Stephen M. Strittmatter - US grants

The axonal growth cone is critical to the development and maintenance of the uniquely complex and specific synaptic connections which underlie nervous system function. GAP-43 is a neuronal protein whose expression is closely correlated with growth cone activity in development and regeneration, and with synaptic plasticity. Homologous recombination will be employed to develop animals and cell lines incapable of expressing GAP-43. This technique relies on crossing over between an exogenous mutated DNA molecule and the endogenous gene to replace a normal allele with a mutated copy. In animals and cell lines with deletion of GAP-43, the importance of the protein for growth cone function will be analyzed. This information will allow creation of models and perhaps rational therapies for some disease states in which neural development, regeneration or plasticity are flawed. Examples include developmental disorders, neurite regeneration in neuropathy and perhaps in recovery from central injuries like stroke, and learning in memory disorders with impaired synaptic plasticity.

Nervous system function is absolutely dependent on the complex pattern of synaptic connectivity formed during development, and maintained during neuronal regeneration. The distal tip of the extending neurite, the growth cone, is in large part responsible for distinguishing between myriad pathways and targets. The growth cone membrane contains very high levels of the GTP-binding protein, G-o This concentration suggests that G-o participates in a signal amplification system allowing a single filopodial contact with a shallow gradient of attractant or repulsive factors to dramatically alter growth cone motility. To test this hypothesis, mutated alpha subunits of G proteins which are constituitively activated or inactivated will be expressed in various neuronal cells and the effect on neurite outgrowth will be evaluated. Intraneuronal proteins which determine a cell's intrinsic growth potential might function via G-o. The protein GAP-43 may be one such molecule, since it is highly induced during neuronal development and regeneration, and is localized to the growth cone membrane. GAP-43 can activate purified G-o and this project will examine whether, within cells, GAP-43 modulates second messenger systems by activating G proteins. The two second messenger systems to be examined are the adenylate cyclase system of epithelial cells, and the phosphoinositide-mediated chloride channel response of the X. laevis oocyte. There are many extracellular molecules which alter growth cone motility. One group of membrane-bound factors acts extracellularly to collapse growth cones at synaptic targets, and preliminary evidence indicates that their mechanism of action may also involve G-o. G protein-coupled receptors for such molecules will be searched for in an expression cloning system. Thus, a focus on G protein transduction in the growth cone may explain the action of known regulators of growth cone function, and allow identification of novel proteins in the growth cone. Such a detailed molecular analysis of the growth cone should have broad clinical applications. Failure of these mechanisms might underlie unexplained developmental disorders of the nervous system. By pharmacologically enhancing the normal function of such pathways, repair of the adult nervous system after multiple types of injury might be increased. It is also probable that the same system contributes to synaptic plasticity in the adult nervous system, and a failure of this system might therefore account for some unexplained degenerative disorders of the nervous system.

The axonal growth cone is critical to the development and maintenance of the uniquely complex and specific synaptic connections which underlie nervous system function. GAP-43 is a neuronal protein whose expression is closely correlated with growth cone activity in development and regeneration, and with synaptic plasticity. Homologous recombination will be employed to develop animals and cell lines incapable of expressing GAP-43. This technique relies on crossing over between an exogenous mutated DNA molecule and the endogenous gene to replace a normal allele with a mutated copy. In animals and cell lines with deletion of GAP-43, the importance of the protein for growth cone function will be analyzed. This information will allow creation of models and perhaps rational therapies for some disease states in which neural development, regeneration or plasticity are flawed. Examples include developmental disorders, neurite regeneration in neuropathy and perhaps in recovery from central injuries like stroke, and learning in memory disorders with impaired synaptic plasticity.

The growth cone at the distal tip of the extending axon is a specialized sensory apparatus which transduces extracellular signals into growth along appropriate pathways to correct synaptic targets. Its proper function is crucial to nervous system development and hence function. This proposal seeks a molecular understanding of the signal transduction mechanisms at the neuronal growth cone. Particular emphasis is focused on the action of collapsin-1, a member of the semaphorin family of proteins recently recognized to inhibit axonal extension and terminal arborization. The essential role of monomeric G proteins of the rho family and of heterotrimeric G proteins in dorsal root ganglion (DRG) growth cone signal transduction will be examined by introducing mutant activated and dominant negative proteins. The hypothesis that GAP-43 augments sensitivity of the growth cone to extracellular signals will be examined in cultured DRG neurons from mice with a targeted deletion mutation in the GAP-43 gene. A recently identified family of neuronal CRMP proteins appear to be required for collapsin-1 inhibition of growth cone function. This project will further define CRMP action by identifying proteins interacting with CRMP, exploring enzymatic activities of CRMP and comparing the properties of different CRMP family members. Neurite outgrowth and collapsin-1 sensitivity of cells overexpressing different forms of CRMP will be examined. A collapsin-alkaline-phosphatase fusion protein will be used to identify collapsin binding proteins which may serve as collapsin receptors in the neuronal growth cone. Once such receptors are identified their interaction with CRMP, GAP-43, and G proteins can be delineated. Together these experiments provide a detailed description of the molecular events which underlie the growth cone responsiveness to extracellular inhibitory signals such as collapsin-1. Knowledge of these pathways provides necessary groundwork for understanding the pathophysiology of human developmental abnormalities of the brain. The same mechanisms are likely to function during adult nervous system injury and improve function in degenerative neurologic diseases.

DESCRIPTION (provided by applicant): Nervous system function is critically dependent on the pattern of connectivity that is defined during development, modified by experience and perturbed in disease. We will focus on three specific aspects of axon growth cone signal transduction in the proposed studies. The first aim is to provide a comprehensive understanding of Sema3 signaling. Because of the central role of class 3 Semaphores as growth-cone-collapsing agents and of the unique nature of the Plexin cytoplasmic domain, this work will contribute in a central way to understanding axon guidance. By exploring RGM/Neogenin signaling in the second aim, the basis of action for a recently defined class of axon guidance factor and the signaling specificity of Neogenin versus DCC will be defined. The understanding of basic axon guidance mechanisms derived from Aims 1 and 2 lays the groundwork for ongoing studies of axonal misdirection and failed growth in human disease states. Any attempts at clinical intervention must draw on such a fund of knowledge. In the third aim, a specific human disease that may be linked to altered axon guidance is considered. Such work may provide an understanding of how genetically determined alterations in axonal connectivity can lead to human disease. While LGI1 mutation in ADPEAF is not a common form of epilepsy, it is likely that parallel pathways are causative in a larger number of sporadic cases of epilepsy. Together these studies will provide novel molecular insights into how nervous system connectivity is assembled or misassembled during development. These findings will have implications for the understanding developmental disorders of the brain arid will aid in the design of therapies based on axonal growth and regeneration

DESCRIPTION (provided by applicant): Disability in clinical neurology frequently results from axonal damage rather than neuronal cell loss. Spinal cord injury is an example where successful axonal regeneration holds the potential for extensive functional recovery. Recent work has uncovered a number of the molecular determinants of axonal regeneration. The CMS myelin proteins, Nogo-A, MAG and OMgp, inhibit axonal growth in tissue culture. All three proteins bind to one axonal receptor, the Nogo-66 receptor (NgR), to initiate a signaling cascade that inhibits axonal extension. Reactive astrocytes secrete chondroitin sulfate proteoglycans (CSPGs) that also limit axonal growth, especially near injury sites. The growth of adult axons is dependent on cell autonomous positive factors as well as the presence of extracellular inhibitory proteins. Peripheral axotomy successfully induces a regeneration gene program, while central axotomy of the same neuron has much less effect. Proteins that are induced selectively after peripheral axotomy but not central axotomy include GAP-43, SPRRIA and Fn14. This project seeks to examine the role of myelin inhibitors and CSPGs in CMS axonal regeneration using genetic and molecular means, with spinal cord injury as a model. The role of SPRR1A and Fn14 in peripheral nerve regeneration will also be assessed genetically, and their mechanism of axon growth enhancement will be probed. Together, these aims seek to advance our molecular understanding of the extrinsic and intrinsic factors that determine the success of axonal regeneration, and have the potential to provide new therapeutic modalities to improve nervous system function after a wide range of chronic neurologic injuries.

DESCRIPTION (provided by applicant): Many nervous system injuries sever distal axons, but leave proximal neuronal cell bodies intact. Recovery of function is largely dependent on the degree of axonal regeneration. After CNS damage, as occurs in spinal cord injury, axonal regeneration is routinely dismal. Nogo was recently identified as a CNS myelin-derived inhibitor of axonal regeneration. An understanding of the mechanism of Nogo action is critical to assessing and modulating its physiologic role. Here, we seek to characterize a Nogo receptor and to define its physiologic role. In preliminary studies, we have characterized a high affinity Nogo-66 binding site and obtained functional evidence that a novel brain-specific leucine-rich repeat protein mediates Nogo action. In the first specific aim, we will characterize this receptor protein by determining structure-function relationships for Nogo binding and axon repulsion, and by analyzing expression patterns before and after neural trauma. The absence of an intracellular signaling domain in this receptor protein implies an interaction with a second transmembrane receptor subunit responsible for transduction. The second aim is to identify this additional receptor subunit through its affinity for the identified Nogo binding receptor protein and by analyzing Nogo-induced tyrosine phosphorylation. Thirdly, a strain of mice lacking the Nogo receptor protein will be generated by homologous recombination. Neuronal development and the degree of anatomical recovery after neuronal trauma in these mice will be characterized. The identification of Nogo and its receptor provides the opportunity for novel and rational therapeutic interventions in spinal cord injury. This research may also prove applicable to a wide range of chronic brain axonal injuries, such as traumatic brain injury, white matter strokes and chronic progressive multiple sclerosis.

DESCRIPTION (provided by applicant): A major neurological complication in very low birth weight infants is the development of periventricular leukomalacia and ventriculomegaly, with subsequent cerebral palsy and cognitive impairment. The primary pathological hallmark of this condition is the loss of myelin and oligodendrocytes. Here we seek to explore the cellular and molecular consequences of oligodendrocyte loss during this stage of neurodevelopment. In particular, we hypothesize that oligodendrocyte loss will lead to secondary changes in axonal morphology and hence behavior. A rodent model for periventricular leukomalacia involving chronic sublethal hypoxia will be characterized in detail for alterations in oligodendrocyte/axon interaction and axonal morphology. These changes will be correlated with behavioral deficits. Preliminary data implicate suppression of the oligodendrocyte proteins Nogo and MAG during hypoxia in the development of ectopic axonal sprouting. To verify the role of these particular proteins, mice lacking Nogo, MAG or the NogoReceptor will be compared with those exposed to chronic sublethal hypoxia. Similarities between the mutant mice and the hypoxic mice will support the hypothesis that these proteins contribute to the pathophysiology of periventricular leukomalacia. In addition, the synergistic effects of hypoxia plus Nogo, MAG or Nogo receptor gene deletion will be analyzed. This may provide further support for the hypothesis that hypoxia-induced deficits in oligodendrocytes lead to maladaptive changes in axonal morphology. Together these studies should advance our current understanding of the interactions between oligodendrocytes and axons under conditions similar to those experienced by very low weight birth weight infants. Such knowledge may lead to the development of novel therapeutic approaches aimed at lessening the long-term neurological sequela of prematurity.

DESCRIPTION (provided by applicant): The functioning of the nervous system depends on its complex pattern of connections. Axonal connections are initially set up during development by cues, which guide axons along specific pathways and then specify connection sites within the target region. In recent years, dramatic progress has been made in identifying such cues molecularly. Based on this progress, current studies are elucidating the molecular logic of extracellular signals that set up connectivity, as well as intracellular pathways that interpret these signals. In the adult central nervous system, damaged axonal connections do not regenerate, even though peripheral nerves can regenerate efficiently. This is due at least in part to CMS inhibitors, some of which have now been cloned. This meeting will bring together the fields of development and regeneration, with the goal of facilitating synergistic overlaps in techniques, molecules and biology. Topics will include basic research on molecular cues, and resulting new approaches toward therapeutic axon repair.

Cerebrovascular accidents of ischemic origin constitute a major cause of persistent neurologic deficits. The development of immediate therapy for brain ischemia has focused on thrombolytic or neuroprotectant mechanisms. A complimentary treatment approach for lessening the stroke-related disability is to promote the rearrangement of axonal connectivity amongst surviving neurons. It is known that the plasticity of adult neuronal connections is quite limited. Previous work has indicated that CNS myelin proteins play a role in limiting the degree of axonal regeneration after traumatic transection in the spinal cord. In particular, the proteins Nogo, MAG and OMgp all bind to a Nogo Receptor (NgR) to inhibit axonal growth. Here, we will examine the relevance of this pathway in recovery from ischemic stroke and we will use this knowledge to develop methods for promoting neurological function after focal brain ischemia. Preliminary data demonstrate that NgR antagonism by genetic or pharmacological means can produce enhanced recovery from stroke. This is separate from neuroprotection. We will extend these studies in . several directions. First, we will consider the therapeutic window for this effect. How long and in what dose is NgR antagonism effective? Are the beneficial effects persistent or reversible and are they age- dependent? We will also explore the mechanism of these effects by characterizing the axonal connectivity of selective fiber systems and cortical maps. Both to develop alternative pharmacologic approaches and to verify the NgR antagonistic mechanism, additional treatments that alter upstream and downstream molecules in the NgR pathway willbe studied. Overall, this work will determine the extent to which enhanced axonal plasticity created by blockade of NgR function improves stroke recovery.

DESCRIPTION (provided by applicant): A major neurological complication in very low birth weight infants is the development of periventricular leukomalacia and ventriculomegaly, with subsequent cerebral palsy and cognitive impairment. The primary pathological hallmark of this condition is the loss of myelin and oligodendrocytes. Here we seek to explore the cellular and molecular consequences of oligodendrocyte loss during this stage of neurodevelopment. In particular, we hypothesize that oligodendrocyte loss will lead to secondary changes in axonal morphology and hence behavior. A rodent model for periventricular leukomalacia involving chronic sublethal hypoxia will be characterized in detail for alterations in oligodendrocyte/axon interaction and axonal morphology. These changes will be correlated with behavioral deficits. Preliminary data implicate suppression of the oligodendrocyte proteins Nogo and MAG during hypoxia in the development of ectopic axonal sprouting. To verify the role of these particular proteins, mice lacking Nogo, MAG or the NogoReceptor will be compared with those exposed to chronic sublethal hypoxia. Similarities between the mutant mice and the hypoxic mice will support the hypothesis that these proteins contribute to the pathophysiology of periventricular leukomalacia. In addition, the synergistic effects of hypoxia plus Nogo, MAG or Nogo receptor gene deletion will be analyzed. This may provide further support for the hypothesis that hypoxia-induced deficits in oligodendrocytes lead to maladaptive changes in axonal morphology. Together these studies should advance our current understanding of the interactions between oligodendrocytes and axons under conditions similar to those experienced by very low weight birth weight infants. Such knowledge may lead to the development of novel therapeutic approaches aimed at lessening the long-term neurological sequela of prematurity.

[unreadable] DESCRIPTION (provided by applicant): The failure of adult CNS axons to sprout and to grow after injury limits recovery in a broad range of neurological conditions, including spinal cord injury (SCI), stroke, head trauma and chronic multiple sclerosis. Over the last 5 years, significant progress has been achieved towards defining the molecular pathways limiting adult CNS axon growth and towards translating this knowledge into therapeutic opportunities. During the last cycle of this project, we identified the Nogo-66 Receptor (NgR) protein as a ligand-binding receptor for the myelin-derived proteins, Nogo and MAG. Pharmacological perturbation of NgR function or genetic disruption of the NgR locus allows an enhanced degree of axonal growth for certain fibers in the post-SCI and post-stroke nervous system. We showed that such CNS fiber growth is associated with improved behavioral recovery of motor function. Success in promoting recovery from injury led us to consider of the physiological rather than pathological role of myelin inhibitors. We found that experience-dependent cortical plasticity is gated by NgR-dependent mechanisms in the absence of injury. While this work has defined one pathway regulating adult CNS axonal sprouting, it has also framed crucial questions about NgR function that will be addressed in the second cycle of this project. In the proposed work, we will examine the anatomical, molecular and temporal specificity of NgR action in plasticity and regeneration. We will consider the cellular basis for NgR-gated brain plasticity. The current hypothesis is that NgR functions to "lock" neuronal elements into place and to prevent anatomical rearrangements. The molecular basis for NgR signal transduction will also be probed biochemically and genetically. Together, these studies should provide critical insights into the mechanism and extent of NgR contribution to axonal plasticity and regeneration in the adult CNS. Such data will inform our understanding of the stability of connectivity within the CNS and illuminate the possibility of harnessing this knowledge for therapeutic interventions. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The Yale Program in Cellular Neuroscience, Neurodegeneration and Repair (CNNR) was co-founded by the PI and Co-PI four years ago with the goal of linking basic research in neuronal cell biology with efforts to advance pathophysiological understanding of neurodegenerative disease and neural repair. We hypothesized that strong synergies will result from bringing together researchers in disease-oriented science with experts in the cell biology of the nervous system. To date we have recruited 5 new faculty into contiguous laboratory space, interconnected them with other neuroscientists at Yale, and established an advanced core facility for imaging and functional genomics of neurons. Here, we propose the recruitment of two additional Assistant Professors into the Yale CNNR in order to fill key unmet needs in our Program. The vigorous research programs already launched by existing faculty will interdigitate with those of the two new Recruits and will foster their success and the overall success of the CNNR. It is anticipated that one Recruit will fill a crucial role by providing electrophysiological expertise at the single cell level coupled with optical techniques. A Recruit using these methods will have a fertile ground for selecting molecular biology collaborators from amongst the existing CNNR faculty. Such a Recruit will also provide a new dimension of ongoing explorations of neuronal function in health and disease. The second Recruit is expected to lead a program centered on the cell biology of Alzheimer's Disease. While this is the most prevalent neurological condition, Yale has no laboratory focused primarily on this disorder. With this Recruit, the Yale CNNR will allow the wealth of nerve cell biology at Yale to be applied to Alzheimer's Disease. Importantly, the CNNR environment will provide the new Assistant Professor with a strong basic science expertise and fresh ideas towards an improved pathophysiological understanding, and eventually a treatment, of this condition. Yale has committed laboratory space and will provide two-thirds of the funds for the new Recruits, but the proposed support is essential to provide the resources necessary for recruitment of two new Assistant Professors into the Yale CNNR.

Damage to axonal connections is responsible for symptoms in a broad range of neurological conditions. The paralysis and sensory dysfunction of spinal cord trauma epitomize the lack of spontaneous recovery from such deficits. Poor recovery is observed despite the strong intrinsic capacity for growth in response to axotomy. The re-extension of injured axons is restricted by inhibitory molecules in the extracellular milieu of the injured CNS, including Nogo and Chondroitin Sulfate Proteoglycans (CSPGs). Blockade of these inhibitors releases a degree of axonal growth and improves functional outcomes from neurological injury in preclinical studies. We hypothesize that modification of additional molecules and combinations of molecules will accomplish greater adult CNS axonal growth and greater functional recovery. Members of the ephrin, RGM and Sema protein families are candidate adult CNS axonal growth inhibitors. We will utilize specific genetic tools to assess their role in vivo in limiting axonal growth and recovery from spinal cord injury. It is crucial to examine the interactions between these proteins in vivo to determine whether they function redundantly, additively or synergistically. By examining mice lacking functional genes for these selected inhibitors, we will reveal whether specific combinations are particularly advantageous for disinhibiting adult CNS axonal growth and improving neurological recovery. Together these studies will define opportunities for enhancing axonal growth in the injured adult brain and spinal cord. This work is critical to optimize the opportunity for development of clinical methods to increase axonal growth and improve functional recovery from neurological damage.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is one of the largest unmet medical need today. Epidemiologic data indicate that this need will mushroom in the coming decade unless new therapeutic options are identified. Pathological and human genetic studies have made substantial progress in supporting an "Amyloid Hypothesis" of AD and efforts to remove amyloid-A[unreadable] (A[unreadable]) immunologically, to block A[unreadable] production by secretases and enhance A[unreadable] degradation are advancing. However, a cell biological understanding of how A[unreadable] is toxic for neurons has lagged. Recent studies have focused attention on soluble oligomers of A[unreadable] as culprits in the disease process in both correlative and functional studies. The neuronal targets by which oligomeric A[unreadable] mediates neuronal dysfunction are unknown but their identification would provide a novel pathway in drug development. In Preliminary Studies, we have identified the cellular Prion Protein (PrPC) as an A[unreadable]-oligomer receptor by expression cloning. Synaptic responsiveness in brain slices from young adult PrP null mice is normal, but the A[unreadable]-oligomer blockade of long-term potentiation (LTP) is absent. Thus, PrPC is a mediator of A[unreadable]- oligomer induced synaptic dysfunction in vitro. Here, we will determine if the same molecular interaction plays a role in A[unreadable]-induced memory dysfunction and neurodegeneration. We will determine the requirements for specificity in this interaction and explore downstream signaling pathways. Together, this work holds the promise of validating a novel therapeutic target for AD, one that is based not on A[unreadable] levels but on preventing the deleterious actions of A[unreadable]-oligomers on neurons through a specific binding site. PUBLIC HEALTH RELEVANCE: Alzheimer's disease extracts a massive health care burden but there are few treatments. Molecular studies have revealed that the amyloid- [unreadable] peptide plays a key role in the brain malfunction. In this project, we test the hypothesis that cellular Prion protein mediates amyloid- [unreadable] action. If validated, this hypothesis predicts that Prion protein will be a novel target for therapeutic development for Alzheimer's disease.

DESCRIPTION (provided by applicant): Dementia afflicts a large and rapidly growing number of individuals and their families. A majority of cases are caused by Alzheimer's Disease (AD), but substantial percentages are attributed to other causes. Frontotemporal dementia (FTD) is the third most common cause of neurodegenerative dementia, after AD and Diffuse Lewy Body Disease. The clinical features of FTD include memory deficits, behavioral abnormalities, personality changes and language impairments. Amongst FTD cases, the most common cause has been recognized recently to be inherited mutation in the Progranulin (PGRN) gene. This genetic insight has the potential to lead to rational and effective therapies for FTD. The success of such translation to clinical benefit requires understanding how PGRN acts in the brain and how its mutation results in disease. The current proposal seeks to define PGRN action using molecular and genetic tools. The long- term goal is the identification of targets for FTD treatment and prevention. In Preliminary Studies, we examined PGRN binding to the cell surface and have identified a high affinity neuronal binding site. The 50% PGRN decrease causative in FTD cases is mimicked in mice lacking one copy of the PGRN gene, and is full corrected by genetic ablation of the binding partner. These findings indicate that the PGRN binding protein has a crucial in determining PGRN levels and hence FTD. In the proposed Specific Aims, the relevance for this binding partner for FTD pathology and neurological dysfunction will be assessed in preclinical studies. This work may provide new avenues for therapeutic intervention for FTDs which currently have not treatment.

DESCRIPTION (provided by applicant): The CNS of adult mammals, as compared to the peripheral nervous system or nervous system of other organisms, has extremely limited capacity for axon regeneration and synapse reformation. Specific factors limiting adult mammalian regeneration of axons and synapses have been identified, but they provide an incomplete explanation for poor adult mammalian CNS regeneration. We propose the first genome-wide shRNA-based screen for endogenous genes limiting the repair of axons and synapses in the mammalian CNS. In the first R21 Phase of this project we will develop a tissue culture screening method for axonal and synaptic regeneration relevant to cortical neurons. In Preliminary Studies, we conducted a pilot loss-of-function screen with 1087 shRNAs targeting mouse phosphatases, monitoring total regenerative axon length. We will confirm activity and assess their interaction with previously identified extrinsic inhibitors of axonal regeneration. We also propose to extend our preliminary analysis of the phosphatome in regeneration by imaging growth cones and categorizing their morphology. In addition, we will assess the reformation of synapses during regeneration, and demonstrate its utility in the phosphatome screen of regeneration. Finally, we will demonstrate that in vivo axonal regeneration is regulated by at least one novel phosphatase identified in the pilot screen. After accomplishing these milestones, we will screen the mouse genome for effects on CNS axonal and synapse regeneration during the R33 Phase. The cortical scrape assay will be probed at the genome-wide level with lentiviral shRNAs. Metrics for axon regeneration, growth cone morphology and synapse reformation from the R21 phase will be employed. The focus will be to identify all genes that naturally limit corticl axon and synapse regeneration, by demonstrating enhanced regeneration after knockdown of individual genes. We will determine if newly discovered endogenous brakes on regeneration are dominant with respect to the action of extrinsic inhibitors. Together, these Aims will provide the first functional genomic assessment of axonal and synaptic regeneration in the adult mammalian central nervous system. The findings will have with high relevance for the development of novel therapeutics for neurological disorders.

DESCRIPTION (provided by applicant): A limited but important degree of neurological recovery occurs after Spinal Cord Injury (SCI). Anatomical plasticity of synaptic connections at multiple levels of the neuraxis from the cerebral cortex to the caudal spinal cord is likely to contribute to this functional recovery. To exploit and optimize plasticity-based recovery of function for SCI, it is essential to define its cellular nature and extent. Anatomical plasticity wthin the cerebral cortex has been examined to a very limited extent after SCI, even though it is a likely site for plasticity. Furthermore, the stability of individual synapses has never been monitored in vivo after SCI. Using time-lapse in vivo two-photon microscopy we will image synaptic connections in the cerebral cortex after SCI to determine the extent of rearrangement at the synaptic level. Importantly, the role of training, CSPG and NgR1 in modulating recovery will be linked to cortical synapse dynamics. Using region-specific conditional gene deletion and optogenetic mapping, we will determine the functional significance of anatomical plasticity in cortical synapses during SCI recovery. The findings have the potential to establish cortically directed therapeutic interventions as cellular targets for SCI rehabilitation.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a neurodegenerative condition with a massive health burden, but no therapeutic option to slow or halt progression. It is estimated that up to 14 million Americans will suffer from AD by 2050, which, apart from the human cost, could have a catastrophic effect on our health care system and the broader economy. This application aims to assess the potential therapeutic benefit of AZD0530 (Saracatinib) for AD. AZD0530 is a selective inhibitor of Src family kinases, and has been developed primarily for the treatment of cancer. However, our data show that Fyn kinase, a member of the Src family kinases, also plays a fundamental role in the pathogenesis of AD. Multiple studies implicate Fyn kinase in the synaptic pathophysiology of AD, with links to both Ass and Tau pathology. For transgenic AD mice, genetic removal of Fyn kinase alleviates, and overexpression of Fyn exacerbates, the impairment of synaptic density and spatial memory. The A beta peptide is thought to trigger AD. Our work, confirmed and extended by others, showed that Cellular Prion Protein (PrPC) acts as a toxic Ass oligomer receptor. Downstream signaling from the A beta-PrPC complex is of major interest, as it might provide for a selective therapeutic target in AD. Recently, engagement of PrPC by A beta was found to activate Fyn kinase, initiating a detrimental signaling cascade with synaptic dysfunction. These data provide a direct mechanistic link between A beta and Fyn kinase in AD. In addition, Fyn kinase interacts directly with Tau, and the synaptic function of Fyn requires dendritic Tau. We hypothesize that blocking Fyn kinase is an effective therapeutic strategy in AD. AZD0530 safely inhibits Src family kinases, including Fyn, with high potency in humans. This application will validate the predicted benefit of AZD0530 in a mouse model of AD while measuring pharmacodynamic parameters, and initiate clinical trials. A Phase 1b trial will determine cerebrospinal fluid drug levels in AZD0530-treated human AD subjects and confirm safety and tolerability. Achievement of milestones for the preclinical and clinical Phase 1 studies will support the initiation of a Phase 2a proof-of concept clinical trial. A total of 159 patients will be studied in a 12-month, double-blind randomized placebo-controlled trial of AZD0530 in mild Alzheimer's disease. The primary outcome measures will include the slowing of a decline in regional brain glucose metabolism as measured by 18F-FDG PET imaging (a validated surrogate marker for clinical progression in AD), as well as assessments of safety and tolerability. Secondary outcomes will include standard clinical efficacy measures, rate of change in MRI volumes, and CSF total Tau and phospho-Tau. The goal of this project is to provide evidence in support of a multi-center Phase 3 trial of AZD0530 in AD.

SUMMARY FOR PROJECT 2 Synaptic communication is at the crux of brain function, and a key feature of AD is synaptic malfunction and synapse loss. A range of genetic and biomarker data indicate that the A? peptide triggers synaptic disease. The mechanisms by which AD-selective forms of A? damage the synapse are not well defined but have the potential to provide multiple sites for therapeutic intervention, which are distinct from regulating APP and A? themselves. Numerous studies implicate Fyn kinase in the synaptic pathophysiology of AD, with links to both A? and Tau pathology. For transgenic AD mice, genetic removal of Fyn kinase alleviates, and overexpression of Fyn exacerbates, the impairment of synaptic density and spatial memory. Our work, confirmed and extended by others, showed that Cellular Prion Protein (PrPC) acts as a cell surface binding site for toxic A? oligomers. Engagement of PrPC by A? was found to activate Fyn kinase, initiating a detrimental signaling cascade with synaptic dysfunction. Using Fyn and PrPC as molecular handles for synaptic pathology at the inner and outer surfaces of the post-synaptic density, we have sought to understand how these proteins are coupled and which signal transduction pathways are dysregulated in AD. Our Preliminary data strongly support a crucial role for mGluR5 and Fyn in AD pathophysiology. Here, we seek to validate the role of an A?o PrPC mGluR5 Fyn pathway in AD, identify biomarkers of this pathology, and explore the utility of mGluR5 agents in AD models. This project will advance the hypothesis that selectively blocking A?o mGluR5 signaling provides effective disease- modifying therapeutic strategy for AD.

PROJECT SUMMARY: OVERALL YALE ADRC The Yale Alzheimer?s Disease Research Center (ADRC) seeks to advance our understanding of Alzheimer?s disease at a cell biological level with the eventual goal of translating laboratory discoveries into novel effective clinical therapies. Seven Cores (Administrative, Clinical, Data, Biomarker, Neuropathology, Imaging and Outreach) and a Research Education Component will work together to achieve this goal. Our unifying theme is a focus on the cell biology of specific neurons, and its disruption in AD, whether measured by genome/proteome-wide methods in Biofluids, imaged diagnostically, manifested clinically in behavioral attributes, detected pathologically in brain tissue at autopsy or observed in cultures of induced pluripotent stem cells. The participation of the seven Cores will accelerate and optimize the ability of individual Research Projects both within and beyond the Yale ADRC to ask and answer specific pathophysiological questions and to translate knowledge to therapies. The breadth of the Core support for particular projects will allow assessments across the heterogeneity of Alzheimer?s disease. The Yale ADRC expects to extend its track record of facilitating Research Projects focused on specific neuronal organelles and specific neuronal subtypes perturbed in disease while making use of human tissue analysis and human subject imaging to evaluate mechanistic hypotheses. The Biomarker Core will develop novel, sensitive and high-throughput mass spectrometry assays and genomic methylation profiles to monitor disease mechanisms. The Imaging Core will develop, integrate and apply novel PET tracers and functional MRI connectivity maps to subjects of the Clinical Core and multiple separately supported Research Projects. A key emphasis will be the translational development of research findings into therapeutic benefit. To support the future strength of Alzheimer?s research, the Yale ADRC will strive to advance the careers of Young Investigators through mentorship from a distinguished Internal Advisory Committee, and through Development Project awards coupled with an extensive educational program developed by the Research Education Component. In addition to collecting clinical data and biospecimens of brain, CSF, DNA, serum, blood cells and iPSCs for analysis by members of the Yale ADRC research team, the Yale ADRC will support other NIH- funded research studies on related topics and contribute materials to national NIA-sponsored research networks. The Outreach Core will connect with the community to provide greater knowledge regarding Alzheimer?s disease and related dementia.

SUMMARY FOR ADMINISTRATIVE CORE The Administrative Core will provide leadership, cohesion and logistics for the entire Yale ADRC to achieve its goal of understanding the cellular basis of Alzheimer's disease and translating this knowledge into effective therapy. The Administrative Core will emphasize the use of proteomic studies for translational science. While specific projects seek to provide a cell biological analysis, the Yale ADRC will also strive to develop the careers of young Alzheimer's researchers and to share Biospecimens and clinical data with Alzheimer and Aging researchers at Yale and within NIA-sponsored networks. The Administrative Core will organize regular meetings amongst Core leaders and with External and Internal Advisory committees. The distinguished Internal Advisory Committee will provide general advice, career mentoring and evaluate Pilot Project proposals. The Pilot Projects will support AD research and will be administered by the Core with the dual goal of pushing the boundaries of Alzheimer research and aiding the development of New Investigator careers.

SUMMARY Central to the pathophysiology of cognitive dysfunction in Alzheimer's disease (AD) is loss of synapses with an impairment of plasticity at surviving synapses. Therapeutic efforts to intervene in AD have focused on the Aß peptide as an upstream trigger for synaptic disease, but clinical trials have been disappointing so far. Additional validated targets for AD therapy are needed, in particular those focused more directly on synaptic deficits. By their very nature, genetic studies of Late Onset AD (LOAD) risk are of direct clinical relevance. The largest GWAS analysis of LOAD identified a short list of genes whose common variants alter risk, providing potential new targets for AD therapy. We considered whether any of these might be directly linked to synaptic dysfunction in AD. Nearly all of the LOAD risk genes are hypothesized to bind Aß, to alter Aß metabolism, to regulate cellular endocytosis, or to modulate immune function. Therefore, their action on synaptic dysfunction must be considered indirect, via Aß levels or via the immune reaction to pathology. From the list of AD genetic risk factors, Pyk2 (also PTK2B or FAK2) is the only gene recognized to encode a protein concentrated at post- synaptic densities with direct effects on synaptic plasticity. Here, we seek to assess the role of Pyk2 in AD genetically and mechanistically, and to evaluate the protein as a therapeutic target focused on synaptic dysfunction. Previously, we have studied the biochemical basis for Aß oligomer (Aßo) toxicity in neurons and these studies have also implicated Pyk2. Using an unbiased genome-wide screening method we searched for Aß oligomer- specific binding sites expressed in brain, and identified PrPC. We defined an Aßo?PrPC?mGluR5?Fyn cascade that damages synapses in AD models. Importantly, the Pyk2 protein physically associates with mGluR5 and Fyn, as well as being implicated in synaptic plasticity. We will evaluate the hypothesis that Pyk2 is essential for manifestations of human familial AD transgene phenotypes in mice, and we will assess Pyk2 dysregulation in human AD samples. We will also target the enzyme pharmacologically. These studies have the potential to couple a validated LOAD genetic risk to synaptic dysfunction in AD, and may establish a connection with the Aßo?PrPC?mGluR5?Fyn cascade. Critically, these data may guide attempts to develop pharmacological tools to target the synaptic Pyk2 pathway for therapeutic intervention in AD.

SUMMARY The CNS of adult mammals, as compared to the peripheral nervous system of mammals or the nervous system of other organisms, has extremely limited capacity for axonal regeneration. Specific factors limiting adult mammalian regeneration of axons have been identified, but they provide an incomplete explanation for poor adult mammalian CNS regeneration. We have completed a genome-wide shRNA-based screen for endogenous genes limiting the repair of axons in the mammalian CNS. We have also conducted experiments to identify conserved genes that affect axon regeneration in the model organism C. elegans. Factors common to both experimental systems are expected to identify fundamental mechanisms in regeneration that are likely to affect the equivalent process in human patients. We aim to study and develop the translational potential of those evolutionarily conserved mechanisms here. From our studies we have selected one evolutionarily conserved pathway identified both in mouse cell culture and in C. elegans axon regeneration. It is bioinformatically the most enriched gene set in the primary mammalian screen data, with multiple family members identified, and also regulates regeneration in C. elegans. The relevance of the pathway will be tested in preclinical models of traumatic spinal cord injury. Multiple steps in the pathway will be assessed in rodent spinal cord injury models. Both gene deletion strains and pharmacological inhibition will be studied to provide a validated pathway for future therapeutic development. While we will focus on one particular pathway regulating membrane traffic in the axon, we will utilize both laser axotomy and mouse spinal cord traumatic injury to explore additional pathways identified in the primary screen. This project builds on genetic screens in the mature mammalian central nervous system and C. elegans to analyze novel mechanisms that promote axon regeneration after mammalian spinal cord injury. The findings will have high relevance for the development of novel therapeutics for neurological disorders.

SUMMARY The National Eye Institute has identified the restoration of vision as an ?audacious goal? requiring the reestablishment of neural connections from a functional retina with the brain. Unfortunately, the regeneration of adult mammalian CNS axons is extremely limited or nil. The reconnection of retinal ganglion cells with lateral geniculate neurons to support vision recovery requires overcoming endogenous limitations on axonal regeneration. A small group of genes limiting regeneration in particular retinal ganglion cell subtypes have been identified, but only a very small fraction of the genome has been tested with regard to a role in limiting axon regeneration. We hypothesize that additional regeneration-regulating factors exist, and that conservation of function across cell types, species and modes of growth will facilitate their discovery. We have completed a genome-wide shRNA-based screen for endogenous genes limiting the repair of axons in the mammalian CNS using cultured mouse cerebral cortex neurons with over 135,000 separate regeneration experiments. Across 17,000 genes, this loss of function screen yielded 500 regeneration genes. Here, our primary goal is to test this selected list for the ability to promote retinal ganglion cell axon regeneration in vivo. We expect that genes with the most robust regeneration-limiting function will have conserved function from cortical neurons to retinal ganglion cells. Amongst the cortical axon regeneration gene list, we will prioritize genes for optic nerve regeneration experiments using two additional datasets. In one experiment, we determined the distinct transcriptional profiles of sprouting versus non-sprouting mouse CST neurons after CNS injury, hypothesizing that the most relevant genes will participate in both sprouting and regeneration, as is the case for previously described regeneration factors. We have also conducted experiments to identify species conservation of regeneration by screening for genes that regulate motor axon regeneration in the nematode C. elegans. Factors common to multiple experimental systems are expected to identify fundamental mechanisms in regeneration that are likely to affect the equivalent process in human visual system. For those newly discovered genes whose loss of function supports optic nerve regeneration, secondary studies will assess synergy amongst positive genes, retinal ganglion cell type specificity of action, and central axonal pathfinding function. This project builds on genetic screens in the mature mouse central nervous system and C. elegans to analyze novel mechanisms that promote axon regeneration after mammalian retinal ganglion cell axotomy.

?DESCRIPTION (provided by applicant): In Alzheimer's disease (AD), an early causative role for Amyloid beta (A?) peptide is supported by pathology, by human genetics and by biomarker studies. More specifically, A? oligomers trigger a toxic cascade that impairs synaptic function and subsequently leads to Tau pathology and progressive cognitive dysfunction. Therapeutic efforts to intervene in the A? pathway have focused on the production or clearance of the peptide, and unfortunately have been disappointing so far. Additional validated targets for AD therapy are needed. Previously, we have studied the basis for A? oligomer (A?o) toxicity in neurons. Using an unbiased genome-wide screening method we searched for A? oligomer-specific binding sites expressed in brain, and identified PrPC. Amongst reported A?o binding sites, only PrPC was identified through an unbiased, genome-wide screen. In the previous grant cycle, we went on to define an A?o-PrPC-mGluR5-Fyn cascade that damages synapses in AD models. Here, we will pursue three aims to expand our knowledge of A?o synaptotoxic signaling, focusing on the PrPC-mGluR5 complex. First, we use conditional deletion of PrPC expression in AD transgenic mice, and show a role for A?o signaling via PrPC in the maintenance and progression of synaptic and memory impairments. Deleting PrPC rescues established deficits. This highlights the need to understand how specific residues in the natively unfolded segment of PrPC recognize oligomeric but not other forms of A? peptide to trigger synaptic symptoms. We will combine biochemical, mutagenesis and NMR analyses to provide molecular insight. Not only are PrPC and mGluR5 required individually for mouse transgenic phenotypes, but preliminary data show that they also interact genetically in linking A?o to intracellular signaling molecules and in generating mouse model synapse and memory loss. Importantly, while mGluR5 interacts with many intracellular polypeptides, PrPC is unique as an extracellular polypeptide interaction. We will examine the basis for the interaction of these two proteins, defining requisite domains and changes in quaternary structure. While we and later others observed that negative allosteric modulators of mGluR5 rescue A?o and AD transgene phenotypes, the therapeutic index is very narrow. Minor increases in dose interrupt endogenous Glu signaling and impair behavioral function. The optimal therapeutic compound would preserve endogenous mGluR5 signaling for Glu but block signaling from A?o-PrPC. Having identified a high potency Silent Allosteric Modulator with this profile, we propose to test its efficacy to bloc neuronal A?o signaling in neurons and in transgenic mice. Together these studies will provide insight into how the PrPC-mGluR5 transduction complex plays a central role in AD related signaling and explore a potential therapeutic approach.

Project Summary Histopathological studies of Alzheimer's disease have consistently demonstrated the presence of inflammation in Alzheimer's disease (AD). Inflammation was previously thought to be a bystander in the pathogenesis of disease however several lines of evidence, most importantly from genetic studies of susceptibility loci have demonstrated a number of genes, most of which are implicated in the function of microglia. Microglia, the main protagonist in the innate immunity of the brain, interacts with constituents of the amyloid plaque and appears to be activated by this interaction. However it is not clear whether this activation is protective, destructive or protective in one setting of disease and destructive in another. Contradictory evidence comes from animal models, epidemiological data and clinical trials. Part of the problem has been the difficulty of in vivo study of central nervous system immunity. The discovery of the transporter protein (TSPO) may represent a breakthrough in the in vivo study of neuroinflammation. TSPO is located in the outer mitochondrial membrane and is upregulated during activation of microglia. Several PET ligands exist which image this molecule and, in particular, 11C-PBR28 is well suited to the study of neuroinflammation in AD. One study using this ligand was able to show increased binding in AD but not mild cognitive impairment, leaving a number of tantalizing questions unanswered. Firstly it is not clear whether the increased baseline activation of microglia in AD is simply due to the presence of amyloid or represents a dysregulation of microglial response to stimulation. Secondly, if this dysregulation is demonstrated, are the inflammatory increases in AD subjects simply due to microglial senescence or represents a disease-specific jump in microglial reactivity. For this reason, we propose to apply a technique to measure inflammatory reactivity using TSPO imaging of the brain at baseline and 180 minutes after the intravenous administration of lipopolysaccharide. We define microglia activation reserve index as the percentage change in TSPO binding (measured as volume of distribution) at baseline and after LPS. We also aim to compare microglia activation reserve index between young healthy individuals, cognitively normal elderly and subjects with AD. We expect to find an increase in the reactivity in the innate immune systems of subjects with AD which exceed that which may be expected from simply the effect of aging. These results, if demonstrated, will have profound diagnostic and therapeutic implication in the management of Alzheimer's disease. Increased MARI with AD may be used as a diagnostic biomarker and point the way to anti-inflmmatory therapies for disease course modification.

SUMMARY Devastating and persistent neurological deficits occur after Spinal Cord Injury (SCI), despite survival of nearly all neurons. The primary cause of disability is disconnection of networks by axon transection. Recovery of some movement would be adequate for patients to gain a level of independence in wheel chair transfers, bowel and bladder management, and locomotion. Today, there is no approved medical therapy for the 300,000 to 1,200,000 individuals in the USA with SCI. The CNS of adult mammals, as compared to the peripheral nervous system of mammals or the nervous system of other organisms, has extremely limited capacity for axonal regeneration. Our axonal growth studies included discovery of Nogo and Nogo Receptor (NgR1). We demonstrated their role in preventing axonal sprouting, regeneration and recovery after injury. We have demonstrated that NgR1(310)-Fc is efficacious for recovery from SCI, even when treatment starts months after damage. It is being developed for human SCI trials now. While specific factors, such as NgR1, limiting axon regeneration have been identified, they provide an incomplete explanation for poor adult mammalian CNS regeneration. We completed a genome-wide shRNA screen for endogenous genes limiting mammalian CNS axon repair. The validity of this method was demonstrated by the identification of INPP5F as a gene limiting neural repair in a pilot screen of phosphatases. One cellular pathway is bioinformatically the most enriched gene set in the mammalian screen, and also regulates regeneration in nematodes. The relevance of this pathway will be tested in preclinical models of traumatic SCI. Both gene deletion strains and pharmacological inhibition will be studied to provide a validated pathway for future therapeutic development. The findings will have high relevance for the development of novel therapeutics for SCI.

SUMMARY This project seeks to develop a novel disease-modifying compound for Alzheimer?s disease (AD). Synapse loss in AD brain has been tightly correlated with cognitive symptoms and is triggered initially by amyloid beta (Aß) peptide accumulation. We have described a pathway in which soluble Aß oligomers bind to Prion Protein, thereby engaging mGluR5 as a co-receptor, and activating PTK2B (Pyk2) and Fyn kinases to couple with Tau pathology and synapse loss. mGluR5 is a GPCR, and multiple groups have shown that interrupting mGluR5 function rescues preclinical AD phenotypes, making it an attractive drug target. However, mGluR5 has a physiological role as a glutamate receptor and full inhibition impairs function. Consequently, typical antagonists have a narrow therapeutic window. Yale has obtained an exclusive license to an extensive mGluR5-directed compound library with robust Intellectual Property protection. Within this chemical collection, we identified a highly potent, orally bioavailable mGluR5 ligand that does not alter basal or glutamate activity, but does block Aßo/PrPC activation of mGluR5. This compound is considered a silent allosteric modulator, or SAM, for mGluR5, meaning ?silent? with regard to glutamate, while antagonistic with regard to Aßo/PrPC. Preliminary studies demonstrate robust efficacy of this SAM compound for multiple preclinical mouse AD phenotypes. Drug treatment recovers synapse density, restores LTP and returns memory performance to WT levels. While closely related mGluR5 ligands have extensive selectivity and DMPK data, these are not yet developed for the SAM compound at this Early Stage, and we propose to obtain them here for this lead candidate. The overall goal is to develop disease-modifying oral therapy effective to slow, halt or partially reverse AD progression both in the MCI state and in mild dementia.

SUMMARY Alzheimer?s Disease (AD) progresses slowly and involves many molecular pathways. In addition to accumulation of Aß plaques and Tau neurofibrillary tangles, there are also inflammatory, vascular and metabolic changes. Human genetic studies of late onset AD (LOAD) risk have identified two major groups of genes, one related to brain inflammation and another with established roles in the endolysosomal pathway. Endolysosomal organelles are utilized by cells to take up, degrade and remove substances from both inside and outside of the cell. The general principles of endolysosomal regulation/dysregulation in AD are not well understood. However, an endolysosomal pathway role is not specific to AD amongst degenerative dementias. In the related condition of Fronto-Temporal Dementia, Progranulin and TMEM106B proteins play key roles, and are linked to the endolysosomal pathway. Here, we seek to leverage knowledge of this FTLD-TDP pathway to understand and modify endolysosomal function in AD and in Tauopathy. Our studies of mice lacking Progranulin suggest that this endolysosomal protein has a pronounced effect on Tau-dependent neuro-degeneration. In Preliminary studies, Tau transgene induced phenotypes are fully rescued by loss of Progranulin. In proposed work, we will characterize this exciting finding with regard to the molecular and cellular details of Tau aggregation, phosphorylation, spreading, metabolism and toxicity. These studies will define a role of Progranulin-dependent regulation of endolysosomes in Tauopathies, including AD. Loss of a second endolysosomal protein, TMEM106B, counteracts loss of Progranulin in certain settings, but not others. For Tauopathy and AD, we will test whether reducing TMEM106B function worsens pathophysiology, and if increasing TMEM106B mimics the loss of Progranulin to rescue Tauopathy. The long-term goal is to define a neuro-degeneration-related endolysosomal pathway that can be targeted to provide disease-modifying therapy for Tauopathies, including Alzheimer?s Disease.

SUMMARY FOR ADMINISTRATIVE CORE The Administrative Core will provide leadership, cohesion and logistics for the entire Yale ADRC to achieve its goal of understanding the cellular basis of Alzheimer?s disease and translating this knowledge into effective therapy. The Administrative Core will emphasize the use of emerging imaging and integrated omic methodologies for translational science. The Yale ADRC will also strive to develop the careers of young Alzheimer?s researchers and to share Biospecimens and clinical data with Alzheimer and Aging researchers at Yale and within NIA-sponsored networks. The Administrative Core will organize regular meetings amongst Core leaders and with External and Internal Advisory committees. The distinguished Internal Advisory Committee will provide general advice, career mentoring and evaluate Development Project proposals. The Development Projects will support innovative Alzheimer?s disease research and will be administered by the Core with the dual goal of pushing the boundaries of Alzheimer research and aiding the development of New Investigator careers.

ABSTRACT Alzheimer?s disease (AD) is the leading cause of dementia (60-80%), affecting tens of millions of people globally and, due to longer lifespans and aging populations, perhaps hundreds of millions more by 2050. AD pathology is first observed in allocortical and limbic areas within the cerebrum, in particular medial temporal cortical regions critical for learning and memory including the hippocampal formation and entorhinal cortex (HIP-EC). Within these areas, pathology exhibits subregional and cell type specificity, with layer 2 of entorhinal cortex and the hippocampal CA1 field (Sommer?s sector) exhibiting pathological hallmarks before dentate gyrus granule cells and other major hippocampal neuronal subtypes. Understanding the molecular basis of this selective vulnerability (and conversely the resilience of other cell types) will provide new insights into the etiology of AD, but to date only limited efforts have been made to understand the molecular signatures differentiating neuronal and non-neuronal cells in HIP-EC. We therefore propose to conduct single nuclear RNA sequencing (snRNA-seq) and single nuclear Assay for Transposase Accessible Chromatin (snATAC- seq) in 5 regions of HIP-EC of AD brains, young/mid adult and aged neurotypical ?control? human brains, and young adult and aged rhesus macaque brains. This will allow us to develop a high resolution cell census of HIP-EC which will in turn allow us to identify enriched genes, gene expression patterns, gene regulatory networks, and biological processes potentially mediating cell type specific differences in the AD and aged HIP-EC.

SUMMARY Alzheimer?s disease (AD) afflicts 5.8 million in the USA, but there remains no disease-modifying therapy. Critical for progression from pre-symptomatic to mild cognitive impairment (MCI) to dementia is the loss of synapses in relevant brain regions. We seek to define the molecular basis of this synapse loss. In the previous cycle, we demonstrated an essential role for aberrant metabotropic glutamate receptor 5 (mGluR5) activation in synapse dysfunction, synaptic loss and memory deficits using cellular assays, brain slices and transgenic mice. Our Preliminary studies here demonstrate that synapse loss is mGluR5-dependent in gene knock-in as well as transgenic models, and that it can be tracked by longitudinal PET imaging. We have discovered a sub-nanomolar potent and orally available mGluR5 silent allosteric modulator (SAM, BMS-984923) that blocks this AD process, while preserving physiological Glu signaling. We now aim to address two key issues regarding this clinically targeted AD synapse loss pathway. Single cell transcriptomic profiling will be utilized to understand how synaptic mGluR5 modulation interacts with diverse molecular pathways and glial cells in AD. Single particle cryo-electron microscope studies will be employed to understand the basis for differential mGluR5 regulation by Aßo/PrPC vs Glu, and for SAM action. Hypotheses generated from the transcriptomic and protein studies will be tested in human iPSC-derived neurons and in mouse gene knock-in models. To maximize relevance, signaling will be triggered by Aßo purified from human AD brain.

For Alzheimer?s disease (AD), a range of pathological, genetic and biomarker data supports the hypothesis that Amyloid beta (A?) peptide triggers the disease process. As AD progresses over decades, subsequent Tau pathology and inflammatory reaction occur during progressive cognitive decline to dementia and death. Symptoms are tightly linked to the loss of neuronal synapses in the brain, but the mechanisms causing synapses to be removed are less clear. Recent data implicate innate immunity, microglia cells and complement proteins in synaptic engulfment. However, the molecular mechanism by which complement proteins accumulate focally and tag specific synapses for removal in AD remains unknown. In addition, the misfolded aggregated peptides, A? and Tau, are known to interact with synapses and cause dysfunction. We hypothesize that the tagging of synapses with microglia-derived complement components for subsequent engulfment is coupled with synapse-specific derangements driven by neuronal interaction with misfolded protein accumulation. We will examine the connection between synaptic damage signals and complement recruitment. Preliminary studies show that interruption of synaptic signaling by PrPC or mGluR5 prevents both C1q tagging and loss of synapses in AD mice, despite persistent microgliosis and complement overproduction. We will examine the specificity, timing and genetic necessity of a link between complement and the A?o/PrPC/mGluR5 complex for synapse tagging and engulfment. Mechanistically, we find that C1q and C1qBP physically associate with hydrogels composed of A?o/PrPC/mGluR5, and this may provide a direct means for C1q selectivity. We will test this possibility by biochemical and cellular analysis, and by genetic studies in mice. Alternatively, synaptic signaling triggered by A? or Tau may regulate other molecules, which in turn recruit C1q. An arrayed expression cloning screen for C1q binding sites revealed a novel high affinity neuronal protein binding C1q. We will test the role of this binding site in C1q tagging of synapses, and in the loss of synapses in neurodegenerative and developmental models.