double-strand breaks (DSBs), the most severe form of DNA damage, in neurons of human amyloid precursor protein (hAPP) transgenic mice, which simulate several aspects of AD. Interestingly, we also discovered that hAPP mice showed a delay in the repair of activity-induced DSBs, when compared to wildtype mice, suggesting deficits in their neuronal DNA repair machinery. We subsequently discovered that breast cancer factor 1 (BRCA1), a well-described DSB repair factor and tumor suppressor, was depleted by approximately 50% in brains of hAPP mice and of patients with AD. Reducing BRCA1 levels in the dentate gyrus of wildtype mice to a similar extent by lentiviral expression of anti-BRCA1 shRNA increased the number of neuronal DSBs in this region and led to cognitive impairments. It is also possible that alterations in other DSB repair factors contribute to AD pathogenesis also. We hypothesize that efficient repair of DSBs is essential for proper neuronal function. We further hypothesize that deficits in neuronal DSB repair critically contribute to the accumulation of neuronal DNA damage in AD and that this process contributes to morphological and functional neuronal alterations that underlie the inexorable cognitive decline this disease causes. To test these hypotheses, we will determine which DSB repair factors are altered in postmortem MCI/AD tissue, examine the pathogenic mechanisms by which DSB repair factors are altered, investigate how DSB repair factors are regulated in neurons, and test whether elevating BRCA1 levels is of therapeutic benefit in AD-related models. Protecting the neuronal genome by increasing DNA repair is a novel strategy that might help prevent or slow the progression of AD and related disorders.

PROJECT SUMMARY  The  autism  spectrum  disorders  (ASDs)  affect  1%  of  the  world?s  population.  The  syndromes  in  this  diverse  family  of  disorders  share  three  core  features:  impaired  social  interactions,  communication  deficits,  and  repetitive  behaviors.  In  the  United  States,  1  in  68  children  are  now  diagnosed  with  ASDs,  a  drastic  increase  over  the  last  few  decades.  Despite  the  perceived  ?epidemic?  of  ASDs,  there  are  few  effective  treatments.  In  roughly  30%  of  patients,  ASD  is  associated  with  epilepsy  that  is  often  also  refractory  to  available  treatments.  Thus, there is an urgent need to develop better treatments for these challenging conditions.   The  core  symptoms  of  ASDs  are  common  in  Dravet  syndrome,  an  intractable  epilepsy  with  onset  in  early  childhood. We recently demonstrated that genetic reduction of tau, a microtubule-­associated protein implicated  in  neurodegenerative  disorders,  prevents  or  markedly  reduces  epileptic  seizures,  cognitive  deficits,  and  premature  mortality  in  a  model  of  Dravet  syndrome  (Scn1aRX/+  mice).  More  surprisingly,  we  found  that  tau  reduction  ameliorated  social  impairments,  communication  deficits,  and  repetitive  behaviors  in  these  mice.  Genetic reduction of tau also ameliorated similar communication deficits and repetitive behaviors in a separate  model  of  ASD  (Cntnap2?/?  mice).  Encouragingly,  genetic  tau  reduction  was  well  tolerated  throughout  the  lifespan,  tau  knockdown  initiated  in  adulthood  also  did  not  cause  obvious  adverse  effects,  and  complete  ablation was not necessary, as even partial tau reduction provided substantial benefit. These data led to our  central  hypothesis  that  tau  reduction  counteracts  ASD  pathogenesis  and  may  be  developed  into  an  effective treatment for several of these conditions.   However,  several  key  issues  must  be  resolved  before  this  strategy  is  ready  for  clinical  development.  To  determine whether ASD subtypes that do not include epilepsy may benefit from such a treatment approach, we  will  examine  whether  genetic  ablation  of  tau  prevents  or  reduces  autism-­like  behaviors  in  a  third  independent  mouse model of genetically determined ASD that does not develop epilepsy, Shank3B?/? mice. In addition, an  ideal ASD therapy would be effective even if it was administered after symptoms become apparent. To test this  possibility, we will knock down cerebral tau expression in Scn1aRX/+ mice with antisense oligonucleotides after  autism-­like behaviors have become manifest. Finally, we will test hypotheses about the molecular, cellular, and  circuit mechanisms by which tau reduction counteracts the core symptoms of ASD. In the long run, our aim is  to enable tau reduction to be developed into a treatment for multiple ASDs. By determining the consequences  of tau reduction on molecular regulators of development, circuit connectivity, and neuronal properties, we may  also  identify  additional  entry  points  for  therapeutic  intervention,  to  the  benefit  of  patients  affected  by  ASDs  or  other devastating diseases associated with tau-­dependent neural network dysfunction.       

PROJECT SUMMARY Our goal is to identify or generate small molecules that safely and effectively lower brain levels of the microtubule- associated protein tau (MAPT) and could be developed into drugs to treat Alzheimer?s disease (AD) and other tauopathies in which tau contributes to neurodegeneration and cognitive decline. Since tau also enables pathogenic effects of other AD-related proteins (e.g., A? and apoE4), reducing tau levels could lessen the effects of multiple drivers of this multifactorial condition and provide a more effective treatment than other monotherapies that have failed in clinical trials. In mouse models, tau lowering in the brain reduces network dysfunction (an early sign of AD) and cognitive decline. Prolonged reduction of cerebral tau levels by up to 75% with antisense oligonucleotides does not elicit overt adverse events in rodents and nonhuman primates. Thus, tau reduction should have limited on-target side effects. Since different forms of tau may contribute to neuropathogenesis, we aim to reduce overall tau levels with small-molecule drugs for the broadest benefit in diverse tauopathies. To discover small molecules that reduce neuronal tau levels, we used high-throughput screening to assess the efficacy of 20,000 structurally diverse compounds in brain neurons. One of these compounds, GL05520, reduced tau protein levels in a dose-dependent manner in rodent and human neurons and reduced neuronal MAPT mRNA levels by up to 75% without affecting transcripts encoding amyloid precursor protein or housekeeping proteins. Preliminary structure-activity relationship (SAR) studies of GL05520 identified three analogs that provide opportunities to improve the physiochemical and pharmacologic properties of the parent compound. Another compound from our primary screen, GL05522, also reduced neuronal tau protein levels but without affecting Mapt mRNA levels, suggesting a different mechanism of action. GL05522 is a potential backup compound if unforeseen liabilities limit the development of GL05520 analogs. Based on the drug-like structures of the tau reducers we identified, we propose a medicinal chemistry plan to improve their potency and pharmacological properties. Optimized compounds will be evaluated in target deconvolution studies to identify their likeliest mechanisms of action, which will facilitate further improvement by an additional round of medicinal chemistry directed at the identified target. Optimized compounds will be tested in standard pharmacokinetic studies of in vivo stability and brain bioavailability. The most promising compounds will be assessed in safety studies. Efficacy studies will test the ability of the lead compounds to reduce brain levels of nonfibrillar and fibrillar tau in rodent models. These studies could pave the way to the development of more effective therapeutics that could transform the treatment of AD and related conditions.

PROJECT SUMMARY Research on Alzheimer?s disease (AD) has strongly focused on pathological alterations detectable by histopathology or radiological imaging such as plaques and tangles. However, drug treatments targeting these alterations have not slowed cognitive decline in AD patients despite clear evidence for target engagement in the brain. Efforts to discover biomarkers for AD have also focused primarily on neuropathological alterations. To pursue novel directions and fill important knowledge gaps in the field, this proposal focuses on functionally relevant neural network abnormalities that likely underlie cognitive deficits and could promote neurodegeneration and AD progression through diverse mechanisms, including dysfunction of microglia, the innate immune cells of the brain. Previously, we found subclinical epileptic activity in patients with AD and in related mouse models. In the patients, the extent of such network abnormalities predicted cognitive decline. In the mouse models, prevention or reversal of the network dysfunction improved survival and reduced cognitive deficits. More recently obtained preliminary data suggest a novel pathogenic link between network and immune cell dysfunction. Suppressing epileptic activity reduced microglial activation in mice with elevated amyloid-b (Ab) levels in the brain. Knock-in mice with microglial dysfunction, induced by a human immune gene variant that increases AD risk, had increased and prolonged epileptic activity after challenge with an excitotoxin. Nonconvulsive epileptic activity in mice with Ab accumulation in the brain correlated with levels of specific inflammatory mediators in plasma. Based on these intriguing findings, we propose to test the novel and unifying hypothesis that aberrant neural network activity and immune dysfunction engage in a vicious cycle that promotes synaptic loss, the likeliest cause of cognitive decline in AD. Conceivably, either of these components can initiate the cycle, possibly at different times in different patients and in response to diverse triggers, including injury-induced surges in amyloid-b (Ab) or tau levels and aging-related alterations. Which of the components is triggered first may depend on a person?s genetics, including genes affecting microglial functions. We will use AD-related mouse models to begin to test these hypotheses at the preclinical level. Specifically, we will determine whether (1) suppression of neural network abnormalities reduces aberrant microglial activation and preserves synaptic density, (2) network dysfunction and synaptic deficits are reflected by immune markers in peripheral blood, and (3) microglial dysfunction contributes to aberrant network activity and synaptic loss. The proposed studies will help delineate the role of immune cells in AD-related network dysfunction and synaptic impairment. They may also identify potential new biomarkers, such as EEG signatures and peripheral blood alterations, and could help identify therapeutic strategies to modulate immune cell activities that may ultimately benefit patients with AD and people at risk of developing this disorder.

Project Summary/Abstract Alzheimer's disease (AD) is a major unresolved public health problem for which there are no effective treatments or means of prevention. Monotherapies aimed at individual molecular targets that have been implicated in the disease by pathological, biochemical or genetic evidence have so far had only minimal, if any, successes in clinical trials, despite reasonable evidence for target engagement. Diverse lines of evidence suggest that AD is a heterogenous disorder with a multifactorial pathogenesis involving diverse factors, including amyloid peptides, tau, aberrant immune cell activities, and in many cases also apolipoprotein (apo) E4, the major genetic risk factor for the disease. These and possibly other factors may ?conspire? to cause the degeneration of vulnerable neuronal populations through complex interactions that are difficult to predict based on current knowledge. We hypothesize that broad and unbiased screens in which many different genes are experimentally disrupted could shed light on these interactions and on the mechanisms underlying AD-related neurodegeneration in general. Such screens have already yielded tremendous insights into diverse biological functions in simple organisms. We hypothesize that the recently established CRISPR/Cas9 technology should make it possible to carry out such screens also in what is widely considered to be the most complex organ of mammalian species, the brain. However, so far, most CRISPR screens have been carried out in cancer cell lines, which probably cannot faithfully recapitulate the specialized functions and selective vulnerabilities of brain cells, which are most relevant to neurodegenerative diseases such as AD. Here we propose to adapt this methodology to enable unbiased genetic screens in brains of AD-relevant rodent models. In Aim 1, we will use CRISPR screening to identify genes that can protect cultured rat neurons against glutamate-induced neurodegeneration, a form of neurotoxicity that is of likely relevance to AD, vascular dementia, and many other brain disorders. In Aim 2, we will establish mixed neuronal/glial brain cell cultures from genetically modified mice expressing human apoE4 and human tau, treat them with A?, and use this model as a platform for a large-scale CRISPR screen to identify genes that can block the neurodegeneration that results from the interaction among these AD-relevant pathogens. In Aim 3, we will use adeno-associated viral vectors to advance this technology toward in vivo screens in brains of mouse models co-expressing human A?, apoE4 and tau. We will determine whether knockout of specific genes can promote neuronal survival in this AD-relevant context. Adapting this technology to perform large-scale, unbiased genetic screens in primary neurons and rodent brains will provide valuable guidance to the scientific community. Beyond that, our studies could identify new mediators of neurodegeneration and open new therapeutic avenues for the treatment of AD and related conditions.

OVERALL ? SUMMARY Alzheimer?s disease (AD) is a major unresolved public health problem. Efforts to prevent or stall this disease have failed, in good part because of inadequate understanding of its complex pathogenesis. Mounting evidence suggest that neural network dysfunction may underlie or promote AD-related cognitive deficits and contribute to disease progression. Yet, the causes and consequences of this dysfunction and the therapeutic potential of counteracting it remain sorely understudied. Therefore, the overarching goal of this program project is to decode the multifactorial etiology of AD-related neural network dysfunction and to leverage the novel mechanistic insights we will gain toward the development of better therapeutic strategies. Through collaborative interactions among four projects and two cores, our program will use systems neuroscience (neurophysiology and behavior) in combination with systems biology (single-cell transcriptomics and epigenomics), as well as neuropathology and improved mouse models, to determine how copathogenic interactions among apolipoprotein (apo) E4, amyloid-b (Ab), and tau cause neural network dysfunctions and cognitive decline in AD. An Administrative Core will coordinate all activities. Projects 1?3 will use novel mouse models of sporadic and familial AD to study interactions of different apoE isoforms with wildtype (WT) human tau (Project 1) or APP/Ab (Project 2), or among apoE4, Ab, and tau that is WT or bears disease-associated amino acid substitutions (Project 3). Project 4 will carry out single-nucleus transcriptomic and epigenomic analyses on postmortem brain tissues from deeply phenotyped human AD cases to gain novel insights into the multifactorial etiology of the human condition, validate leads from mouse studies, and encourage backtranslation into the models. An Integrative Data-Science Core will help us integrate results from all projects through innovative statistical modeling. This approach will reveal which aspects of human AD are most faithfully reproduced in the mouse models and help establish the causal drivers of cell-specific alterations in the human tissues, increasing the mechanistic resolving power of the latter studies. Therapeutic interventions in mouse models will determine whether reducing apoE4 expression in specific cell types can block copathogenic effects of apoE4 and tau on brain functions (Project 1), modulating the activity of specific interneurons can counteract copathogenic effects of apoE4 and APP/Ab (Projects 2 and 4), and knocking down tau can prevent and reverse brain dysfunction in models expressing all three pathogenic factors (Project 3). Through these highly cohesive efforts, our program will dissect the multifactorial interactions among AD-related pathogenic factors, define their relative contributions to the complex pathogenesis of brain dysfunctions, and help distinguish among neuropathological alterations that cause, result from, or are coincidental to neural network dysfunctions and cognitive decline. Sharing the diverse data sets we will generate and disseminating the novel integrative approaches we plan to develop for their analysis could enhance the progress of many other groups working in AD research and drug development or biomedicine in general.

CORE A ? SUMMARY The Administrative Core (Core A) will oversee and coordinate all administrative and research activities within this program. All project leaders have primary faculty appointments in the Gladstone Institute of Neurological Disease (GIND), which Dr. Mucke has directed since its inauguration in 1998. His leadership of this institute, joint appointments in the Department of Neurology and various graduate training programs at UCSF, and long record of service on relevant national and international advisory committees put him in an excellent position to lead this core. He has collaborated with Drs. Yadong Huang (Project 1) and Jorge Palop (Project 2) on Alzheimer?s disease (AD) research for roughly two decades, as highlighted by many widely cited, coauthored publications in this field, and recently recruited Dr. Ryan Corces (Project 3 and Core B) to Gladstone and UCSF. For many years, he has also interacted closely with Drs. Katie Pollard and Alex Pico (Core B) through their leadership roles in Gladstone?s Bioinformatics Core. The well-established relationships among project and core leaders will greatly help Core A to accomplish its overall goal of overseeing, coordinating, and supporting studies proposed in all projects and cores. This program project will focus squarely on AD and places great emphasis on cohesiveness and collaborative interactions among projects. Core A will critically support and enforce these features and help ensure that our program not only addresses truly important biomedical problems, but also creates diverse opportunities for scientific growth and advancement in AD-relevant research for all of its members, ranging from graduate students and postdoctoral fellows to junior and senior faculty. Specifically, Core A will develop and implement program-wide procedures to ensure optimal levels of scientific rigor, data safety, cost effectiveness, and accountability across all four projects and the scientific core; encourage and facilitate collaborative interactions among all project and core leaders as well as between program members and other groups with related interests at Gladstone, UCSF, and beyond; survey relevant resources and fields of research and alert project and core leaders to conceptual or technological developments that could enhance the pursuit of their specific aims; form an external advisory committee and arrange regular meetings to review the program and provide constructive feedback to all project and core leaders; prepare progress reports; coordinate publications among program members; and collaborate with Core B to ensure the efficient dissemination of program-generated data, reagents, and research findings to the broader scientific community.

PROJECT 3 ? SUMMARY Tau contributes to Alzheimer?s disease (AD) and many other brain diseases. However, it is uncertain how tau causes neuronal dysfunction and degeneration, in part because experimental models are not optimized to compare the relative pathogenicity of different tau species in disease-relevant contexts. Mutations in MAPT, the gene encoding tau, cause frontotemporal lobar degeneration (FTLD) instead of AD. In contrast, the rare A152T variant of tau increases risk for both types of diseases. These associations merit further exploration, especially as models expressing FTLD-mutant tau are widely used to study tau in AD and to develop novel AD treatments. Clinical AD onset is preceded by abnormal accumulations of amyloid-b (Ab) peptides in brain, and many AD patients have at least one apolipoprotein (apo) E4 allele, the most important genetic risk factor for AD. Therefore, we will generate new mouse models combining human Ab and apoE4 expression with near-physiological levels of human tau that is (1) wildtype, as in most AD patients, (2) carries the A152T substitution, which increases AD risk, or (3) bears the P301S mutation, which causes FTLD and is widely used in overexpression models. Comprehensive functional, pathological, and transcriptomic analyses of the new models, in collaboration with Projects 1, 2, and 4 and Core B, should yield new insights into differential effects of these tau species and their roles in the pathogenesis of dementia. We hypothesize that tau species that increase AD risk or cause FTLD differ in their effects on the integrity and function of neurons and neural networks, especially when combined with A? and apoE4. Until we know which forms of tau are most pathogenic in different conditions, the most pragmatic therapeutic approach to tau in our view is partial reduction of overall tau levels, which is well tolerated and has benefits in conventional models. We will therefore use tau-targeting antisense oligonucleotides (ASOs) to (1) determine whether reducing human tau can diminish neural network dysfunction, neurodegeneration and cognitive decline in models co-expressing human Ab and apoE4, (2) define the optimal timing for this intervention, and (3) reveal the most critical co-pathogenic mechanisms of tau. We hypothesize that ASO- mediated tau reduction will diminish not only tau pathology in one or more of the new models, but also synaptic deficits, neural network dysfunction, and cognitive deficits, even though it is unlikely to reduce amyloid deposition or plaque-associated microgliosis. This experiment should help determine when tau reduction must be initiated relative to the onset of cognitive deficits for it to have therapeutic benefits in AD-relevant contexts. Single- nucleus/single-cell transcriptomic analyses will be used to identify cell-type-specific gene expression changes and novel molecular and cellular mechanisms that may mediate pathogenic effects of tau or beneficial effects of tau reduction. These analyses will also help Projects 1 and 2 distinguish between pathogenic mechanisms of apoE4 and Ab that do or do not depend on tau and could identify novel molecular and cellular mechanisms that mediate tau sequence-specific effects in the absence or presence of AD-relevant co-pathogens.