Salvatore Oddo, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Alzheimer disease is marked by the accumulation of amyloid plaques and neurofibrillary tangles (NFTs). Clinically, AD patients show a progressive deterioration of memory and other cognitive functions. Recent evidence points to soluble Ap as an excellent candidate for the initial trigger of memory loss. A focus of this proposal is to elucidate the pathways by which Ap and tau interact. We are uniquely position to address this question, as we have generated a transgenic model of AD (3xTg-AD) that develops both plaques and tangles. The goal of the studies proposed for the mentored phase is to elucidate the temporal relationship between Aft and tau in the 3xTg-AD mice. Two specific aims are proposed: Aim 1 will determine if active A[unreadable] immunization prevents or delays the development of NFTs. Our earlier results indicate that passive A[unreadable] immunotherapy suffices to remove early but not late hyperphosphorylated tau lesions. Here we propose to determine if the temporal development of the tau pathology is altered by actively immunizing young, pre- pathological 3xTg-AD mice. Aim 2 will determine if genetically shifting A[unreadable] production from predominantly A[unreadable] 42 to Ap40 impacts the plaque burden and tau load and cognitive deficits. In this aim, we will use a genetic approach to lower A[unreadable] 42 production to determine the consequences of reducing A[unreadable] 42 production on the onset and progression of A[unreadable] and tau pathology and cognitive deficits in the 3xTg-AD mice. The main focus of the independent phase will be to identify molecular mechanisms underlying the A[unreadable] -induced cognitive decline. In particular two additional aims are proposed: Aim 3 will elucidate the role of AKT/CREB in the A[unreadable]-induced learning deficits. This aim follows up on our preliminary data showing that 4-month old 3xTg-AD mice have significantly reduced CREB activation compared to age- and gender-matched NonTg mice, following training in the MWM. Thus, we hypothesize that A[unreadable] 42 blocks CREB activation by directly or indirectly interfering with AKT activity. To test this hypothesis, we will use a genetic and immunological approach to block AP accumulation and determine if CREB and AKT activation deficits are restored following learning. In addition, we will directly increase CREB function to determine if cognitive deficits can be restored in the presence of A[unreadable]. Aim 4 uses a candidate approach to determine other molecular pathways underlying A[unreadable]-induced cognitive decline, and is part of our efforts to define the molecular pathways that link A[unreadable] to cognitive decline. Combined the proposed aims will help to elucidate the underlying molecular pathways linking A[unreadable] to cognition. The identification of pathways leading to cognitive decline may point to new therapeutic targets. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The Alzheimer disease (AD) brain is characterized by two types of protein aggregates, neurofibrillary tangles (NFTs), comprised of hyperphosphorylated tau, and amyloid plaques, comprised of amyloid-[unreadable] (A[unreadable]). Clinically, AD patients show a progressive deterioration of memory and other cognitive functions. Recent evidence points to soluble A[unreadable] as an excellent candidate for the initial trigger of memory loss;however, the molecular mechanisms underlying A[unreadable] -induced cognitive decline remain elusive. In our preliminary studies, we have identified the mammalian target of rapamycin (mTOR) as a potential molecular link between A[unreadable], tau and cognitive decline. Additionally, we show that A[unreadable] oligomers increase mTOR signaling, an event mediated by the [unreadable]2 adrenergic receptors ([unreadable]2ARs). To identify the mechanistic link between mTOR signaling and A[unreadable], tau and cognitive decline, three Specific Aims are proposed: Specific Aim 1 will test the hypothesis that the accumulation of A[unreadable] oligomers increases mTOR activity by a mechanism mediated by [unreadable]2ARs. mTOR plays a key role in regulating protein homeostasis;thus, unveiling the molecular pathways leading to its deregulation in AD will lead to a better understanding of the disease pathogenesis. Here we will dissect the molecular pathways that link the A[unreadable] accumulation to changes in [unreadable]2ARs/mTOR signaling. Specific Aim 2 will test the hypothesis that the A[unreadable] -induced increase in mTOR signaling further increases A[unreadable] pathology and exacerbates cognitive decline. Our preliminary data show that mTOR signaling is increased in 3xTg-AD and Tg2576 mice. Additionally, we show that reducing mTOR signaling with rapamycin, a selective mTOR inhibitor, rescues the early neuropathological and behavioral phenotypes in 6-month-old 3xTg-AD mice. Growing evidence shows that rapamycin may have mTOR-independent effects. To directly address the role of mTOR in AD, we will use a genetic approach and knockout mTOR in the brain of the Tg2576 mice. Specific Aim 3 will test the hypothesis that the increase in mTOR signaling directly contributes to the tau pathology. Our preliminary data show that restoring mTOR signaling in the 3xTg-AD mice suffices to reduce A[unreadable] and tau pathology. However, the tau pathology in these mice is highly dependent on A[unreadable] levels;therefore, it remains to be established whether the effects of restoring mTOR signaling on tau pathology are mediated by a direct interaction between mTOR and tau or are simply due to a decrease in A[unreadable] levels. Using a mouse model overexpressing wild type tau, we will use genetic and pharmacological approaches to decrease mTOR signaling and test the mechanistic link between mTOR signaling and tau pathology. Overall, the proposed Specific Aims will elucidate the underlying molecular pathways linking A[unreadable], tau and cognitive decline. The identification of the pathways that lead to cognitive decline may point to new therapeutic targets. PUBLIC HEALTH RELEVANCE: Alzheimer disease is the most common form of dementia among the elderly and the seventh leading cause of death in the United States. Our studies are aimed at understanding the molecular basis underlying memory loss and cognitive in AD decline and will facilitate the identification of potential therapeutic targets for this insidious disorder.

Part 1: Non-technical Description
This project is supported under the SBE Postdoctoral Research Fellowships (SPRF) program. Age-related cognitive decline, hereafter referred to as cognitive aging, is a fact of life. To this end, structural and functional brain changes invariably lead to decreased cognitive functions even in otherwise healthy individuals. Although there is a large body of work on ways to reduce cognitive deficits associated with disease states, little is known about the mechanisms underlying cognitive aging. This is concerning given that life expectancy is increasing and cognitive aging leads to a deterioration of general health for the aging population. One option to reduce cognitive aging is the supplementation of the maternal diet with choline (MCS), an essential nutrient grouped with the vitamin B complex. Preliminary work has demonstrated that MCS leads to amelioration of cognitive aging. The primary goal of this postdoctoral project is to elucidate the underlying neural and molecular mechanisms linked to MCS benefits. Additionally, the PI plans to examine genetic targets within brain structures associated with memory formation to isolate genes that are differentially altered by MCS and aging. This will likely reveal various gene targets that will be the focus of future cognitive aging studies. Because choline is a non-toxic nutrient found in food and can be easily supplemented orally, the research team believes that any modifications to the recommended daily intake amount to reduce cognitive aging will be expedited. To this end, the results of this application may help establish new guidelines on how a diet regimen of MCS should be implemented in expecting women to reduce cognitive aging in their offspring. Data will be shared with both the scientific and general community through presentations at conferences and public forums.

Part 2: Technical Description
The loss of cognitive function is a pervasive and often debilitating feature of the aging process. To this end, structural and functional brain changes invariably lead to decreased cognitive functions even in otherwise healthy individuals. Recent work has shown that supplementation of choline, an essential nutrient grouped with the vitamin B complex, in the maternal diet (MCS) reduces cognitive aging. However, the molecular mechanisms linked to MCS benefits remains elusive. Elevated homocysteine levels correlate with cognitive aging, and aberrant gene expression mediated by reduced DNA methylation may contribute to cognitive aging. Choline is the major dietary source of methyl groups for the conversion of homocysteine to methionine, and for the production of S-Adenosyl methionine (SAM). SAM is a key substrate for epigenetic mechanisms, such as DNA methylation. Therefore, we hypothesize that MCS may reduce cognitive aging deficits by (1) reducing the buildup of homocysteine levels, and (2) by altering fetal epigenetic mechanisms during development leading to functional improvements in late life. Herein, we will breed 2-month-old C57Bl/6 mice. One-third of the breeding pairs will be kept on a CTL diet (choline normal diet, with standard choline content of 1.1 g/kg choline chloride), while the remaining mice will be kept on a maternal choline supplemented (MCS) diet (5 g/kg choline chloride), from conception through postnatal day 21. The offspring will be kept on the same choline diet as the parents until weaning at postnatal day 21. Notably, a group of dams from the MCS groups will be injected every other day with a betaine-homocysteine S-methyltransferase blocker S-(ä-carboxybutyl)-DL-homocysteine (CBHcy) that prevents the choline-mediated decrease in homocysteine levels. Thus, we will be able to determine whether the benefits of MCS are directly linked to homocysteine levels. Mice will be tested behaviorally using a longitudinal and a cross sectional strategy at 2, 8, 15 and 18 months of age to collect data at multiple time points and control for re-test effects. Tissue will be processed to (1) examine dendritic spine number and morphology within the hippocampus and (2) to examine alterations of DNA methylation in the promoter region of neuronal dendritic morphology-related genes (Dlg4, Rac1, RhoA, Doc2b). We will complement this work by using an unbiased approach to identify genes that are differentially methylated by MCS. These experiments will be done exclusively in hippocampal CA1 neurons isolated by laser-capture microdissection. If successful, our results would dissect the underlying molecular mechanisms whereby choline supplementation reduces cognitive aging. Understanding MCS benefits at the behavioral, neural and molecular level may lead to a modification in the recommended amounts of choline required for pregnant mothers for optimal cognitive functioning and prevention of cognitive aging.

? DESCRIPTION (provided by applicant): Converging data suggest that in Alzheimer's disease (AD), the accumulation of amyloid-? (A?) and tau leads to a progressive deterioration of memory and other cognitive functions. However, the molecular pathways linking the buildup of A? and tau to cognitive deficits remain elusive. During the current grant cycle, we have shown that the mammalian target of rapamycin (mTOR) is hyperactive in neurons and astrocytes of human AD cases and in animal models of AD. We found that reducing mTOR signaling improved AD-like pathology in mice by restoring deficits in protein synthesis and by increasing A? and tau turnover. Furthermore, our preliminary data suggest that hyperactive mTOR signaling contributes to neurodegeneration in AD by facilitating necroptosis, a programmed form of necrosis. This novel and exciting finding may answer a key, and yet unresolved question: which mechanisms govern cell loss in AD. The overall hypothesis of this application is that hyperactive mTOR contributes to AD pathogenesis by disrupting protein homeostasis in neurons and glia leading to cell loss. To this end, we propose three Specific Aims. Specific Aim 1 will test the hypothesis that hyperactive S6K1, a downstream effector of mTOR, contributes to AD pathogenesis by altering protein translation. Our preliminary data implicate S6K1 hyperactivity as a previously unidentified mechanism underlying synaptic and cognitive deficits in AD. Indeed, reducing S6K1 hyperactivity improves AD-like pathology in 3xTg-AD mice. Here we will use complementary approaches to dissect the mechanisms downstream of mTOR/S6K1 that link this pathway to AD pathogenesis. Specific Aim 2 will test the hypothesis that hyperactive mTOR contributes to neurodegeneration in AD by facilitating necroptosis. Our preliminary data indicate that necroptosis, a programmed form of necrosis, contributes to neurodegeneration in AD. Consistent with our hypothesis, data from the literature show that mTOR plays a key role in regulating necroptosis. To test our hypothesis, we will systematically modulate necroptotic signals in animals and cells with different levels of mTOR activity. Specific Aim 3 will test the hypothesis that hyperactive mTOR in astrocytes contributes to A? accumulation, cognitive dysfunction, and neurodegeneration. Our preliminary data show that mTOR is hyperactive in astrocytes of AD mice as well as of human AD cases. This is extremely exciting not only because mTOR regulates the scavenger functions of astrocytes but also because activated astrocytes are known to secrete toxic factors that may induce necroptosis. We will use newly developed genetic tools to modified mTOR in animal models of AD and in human primary astrocytes isolated from human AD cases. Taken together, the experiments proposed in this application will identify the mechanistic links among mTOR, A? and tau accumulation, as well as neurodegeneration and cognitive deficits. Furthermore, given the role of mTOR signaling in aging, our results may unveil new mechanisms by which aging contributes to the development of AD. Elucidating these mechanisms will likely identify several novel putative therapeutic targets.

Abstract Tau is a protein involved in several neurodegenerative disorders, including Alzheimer's disease (AD), frontotemporal dementia, Pick's disease, and corticobasal degeneration. Growing evidence points to tau as a valid therapeutic target for mitigating some of these disorders. However, the role of tau in the adult brain remains elusive. We used homologous recombination to flox exon 4 of the mouse tau gene. By combining these tau floxed mice with an inducible, neuronal-specific, CRE line, we are uniquely positioned to selectively knockout Mapt in adult neurons. In this application, we will test the hypothesis that tau is necessary for learning and memory in the adult brain. Specifically, we will leverage these newly developed tau conditional knockout mice by removing tau from the brains of 6-month-old mice. This will be accomplished by crossing the tau floxed mice with a tamoxifen-inducible Cre recombinase, whose expression is controlled by a neuronal specific promoter. We will assess the acute and chronic effect of knocking out tau by testing mice in a battery of cognitive and non-cognitive behavioral tests, one week and two months after the Cre-mediated removal of Mapt. We will also perform rescue experiments using the three major murine tau isoforms. These experiments will determine whether removing tau in the brain of adult mice has an effect on cognitive and motor function. This is a critical step towards the development of anti-tau therapies for AD and other tauopathies. In summary, we have generated the much needed tau conditional knockout mice, which will allow us to selectively and inducibly ablate Mapt in the adult brain. These mice may have a long-lasting impact on the field and may represent an invaluable tool to evaluate the role of tau and study potential anti-tau therapies in AD and other tauopathies.  

Abstract Severe neuronal loss characterizes Alzheimer's disease (AD). However, the mechanisms by which neurons die remain elusive. Additionally, it is also unclear why some neurons within the same brain region are more resistant to neurodegeneration than others. In this application, we will attempt to address these two critical issues. We focus on necroptosis, a programmed form of necrosis, triggered by receptor-interactive protein kinases (RIPK) 1 and 3 and executed by the mixed lineage kinase domain-like (MLKL) protein. Upon activation by multiple inflammatory stressors, RIPK1 can trigger cell survival or cell death pathways, with the former being a default response to inflammatory stimuli. This default survival pathway is regulated, among others, by the Lys63- deubiquitylating enzyme cylindromatosis (CYLD) and MAPKAP kinase-2 (MK2), which ubiquitinate and phosphorylate RIPK1, respectively. We provide compelling evidence showing that necroptosis is activated in postmortem human AD brains and it correlates with Braak stage, brain weight, and tau pathology. We further show that blocking necroptosis in a mouse model of AD reduces neuronal loss. These novel and exciting data, together with the experiments proposed here, may answer two key but unresolved questions: 1. Which mechanisms do govern cell loss in AD? 2. What does make some neurons more susceptible to neurodegeneration than others? Our overarching hypothesis is that necroptosis contributes to neurodegeneration and selective neuronal vulnerability in AD. Specific Aim 1 will identify the mechanisms linking RIPK1 activation to necroptosis induction in AD. Specific Aim 2 will identify the mechanistic link between RIPK1 and tau. Specific Aim 3 will determine the role of RIPK1 in the gene expression dysregulation observed in AD. Impact: Neuronal loss is a cardinal feature of AD and invariably affects multiple brain regions. Despite this indisputable evidence, the mechanism by which neurons die is still unknown. We propose that necroptosis is a key mechanism by which neurons die in AD and propose experiments to dissect the role of this pathway in AD fully. Our results will open new opportunities for research and interventions for this insidious disorder. From a basic biology perspective, these studies will uncover new and critical knowledge into the pathogenesis of this disease. From a therapeutic perspective, these studies will determine to what extent targeting necroptosis might be a valid approach to mitigate neuronal loss in AD.

Severe neuronal loss characterizes Alzheimer's disease (AD); however, the mechanisms by which neurons die remain elusive. Elucidating the mechanisms underlying neuronal loss in AD will be invaluable to the development of new therapeutic approaches. In this application, we focus on necroptosis, a programmed form of necrosis, triggered by receptor-interactive protein kinases (RIPK) 1 and 3 and executed by the mixed lineage kinase domain-like (MLKL) protein. Upon activation by multiple inflammatory stressors, RIPK1 binds to RIPK3 to form a multiprotein complex known as necrosome, which is sufficient for necroptosis activation. The necrosome forms on the membrane of autophagosomes highlighting a close interaction between necroptosis and autophagy. We provide compelling evidence showing that necroptosis is activated in AD where it may contribute to neurodegeneration. Consistently, we and others have reported that in AD brains RIPK1 is upregulated in microglia and neurons. We also generated a causal gene regulatory network to model RIPK1 interactions in AD and found that RIPK1 activity may explain a significant portion of transcriptomic changes in AD. We further show that pharmacologically decreasing RIPK1 activity reduces neuronal loss in 5xFAD mice. In this application, we will test the overarching hypothesis that RIPK1 contributes to neurodegeneration in AD by activating necroptosis. Specific Aim 1 will identify the role of microglial and neuronal RIPK1 in AD. Specific Aim 2 will determine the mechanisms of RIPK1-mediated necroptosis activation in AD. Specific Aim 3 will identify new strategies to block RIPK1-mediated neurodegeneration. Impact: The mechanisms by which neurons die are still unknown. We propose that RIPK1-mediated necroptosis is a crucial mechanism of neurodegeneration in AD and propose experiments to dissect the role of this protein kinase in this insidious disorder. Our results will open new opportunities for research and interventions for AD and will identify new highly translational compounds to block necroptosis activation.

Abstract Aging is the primary risk factor for Alzheimer's disease (AD) and related disorders. Nevertheless, the mechanisms by which aging contributes to the onset of the disease remain elusive. In this application, we will attempt to identify critical signaling pathways that might link aging to AD pathogenesis. We focus on the mammalian target of rapamycin (mTOR), a ubiquitously expressed protein with an established link to aging. For example, reduction of mTOR signaling in mice extends lifespan and improves age-dependent motor dysfunction, insulin sensitivity, obesity, and immune system function. A large body of evidence also points to mTOR as playing a pivotal role in regulating microglia function during physiological and pathological conditions. For instance, reducing mTOR signaling in microglia reduces secretion of pro-inflammatory cytokines, reactive oxygen species, and other toxic compounds from activated microglia. We and others have shown that mTOR signaling is increased in postmortem human AD brains. In addition, we show that genetic and pharmacological reduction of mTOR ameliorates amyloid-? and tau pathology, and improves synaptic function and cognition in multiple animal models AD. Mechanistically, we identified the Solute Carrier Family 8 Member 2 (SLC8A2), a neuronal Na+/Ca2+ pump, as a possible link between mTOR and AD. These novel and exciting findings led us to the following hypothesis: mTOR represents a link between aging and AD. Specific Aim 1 will identify the role of microglial mTOR hyperactivity in AD. These experiments will lead to a better understanding of how mTOR modulates cognition and neurodegeneration in AD. Given the role of mTOR in aging, this aim is a critical step toward unveiling the mechanisms linking aging and AD. Specific Aim 2 will elucidate the signaling pathways linking mTOR to AD pathogenesis. In addition, if successful, the results of this aim will corroborate SLC8A2 as a novel molecular target for AD and related disorders. Specific Aim 3 will identify the role of mTOR in the gene expression dysregulation observed in AD. The results of this Aim will provide a detailed mTOR gene regulatory network in the context of aging and AD and identify an mTOR-mediated gene expression signature that is unique between aging and AD. Impact: This application will define the mechanistic links between mTOR and AD. Furthermore, given the role of mTOR signaling in aging, our results may unveil new mechanisms by which aging contributes to the development of AD. Elucidating these mechanisms will likely identify several novel putative therapeutic targets for AD.