Geoffrey G. Murphy, Ph.D. - US grants

The long-term objective of this project is to elucidate the neuronal mechanisms of learning and memory in the marine mollusk Aplysia californica. The defensive withdrawal reflex of this animal constitutes a useful system for this purpose. The reflex exhibits several forms of learning, including classical conditioning. The neuronal circuitry which underlies the withdrawal reflex, particularly its monosynaptic component- the synapse between the sensory and motor neurons-is relatively simple and well understood. The proposed experiments will focus on a form of synaptic plasticity known as Hebbian long-term potentiation (LTP). This form of synaptic plasticity, particularly its manifestation in the CA1 region of the hippocampus, has been prominently implicated in vertebrate memory and cognition. Recently, a type of LTP has been described for Aplysia sensorimotor synapses in cell culture (apLTP) which appears to be mechanistically quite similar to LTP of CA1 synapses. The specific aim of this proposal is to deterMine whether an apLTP-related process is involved in learning in Aplysia. The proposed experiments will utilize preparations comprising the central nervous system of Aplysia in electrophysiological experiments involving a cellular analogue of classical conditioning of the withdrawal reflex. The proposed research will serve as the basis for improving our understanding of human learning and memory. It may thereby contribute to ameliorating human memory- associated diseases, such as Alzheimer' s.

The long-term objective of this project is to elucidate the neuronal mechanisms that underlie age-related deficiencies in learning and memory. Recently, our laboratory has created a strain of mice in which the Kvbeta1.1 subunit of the potassium channel has been deleted. Deletion of the Kvbeta1.1 subunit produced impairments in hippocampus-dependent learning in young Kvbeta1.1 mutants. Remarkably, aged (greater than 18 mo.) Kvbeta1.1 mutants showed no such impairment and do not exhibit the age-related memory deficits normally seen in age-matched wild-type mice. The specific aims of this proposal are to 1. Further characterize the learning behavior of these mice, using a combination of well established behavioral paradigms (i.e. Morris water maze and Pavlovian fear conditioning) and 2. Investigate the neuronal mechanisms that underlie the absence of age-related learning deficits in the aged Kvbeta1.1 mutants. Specifically, electrophysiological experiments will be conducted to explore the possibility that the Kvbeta1.1. mutants might exhibit alterations in neuronal excitability and how these alterations might affect specific forms of synaptic plasticity thought to mediate learning and memory. The proposed research will serve as the basis for improving our understanding of age-related changes in human learning and memory and thereby help in the amelioration of cognitive decline in the elderly.

DESCRIPTION (provided by applicant): Deficits in learning and memory which arise independent of overt pathology are considered to be a normal component of aging. It is estimated that about 40% of people over the age of 65 years suffer from some sort of age-related cognitive impairment. The exact nature of the underlying neuronal changes that give rise to these naturally occurring age-related learning and memory deficits remains unknown. However, in recent years several promising hypotheses have emerged. Prominent among them is the "calcium dysregulation hypothesis of brain aging and neuro-degeneration." This hypothesis asserts that a number of age-related changes in neuronal function are the result of a dysregulation in the homeostasis of cytosolic free calcium. Indeed there is significant experimental evidence demonstrating an elevated level of intracellular calcium in the neurons of aged animals during neuronal activity. Furthermore, there are several lines of converging evidence that suggest that the increase in calcium is due to an increase in the number of L-type voltage-sensitive calcium channels (L-VSCC), and this increase in calcium channel density is correlated with a decrease in performance in spatial learning ability and working memory. Taken collectively, the evidence outlined above is compelling; however, the hypothesis that an age-related up-regulation of neuronal LVSCC expression can actually cause cognitive impairment has yet to be tested directly. To directly test this hypothesis, we have generated transgenic mice in which the neuronal L-type calcium channel subunit Cav1.3 can be over-expressed in an inducible, cell-type and region-specific manner. In young transgenic animals, over-expression of Cav1.3 would be expected to produce a selective and premature cognitive impairment. The overall effort of this proposal will be to: 1) complete the initial characterization of these mice at the molecular and biochemical level, and 2) complete the initial behavioral characterization. These transgenic mice are expected to advance our knowledge on two fronts. First, they will allow us to test directly the hypothesis that age-related increases in calcium channels can give rise to the cognitive impairments that often accompany aging. Second, if this hypothesis is indeed correct, these mice will then become an invaluable tool in the development of therapies targeting the amelioration of age-related cognitive impairments that arise from up-regulation of calcium channel expression.

[unreadable] DESCRIPTION (provided by applicant): Ablation of the distal end of the short arm of chromosome 1 (1p36 deletion syndrome) is one of the most commonly occurring terminal deletion syndrome in humans, occurring in about 1 in 5000 newborns. Subjects with 1p36 deletion syndrome (1p36DS) exhibit a wide range of clinical features including growth delay, congenital heart defects and craniofacial dysmorphism. In addition, individuals with 1p36DS have rather profound neurological disorders: moderate to severe mental retardation and seizure activity are common clinical features. Although there is significant variability with regard to the extent of the deletion, several genes have been mapped to region 1p36 as candidate genes that may underlie the neurological phenotype in 1p36DS. One such gene-KCNAB2-which encodes the potassium channel auxiliary subunit Kv[unreadable]2, has recently been linked to epilepsy in patients with 1p36DS. To determine to what extent loss of Kv[unreadable]2 contributes to the neurological phenotype of 1p36DS we have begun to examine mice in which the mouse homologue of KCNAB2 has been deleted by homologous recombination (Kv[unreadable]2 KO mice). Our preliminary experiments reveal that the Kv[unreadable]2 KO mice exhibit hippocampal-dependent learning/memory impairments and spontaneous seizures. These neurological abnormalities occur in the absence of alterations in basal synaptic transmission within the hippocampus. In this Exploratory/Developmental application we seek to build upon these studies to further develop this mouse model. The experiments outlined in Specific Aim I will further explore the learning/memory impairments in the Kv[unreadable]2 KO mice in two additional hippocampal-dependent learning/memory tasks: the Morris water maze and the Olton 8-arm radial maze. In Specific Aim II standard video/EEG methodology will be utilized to define the frequency, severity and prevalence of the spontaneous seizures in the Kv[unreadable]2 KO mice. In Specific Aim III we will determine to what extent long-term potentiation and intrinsic neuronal excitability are altered by deletion of Kv[unreadable]2. We anticipate that the results obtained from this Exploratory/Developmental will not only provide important insights into the relative contribution of KCNAB2 deletion to the neurological phenotype observed in 1p36 DS but will also help to elucidate the neuronal function of Kv[unreadable]2. ject Narrative 1p36 deletion syndrome is a chromosome disorder in which the end of the short arm of either copy of chromosome 1 is deleted. Patients with 1p36 exhibit a wide variety of symptoms including several neurological abnormalities. This application proposes to use genetically engineered mice to examine the neurological impact of deleting a specific gene (KCNA2B) known to be lost in 1p36 deletion syndrome. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Advancing age is often accompanied by cognitive impairments characterized by substantial deficits in learning and memory. These deficits adversely effect day to day living, reduce the quality of life and impose a financial and social burden on the affected and their families. The exact neuronal mechanism that gives rise to these age-related impairments remains unknown. However, data from experiments using a variety of model systems, suggests that dysregulation of neuronal Ca2+ homeostasis is a significant contributing factor to these cognitive impairments. Specifically, there appears to be an age-related increase in intracellular calcium [Ca2+]i in neurons observed during neuronal activity. This increase in activity-driven accumulation of [Ca2+]i adversely impacts neuronal excitability and long-term potentiation (LTP), processes that have both been implicated in learning &memory. It has been suggested that the increase in [Ca2+]i is the result of an age-related increase in expression of L-type voltage-gated calcium channels (L-VGCCs). This has led to the hypothesis that an age-related increase in L-VGCCs expression leads to a decrease in neuronal excitability which in turn reduces LTP. The resulting decrease in LTP disrupts the ability of the hippocampus to encode new information. In this proposal we will use a multidisciplinary approach to test key elements of this hypothesis. In Specific Aim I we will use L-VGCC knockout (KO) mice to determine whether L-VGCCs are necessary for the age-related decrease in neuronal excitability and LTP. In Specific Aim II, using organotypic hippocampal slice cultures and epitope-tagged recombinant L-VGCC pore forming subunits, we will determine whether over-expression of L-VGCCs is sufficient to produce changes in neuronal excitability and LTP similar to that observed during normal aging. Finally, in Specific Aim III we will utilize L-VGCC KO mice to determine to what extent L-VGCCs contribute to the learning &memory deficits normally observed in aged mice. Results from these studies will provide us with valuable insights into the neurobiology of aging and will aid in the identification of targets for the therapeutic intervention, designed to ameliorate cognitive impairments that arise from alterations in age-related calcium homeostasis.

DESCRIPTION (provided by applicant): Advancing age is often accompanied by cognitive impairments characterized by substantial deficits in learning and memory. These deficits adversely effect day to day living, reduce the quality of life and impose a financial and social burden on the affected and their families. The exact neuronal mechanism that gives rise to these age-related impairments remains unknown. However, data from experiments using a variety of model systems, suggests that dysregulation of neuronal Ca2+ homeostasis is a significant contributing factor to these cognitive impairments. Specifically, there appears to be an age-related increase in intracellular calcium [Ca2+]i in neurons observed during neuronal activity. This increase in activity-driven accumulation of [Ca2+]i adversely impacts neuronal excitability and long-term potentiation (LTP), processes that have both been implicated in learning & memory. It has been suggested that the increase in [Ca2+]i is the result of an age-related increase in expression of L-type voltage-gated calcium channels (L-VGCCs). This has led to the hypothesis that an age-related increase in L-VGCCs expression leads to a decrease in neuronal excitability which in turn reduces LTP. The resulting decrease in LTP disrupts the ability of the hippocampus to encode new information. In this proposal we will use a multidisciplinary approach to test key elements of this hypothesis. In Specific Aim I we will use L-VGCC knockout (KO) mice to determine whether L-VGCCs are necessary for the age-related decrease in neuronal excitability and LTP. In Specific Aim II, using organotypic hippocampal slice cultures and epitope-tagged recombinant L-VGCC pore forming subunits, we will determine whether over-expression of L-VGCCs is sufficient to produce changes in neuronal excitability and LTP similar to that observed during normal aging. Finally, in Specific Aim III we will utilize L-VGCC KO mice to determine to what extent L-VGCCs contribute to the learning & memory deficits normally observed in aged mice. Results from these studies will provide us with valuable insights into the neurobiology of aging and will aid in the identification of targets for the therapeutic intervention, designed to ameliorate cognitive impairments that arise from alterations in age-related calcium homeostasis.

? DESCRIPTION (provided by applicant): It is estimated that about 40% of people over the age of 65 suffer from some sort of age-related cognitive impairment that significantly impact quality of life. For many, these impairments will occur in the absence of overt clinical symptoms or neuropathology associated with Alzheimer's disease. While the exact cellular substrates that underlie these age-related alterations in cognition remain unknown, it has been previously shown that dysregulation of cytosolic free calcium ([Ca2+]i) homeostasis leads to altered neuronal function in aged animals. Furthermore, it has been suggested that one of the initial triggers for the sequelae of events that leads to altered calcium homeostasis is a paradoxical age-related increase in the expression of L-type voltage-gated calcium channels (LVGCCs). Furthermore, the increase in LVGCC expression has been correlated with decreased neuronal excitability and altered synaptic plasticity in aged animals. While these experiments suggest a correlation between age-related increases in LVGCC expression, learning deficits and altered neuronal function, it has yet to be demonstrated directly that increased LVGCC expression can actually disrupt neurocognitive function. Therefore, the primary objective of this proposal is to determine to what extent increased LVGCC expression impacts cognition and neuronal function. Our central hypothesis is that overexpression of LVGCCs can produce cognitive impairments and altered neuronal function in young mice similar to that which is observed during aging. Using a reverse genetics approach, we will test our central hypothesis directly by examining cognition and neuronal function in mice that have been genetically engineered to overexpress LVGCCs in the forebrain. Additionally, we propose to refine the current mouse model by generating two new transgenic lines that will provide greater regional and temporal specificity. The proposed experiments will provide us with valuable insight into the extent that dysregulation of intracellular calcium homeostasis contributes to age-related cognitive decline and will also provide a framework from which future investigations will advance targeted therapies intended to ameliorate cognitive impairments in the elderly.

Project Summary / Abstract: There is an urgent need for disease-modifying treatment of Alzheimer's disease (AD) starting at its very onset. This knowledge gap remains because conventional approaches cannot measure in vivo brain region-specific biomarkers of the earliest relevant dysfunction underlying abnormal behavior. Often, spatial disorientation is observed during prodromal AD, and its occurrence predicts later dementia. A brain region contributing to this spatial confusion is the CA1 subfield of hippocampus because of its essential role in encoding spatial information. HC oxidative stress is most commonly identified at the very start of AD, and in experimental models of AD. Yet, it has not been possible to prove that prodromal oxidative stress in the relevant CA1 subfield plays a pathogenic role in at-risk patients showing impaired spatial memory because conventional methods only measure oxidative stress from post-mortem tissue. Addressing this major knowledge gap requires a new paradigm that compares antioxidant treatment efficacy in HC CA1 subregions in vivo with improved spatial learning and memory in experimental models, and that can then be translated into patients. In this proposal, we present a transformative solution to this problem based on a novel method recently discovered by our lab: QUEnch-assiSTed MRI (QUEST MRI). QUEST MRI is a robust and sensitive tool that has been validated against ?gold standard? methods and maps in vivo excessive free radical production in, for example, murine dorsal CA1. The QUEST MRI index of abnormally high production of paramagnetic free radicals in specific brain regions is a greater- than-normal spin-lattice relaxation rate R1 (1/T1) that can be returned to baseline after acute antioxidant administration. Our QUEST MRI studies have confirmed dorsal HC CA1-specific oxidative stress in spontaneous and familial AD mouse models with declines in spatial learning and memory in conjunction with HC CA1 oxidative stress measured ex vivo. We also find downstream consequences of oxidative stress such as greater-than-normal amounts of the lipid peroxidation product 4-hydroxynonenal (HNE), dorsal HC CA1 calcium dysregulation and reductions in dorsal HC CA1 calcium-dependent afterhyperpolarization (AHP). To improve statistical power, this proposal is tightly focused on uniquely testing a specific working hypothesis that oxidative stress in dorsal CA1 in vivo causes deterioration of spatial memory in experimental models. Our highly innovative studies by an experienced team of experts will validate a new bridging tool for testing in vivo antioxidant therapeutic strategies to mitigate a clinically important early decline in spatial memory preceding later loss of personhood in AD.

Program Summary/Abstract This U01 proposal is designed to provide the preclinical framework required to advance a novel stem cell therapy to human clinical trials as an effective treatment for Alzheimer's disease (AD). AD is the most prevalent age-related neurodegenerative disorder and leading cause of dementia, affecting an estimated 5.3 million people in the U.S. There is no cure and no means of prevention. To date, a handful of traditional, single-target pharmacological approaches have produced only marginal clinical improvements - there is a critical need for more effective therapies. Therefore, the long-term goal of our research is to develop a disease-modifying cellular therapy for AD that will have a meaningful impact on patients' lives. Cellular therapies target multiple disease mechanisms and provide a multifaceted approach to treat the complex pathologies associated with AD. In collaboration with Neuralstem, Inc., we have developed a unique line of human cortex-derived neural stem cells (NSCs) that produce several neuroprotective growth factors. Our findings to date, as well as proof- of- concept studies by others, show the benefit of cell therapies in AD models and indicate that efficacy is enhanced when coupled with delivery of trophic factors. In our proposed approach, NSC transplantation will combine the multifactorial therapeutic potential of a cellular therapy with sustained and directed delivery of neurotrophic factors, providing increased benefit compared to traditional approaches and improving outcomes in AD. Our preliminary data in a mouse model demonstrate that NSC transplantation is safe and effective, significantly impacting cognition and reducing A? plaque burden. In this proposal, we will determine the maximum tolerated dose and assess NSC bio-distribution and tissue tropism in two well-established and highly relevant mouse models: 5XFAD and rTg4510. We will then perform large-scale efficacy testing of NSCs in these mouse models and complete a dose-response feasibility study in non-human primates, which are anatomically and cognitively more relevant to human clinical testing. Overall, our proposal will have a significant impact on AD by providing proof-of-concept efficacy data for a well-characterized cellular therapy in two relevant mouse models and safety data in a large animal with a brain structure that is more analogous to humans. Completion of our proposed IND-enabling studies, as well as our laboratory's unique track record of translating proof-of-principle animal studies to human trials, will enable this stem cell therapy to progress into an attainable disease-modifying intervention for AD patients.

ABSTRACT The pathophysiology of Alzheimer?s disease (AD) is complex and represents one of the most difficult and pressing challenges facing modern neuroscience. Intracellular neurofibrillary tangles (NFTs) comprised of hyperphosphorylated tau and senile plaques consisting of aggregated extracellular amyloid beta (A?) are the hallmark pathological features of AD. However, it is clear that other factors likely contribute to the neural system failure and cognitive impairments associated with AD. Mounting clinical and experimental data suggest that cardiovascular disease (CVD) risk factors that promote vascular remodeling and dysfunction are associated with cognitive impairment, and are significant risk factors for the development of AD dementia. These studies include observations directly linking pathways associated with vascular injury such as hemostasis, angiogenesis, and hypertension to AD, leading to the hypothesis that CVD risk factors may act via common mechanisms to promote AD development or progression. For example, current data show that plaques, tangles, and CVD risk factors all upregulate expression of plasminogen activator inhibitor 1 (PAI-1), an independent CVD risk factor. Recent studies also suggest that PAI-1 may be a diagnostic biomarker and/or a risk factor for clinical AD, and PAI-1 expression increases with age, the most significant risk factor for AD dementia. PAI-1 is best understood for its role regulating fibrinolysis and wound, and in mouse models of AD PAI-1 deficiency is correlated with improved outcomes. In preliminary data presented here in the 5XFAD amyloidogenic mouse model we find that significant vascular remodeling occurs concurrently with amyloid plaque development and cognitive impairment. These changes are associated with reductions in cortical blood flow and increased PAI-1 expression. RNA-Seq and pathway analysis identify highly significant increases in gene expression in pathways known to be involved in vascular remodeling in the 5XFAD mice compared to Wt littermates, including pathways associated with angiogenesis and cardiovascular development. We also find that pharmacologic inhibition of PAI-1 in 5XFAD mice reduces abnormal vascular remodeling and improves cognition in the 5XFAD mice, without reducing plaque burden; and importantly that expression of genes within the vascular remodeling pathways are dramatically reduced in 5XFAD mice receiving the PAI-1 inhibitor. Based on the current literature and our preliminary data we will test the novel hypothesis that there is a causal relationship between vascular remodeling, and impaired cognition in the context AD, and that PAI-1 plays a critical role promoting pathologic vascular remodeling during AD development and progression.