Grace E. Stutzmann - US grants

[unreadable] DESCRIPTION (provided by applicant): Neuronal Ca2+ signaling through endoplasmic reticulum (ER)-localized inositol trisphosphate (IP3R) and ryanodine receptors (RyR) must be tightly regulated to maintain cell viability, both acutely and over the lifetime of an organism. Exaggerated ER Ca2+ release (up to 4-fold) has been associated with Alzheimer disease (AD) mutations expressed in cell cultures and young mice, but little is known of Ca2+ dysregulations during the normal and pathological aging processes using adult and aged models. The hypothesis is that early intracellular Ca2+ dysregulation represents a unique 'calcium-opathy' that contributes to later progression of AD, and is not an accelerated component of normal aging. Aim I of this study will determine and differentiate the distinct roles of neuronal IP3 and Ry Ca2+ channels in a non-transgenic control mouse and the 3xTg-AD mouse model of AD. Aim II will analyze the effects of age and AD mutations on the magnitude of the exaggerated ER Ca2+ signals, determine downstream effects on electrophysiological membrane properties and synaptic activity, and parse the contributions of PS1, APP, and tau mutations by comparing 3xTg-AD, APP/Tau, PS1KI and NonTg control mice. Aim III will seek to pharmacologically reverse the exaggerated ER Ca2+ release in the 3xTg-AD neurons and measure effects on amyloid plaque deposition. Likewise, amyloid plaques will be cleared in older 3xTg-AD mice using immunotherapy techniques, and establish if there is a functional relationship between the early Ca2+ dysregulation and AD histopathology. These studies combine electrophysiological recording in brain slices, 2-photon Ca2+ imaging, and flash photolysis of caged compounds from control (non-transgenic), 3xTg-AD, APP/Tau and PS1KI mice at young, adult, and old ages. Immunohistochemical techniques will be used to map and quantify changes in the expression of IP3R and RyR subtypes, and extent of AD histopathology. These findings will elucidate intracellular signaling changes and downstream effects on neuronal physiology that occur both in normal aging, and in neurodegenerative disorders such as AD. PUBLIC HEALTH RELEVANCE: The objective of this study is to determine the functional relationship between early changes in neuronal Ca2+ signaling, and later pathophysiology associated with aging and Alzheimer's disease (AD). The results of this study will have scientific and clinical relevance by differentiating between neuronal signaling changes associated with normal aging and those associated with AD pathogenesis. Benefits to public health include the prospect for earlier AD diagnosis and novel therapeutic intervention, long before the onset of cognitive decline and irreversible histopathology. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A major problem in treating cocaine addiction is the high likelihood of relapse, even after months of abstinence. The persistence of relapse vulnerability suggests that it is maintained by long-lasting adaptations in the brain. There is an urgent need for therapeutic strategies to combat this problem. To this end, we have studied a rat model in which cue-induced cocaine craving progressively intensifies (incubates) over the first 2 months of withdrawal from extended access cocaine self-administration (SA). We showed previously that enhanced craving after ~1.5 months of withdrawal is mediated by Ca2+-permeable AMPARs (CP-AMPARs) that accumulate in nucleus accumbens (NAc) synapses. Thus, detailed information exists on the time-dependency of AMPAR and behavioral plasticity during incubation. In contrast, no information exists about plasticity of spines or NMDAR transmission in this model. The vast majority of studies on these latter endpoints have been performed after non-contingent cocaine regimens which are not directly useful for assessing cocaine craving, and most have examined only one withdrawal time. The objective of our proposal is to evaluate spine density and morphology, and NMDAR transmission at the single spine level, at 5 withdrawal times (15, 25, 35, 60 and 180 days) after discontinuing cocaine SA or saline SA (a control condition). Our central hypothesis is that there are increases in silent synapses early in withdrawal corresponding to an increased number of immature spines; later in withdrawal, silent synapses dissipate, the number of immature spines normalizes, and an increase in mature spines occurs coincident with CP-AMPAR insertion. This hypothesis will be tested by pursuing 3 Aims: 1) Characterize dendritic spine density and morphology in NAc neurons during the incubation of cocaine craving. Single NAc neurons will be filled with Lucifer yellow, imaged with confocal microscopy, and analyzed with NeuronStudio. 2) Investigate silent synapse formation and the contribution of NR2B-containing NMDARs to silent synapses during the incubation of cocaine craving. Silent synapses, particularly those expressing NR2B subunits, are preferentially primed for long term plasticity and represent a potential substrate fo AMPAR insertion. Patch clamp recordings, using minimal stimulation and coefficient of variation analyses, will assess the number of silent synapses; pharmacological approaches will assess the contribution of NR2B. 3) Determine Ca2+ signaling dynamics mediated by NMDARs and the relative proportion mediated by NR2B- containing NMDARs in NAc spines during the incubation of cocaine craving. To evaluate functional changes in NMDARs, NMDAR-mediated Ca2+ influx will be measured in single spines using patch clamp electrophysiology, 2-photon Ca2+ imaging, and flash photolysis of caged glutamate. Overall, by comparing the time-course of changes in cocaine craving, spine plasticity, silent synapse formation, and measures of Ca2+ influx, this exploratory proposal will generate necessary data that will enable us to formulate hypotheses about causality and ultimately develop strategies for reversing craving-related changes in synaptic function.

? DESCRIPTION (provided by applicant): Currently, there are no effective strategies or treatments to preserve cognitive function in AD patients. The recent series of failed clinical trias designed to target A? processing or inflammatory pathways highlight the need to explore alternative pathways. Novel compounds that can effectively preserve cognitive function and prevent disease progression in a manner distinct from previous approaches could provide new therapeutic opportunities. To this end, we developed >100 small molecule compounds designed as allosteric modulators of the ryanodine receptor (RyR), a large conductance calcium channel found on the ER membrane, as candidates for clinical testing in early AD or MCI patients. In both human AD patients and AD mouse models, increased RyR2 expression precedes the amyloid deposition, neuronal loss, and cognitive impairments. In AD mouse models, increased RyR-evoked calcium release is greatest in dendritic spines and synaptic compartments, and contributes synaptic pathology, increased amyloid and tau pathology, disrupted memory function, and other AD-defining features. We and others have recently demonstrated that treating AD mice with dantrolene, a RyR channel stabilizer, resulted in exciting therapeutic effects. Although our treatment regimens differed, the consistent results demonstrate normalized calcium signaling (Chakroborty et al., 2012a; Oules et al., 2012; Stutzmann et al., 2006), normal synaptic transmission and plasticity expression (Chakroborty et al., 2012a), restored synaptic integrity (Stutzmann lab), reduced A levels (Chakroborty et al., 2012a; Oules et al., 2012; Peng et al., 2012), restored RyR isoform levels (Chakroborty et al., 2012a; Oules et al., 2012), and improved performance on memory tests (Oule et al., 2012; Peng et al., 2012). These data support a strong case for stabilizing RyR function, with a focus on RyR2, as a therapeutic strategy. The objective of this study is to test and optimize compounds that will function as RyR channel regulators, serving to suppress excessive calcium release while maintaining physiological functions. The central hypothesis is that stabilizing RyR-mediated calcium release with novel small molecule compounds will normalize calcium signaling, preserve synaptic function, and reduce histopathology, thus serving as an effective therapeutic strategy to prevent cognitive decline in AD. This will be accomplished with the following Aims: 1. Identify optimal RyR2 stabilizing compounds in model cells and iPSC from human AD patients using calcium imaging, electrophysiological and immunoassay techniques. 2. Demonstrate broad efficacy of successful novel compounds on calcium signaling, synaptic plasticity and histopathology in chronically treated 3xTg-AD mouse models. The significance to public health is the development of an effective and novel treatment for AD.

? DESCRIPTION (provided by applicant): Currently, there are no effective strategies or treatments to preserve cognitive function in AD patients. The recent series of disappointing clinical trials highlight the need to explore alternative pathways. Novel compounds that can preserve cognitive function and prevent disease progression in a manner distinct from previous approaches could provide new therapeutic opportunities. To this end, we are developing and testing small molecule compounds designed as allosteric modulators of the ryanodine receptor (RyR), a large conductance calcium channel found on the ER membrane, as candidates for clinical testing in early AD or MCI patients. In both human AD patients and AD mouse models, increased RyR2 expression precedes the amyloid deposition, tau histopathology, neuronal loss, and cognitive impairments. In AD mouse models, increased RyR-evoked calcium release is greatest in dendritic spines and synaptic compartments, and contributes to synaptic pathology and dysfunction, increased amyloid and tau pathology, disrupted memory function, and other AD-defining features. We and others have recently demonstrated that treating AD mice with dantrolene, a RyR channel stabilizer, resulted in exciting therapeutic effects. Although our treatment regimens differed, the consistent results demonstrate normalized calcium signaling (Chakroborty et al., 2012a; Oule et al., 2012; Stutzmann et al., 2006), normal synaptic transmission and plasticity expression (Chakroborty et al., 2012a), restored synaptic structure and integrity (Briggs et al., 2014), reduced A? levels (Chakroborty et al., 2012a; Oule et al., 2012; Peng et al., 2012), restored RyR isoform levels (Chakroborty et al., 2012a; Oule et al., 2012), and improved performance on memory tests (Oule et al., 2012; Peng et al., 2012; Stutzmann lab, unpublished data). These data support a strong case for stabilizing RyR function, with a focus on RyR2, as a novel therapeutic strategy for AD. The objective of this study is to design, test, and optimize compounds that will function as RyR channel negative allosteric modulators, serving to suppress excessive calcium release while maintaining physiological functions. The central hypothesis is that development and optimization of small molecule RyR stabilizers will generate therapeutic leads for clinical testing in early AD and MCI patients, and through the preservation of calcium homeostasis and synaptic function, will protect cognitive abilities. This will be accomplished with the following Aims: 1. Compound development and medicinal chemistry optimization. This will use iterative medicinal chemistry procedures and bioactivity assays in mice. 2. Rapid screening assay in cell culture systems and neurons from AD mice. Initial screening will use automated fluorometric testing of RyR-evoked calcium signals in cultured N2A cells in 96-well plates, followed by screening in primary neurons from control and AD mice. 3. In vivo verification in mouse models. Sub chronic treatment in AD and control mice, followed by physiological and biochemical assays, will then be used to identify and finalize the optimal compounds. The significance to public health is the availability of an effective and novel treatment for AD.

Abstract/Summary Alzheimer?s disease (AD) currently has no cure, nor do we understand the initiating disease mechanisms, particularly those causing memory loss. This aspect of the disease is potentially mediated through synaptic deficits. To date, detailed investigations into early pathogenic mechanisms contributing to synaptic pathophysiology have been lacking. In part, this may reflect technical challenges in measuring real-time signaling events within synapses, and difficulties in translating findings from model animal systems to human neurons. Here, we address these gaps and identify cellular and molecular signaling mechanisms of AD pathogenesis that contributes to synaptic impairments. The objective of this proposal is to detect early features of synaptic pathology in AD mouse models and human neurons, and identify targetable mechanisms that emerge early in the disease process. To this end, we have identified dysregulated Ca2+ signaling as a candidate mechanism, and our studies test assertion this by characterizing abnormal Ca2+ responses within synaptic compartments generated by evoked activity at the hippocampal Schafer collateral synapse, detailing structural deficits and functional consequences in pre- and postsynaptic compartments, characterizing short- term plasticity and synaptic transmission deficiencies and linking this to protein alterations mediating signaling abnormalities in two AD mouse models and in human neurons where feasible. The underlying premise is based on findings in human patients and AD mouse models demonstrating early changes in ryanodine receptor type 2 (RyR2) expression and marked increases in RyR-Ca2+ release within dendritic compartments such as spines. These events precede the amyloid deposition, neuronal loss, and cognitive impairments that define AD. As tightly regulated Ca2+ signals are essential to synaptic functionality, sustained Ca2+ dyshomeostasis stands to be a significant factor in AD-associated synaptic pathology and cognitive decline. Our central hypothesis is that there are prodromal deficits in synaptic structure and function which are caused by early dysregulation of intracellular Ca2+ signaling. Using whole cell and field potential electrophysiological approaches, 2-photon (2P) and ratiometric imaging Ca2+ imaging, electron microscopy (EM), immunoassays and protein biochemistry approaches in AD mouse models, and in human neurons (iN) derived from AD patients, we will examine how dysregulated signaling manifests and drives synaptic pathology in AD. Aim I investigates the emergence and nature of early synaptic transmission and plasticity encoding deficits in AD mice and human iN. Aim II identifies structural abnormalities within and between synaptic compartments in AD brains and iN, and explores the protein modifications driving RyR dysregulation in spines. Aim III will determine if normalizing intracellular Ca2+ signaling preserves synaptic structure and function using siRNA and pharmacological approaches. The public health significance is that this study will identify a proximal source of synaptic degeneration in AD which directly links to processes causing memory and cognitive loss.

Alzheimer?s disease (AD) is a devastating neurodegenerative disorder characterized by aggregation of ?-amyloid (A?) peptides, neurofibrillary tangles composed of hyperphosphorylated tau, and a progressive loss of cognitive function. While much is known regarding the biochemical composition and structure of amyloid and tau in AD, relatively less focus has been placed on the intracellular handling of detrimental protein products, and more specifically, how upstream deficits in organelle function can accelerate the disease process and directly contribute to memory impairments. While neurons rely on dedicated organelles to execute specific functions and sustain health, several in particular have been linked to AD pathophysiology, including the ER, which is important for protein assembly and intracellular calcium signaling; lysosomes, which are critical for breaking down and removing the cellular debris and misfolded proteins collected by authophagosomes; and mitochondria, which are responsible for the bioenergetics of the cell (Mustaly et al., 2018). In the global operations of maintaining neuronal viability, the functions of these organelles are highly inter-dependent, and they are often physically coupled to one another. Despite the close coupling, their respective roles in contributing to AD have typically been studied in isolation. For example, there are compelling studies detailing aspects of ER, lysosome, or mitochondrial dysfunction in AD, yet substantially less is understood about how altered interactions among these organelles can lead to pathogenic cascades. This isolationist approach may lead to critical oversights in understanding key processes in AD and missing potential therapeutic opportunities. Thus, the overall goal of this study is to identify mechanisms underlying deficiencies in specific organelle functions and examine how this affects their interactions, characterize how this potentiates AD pathology, and establish the upstream drivers of this cascade for consideration as a therapeutic target. This will be accomplished through the following Aims: Aim I: Determine if the AD-associated disruption in ER function disrupts lysosomal dynamics and clearance of aggregated proteins. Aim II: Determine the mechanism by which excess ER-Ca2+ release disrupts mitochondrial function and degradation. Aim III: Establish upstream drivers of intracellular pathogenic cascades and determine if targeting ER-homeostasis will resolve lysosomal and mitochondrial defects. The proposed study will have a significant impact on the field as it will provide new mechanistic information about how misaggregated proteins accumulate in AD and are associated with altered ER signaling. Moreover, targeting specific intracellular organelles may reveal effective new treatment strategies for AD.