Nigel T. Maidment, Ph.D. - US grants

A large body of literature exists supporting a role for central dopamine systems in mental illness. The identification of the neuropeptides - cholecystokinin and neurotensin - in sub-populations of mesolimbic dopamine neurons provides possible targets for future, more selective, drug therapy. The broad objective of this proposal is to elucidate the functional significance of the co-existence phenomenon as it pertains to the dopamine neurons in order that such drug development can proceed in a logical fashion. The focus is on understanding the interactions between these three transmitters both at the pre-synaptic level in terms of the regulation of their co-release and post-synaptically in relation to the integration of their actions once released into the synapse. The approach taken - that of measuring neurotransmitter release directly from discrete brain regions in vivo and in tissue culture - takes advantage of recent developments in the P.I.'s laboratory allowing such measurements to be extended to neuropeptides. Thus, the techniques of microdialysis, in vivo voltammetry and dopamine cell culture will be applied in concert to achieve the following specific aims: 1. To test the hypothesis that, in regions where they are co-localized within the same neurons, dopamine, neurotensin and cholecystokinin are differentially released in vivo under specific patterns of neuronal stimulation. 2. To test the hypothesis that the in vivo release of dopamine, cholecystokinin and neurotensin from terminals in the forebrain is differentially controlled by autoreceptors for these transmitters on the cells of origin in the midbrain. That is: a)Does dopamine somatodendritic autoreceptor stimulation or blockade differentially modulate coexisting neuropeptide release? b)Is the release of the neuropeptides similarly under the control of somatodendritic neuropeptide autoreceptors? 3. To elucidate the functional terminal presynaptic interactions between dopamine, cholecystokinin and neurotensin in the mesolimbic and nigrostriatal pathways at the level of transmitter release by addressing the following questions: a)Is dopamine release and turnover controlled by activation of terminal receptors for these peptides? b)Is the release of neurotensin and cholecystokinin influenced by terminal dopamine receptors? c)Is neurotensin and cholecystokinin release self-regulated by terminal autoreceptors for these peptides? 4. To test the hypothesis that co-released dopamine, neurotensin and cholecystokinin in the nucleus accumbens can act independently at the post- synaptic level to differentially modulate enkephalin and GABA release in this region and in the ventral pallidum.

The ability of a drug to induce an addictive behavior, characterized by obsessive drug-seeking and drug-taking in both animals and humans, necessitates an interaction between the drug of abuse and specific populations of cells in the brain. Our desire to understand the neuronal basis of this complex, self-perpetuating, and sometimes fatal drug-induced behavior, has fueled an intense research effort at all levels of neuroscience from molecular to behavioral. Much of this research has focussed on the mesocorticolimbic dopamine system as the site of action for the rewarding properties of many classes of abused drugs. However, endogenous opioid peptides have also been implicated as mediators of this phenomenon. Moreover, these peptides, together with GABA, are present in a major output of the nucleus accumbens projecting to the ventral pallidum - a region of the brain strongly implicated in the reward process. This proposal describes experiments designed to provide in vivo neurochemical evidence for a role of these transmitters in mediating both cocaine and opiate reward. Recent developmental work in the P.I.'s laboratory has demonstrated the feasibility of using microdialysis to monitor opioid peptide release in the basal ganglia and limbic system of freely behaving rats. Based on preliminary data, demonstrating a morphine- and cocaine-induced stimulation of opioid peptide release in the pallidum, the proposal will address the following specific aims. 1. To determine the pharmacological and neuroanatomical basis of acute opiate-induced endogenous opioid peptide release in the pallidum using receptor sub-type selective drugs and central administration. 2. To characterize the effects of repeated forced- and self-administration of opiates on opioid peptide release and to examine the effects of withdrawal on extracellular opioid peptide levels. 3. To elucidate the pharmacological and anatomical basis of acute cocaine effects on opioid peptide release in the nucleus accumbens - pallidal system. 4. To investigate the effects of self-administered and long-term cocaine treatment on extracellular opioid peptides. These studies will provide valuable information concerning the mechanism of opiate and cocaine addiction which could ultimately lead to the development of new therapeutic strategies for treatment of substance abuse.

DESCRIPTION (Adapted From The Applicant's Abstract): Previous research has implicated the mesotelencephalic dopaminergic system as the primary substrate mediating the reinforcing effects of many abused drugs. However, endogenous opioid peptides have also been implicated in this phenomenon. The presence of enkephalins in the nucleus accumbens-pallidal projection, and the demonstrated importance of this pathway in reinforcement, provides an anatomical basis for such a hypothesis. Previously, using microdialysis linked to a solid-phase radioimmunoassay, we demonstrated that opiate administration elevates extracellular concentrations of enkephalins in the pallidum. The first part of the current proposal will examine further the mechanisms underlying this effect. Repeated intermittent administration of opiate and psychostimulant drugs induces a state of behavioral sensitization believed to be largely dependent on a sensitized mesotelencephalic dopamine system. This phenomenon has been implicated in the attribution of 'incentive salience' to environmental cues associated with drug administration. However, not all the evidence is in favor of such a hypothesis and the possible role of other neurotransmitters/neuromodulators in this process needs to be investigated. Our preliminary data suggest that heroin-induced pallidal enkephalin release may exhibit acute sensitization. We will therefore examine if repeated intermittent administration of heroin induces sensitization to the enkephalin-releasing effect of this drug. We will determine for how long such sensitization persists and whether its expression is dependent on conditioned environmental cues. The final part of the proposal will combine microdialysis with a modified conditioned place-preference paradigm to examine if environmental cues themselves elicit a pallidal enkephalin release response in animals conditioned to associate such cues with heroin administration. These studies will provide valuable information concerning the mechanisms underlying heroin addiction.

abnormal involuntary movement; glutamate receptor; dopamine receptor; Parkinson's disease; GABA receptor; subthalamus; neurotransmitter transport; dihydroxyphenylalanine; lenticular nucleus; substantia nigra; basal ganglia; brain electrical activity; glutamates; receptor expression; neurochemistry; behavioral /social science research tag; laboratory rat; microdialysis;

The identification of genetic mutations responsible for familial forms of Parkinson's disease (PD) offers the potential to glean insight into the mechanisms underlying the sporadic form of the disease. Currentmouse models based on deletions or mutations in two such genes, parkin and alpha-synucein, do not exhibit dopaminergic neuron degeneration. However, close scrutiny of two of these models within our Center, has revealed behavioral (Project 1), neurochemical (Project 2) and electrophysiological (Project 3) deficits, which likely model early stages of the progressive human disease process, prior to neuronal degeneration. The goal of the Center is to build on this multidisciplinary approach to determine the time-course of progression of cell dysfunction in multiple genetic models of PD in order to identify common deficits, since these are most likely to be of relevance to sporadic PD. By elucidating the mechanisms responsible for these deficits we hope to uncover therapeutic targets for preventing disease progression, prior to the loss of significant numbers of dopamine (DA) neurons. This project builds upon our observation that striatal extracellular DA levels are elevated in parkin exon 3 KO and alpha-synuclein over-expressing mice, a finding of significance given the potential of DA to promote oxidative stress and, ultimately, cell death. We will determine: 1) if this observation generalizes to parkin exon 2 KO mice, to mice, produced by the Mouse Genetics Core, expressing a parkin mutation shown to cause DA cell death in flies, and to other models of alpha-synuclein over-expression 2) whether transmitter systems other than DA are also disrupted, given the recognized importance of non-motor symptoms in PD, studied in Project 5, and modeled in Project 1; 3) if the increased extracellular DA results from dysregulation of vesicular release, reuptake, reverse transport or metabolism; 4) whether, given the association of parkin and synuclein with components of synaptic vesicles (Project 4), our observations can be explained by disruption of synaptic vesicle cycling.

DESCRIPTION (provided by applicant): Endogenous opioid systems are increasingly recognized as important regulators of basal hedonic homeostasis and as significant factors mediating acute and chronic responses to multiple drugs in addition to opiates. Our previous work has emphasized the role of enkephalins, acting through mu opioid receptors in mediating the opioid component of basal "hedonic tone". We propose to extend these studies to examine the role such systems play in controlling acute behavioral responses to cocaine, whether administered non-contingently or self-administered. The rewarding and reinforcing effects of cocaine will be studied in mu and delta opioid receptor null mice and in mice deficient in pro-enkephalin or B-endorphin using the conditioned place preference and operant self- administration paradigms. In addition to evaluating the acute effects of cocaine in such opioid-deficient mice, we will examine these mice for anomalies in sensitized motivational responses to repeated non-contingent cocaine administration and for propensity to reinstate drug-seeking following prolonged extinction of such behavior. The loci within the brain wherein these endogenous opioid-mediated phenomena originate will be explored by two parallel approaches focusing initially on the ventral pallidum and nucleus accumbens: 1) attempts to reverse phenotypes observed in opioid receptor null mice will be made by local, lentiviral-mediated, expression of the receptor and 2) evidence of changes in opioid peptide release in these structures during various stages of cocaine exposure will be explored using microdialysis combined with mass spectrometry.Project Narrative These studies will increase our understanding of the neurochemical underpinnings of relapse following prolonged abstinence. These are key issues for the development of effective medications to treat cocaine addiction and the information gleaned from these studies will likely generalize to other classes of abused substances.

DESCRIPTION (provided by applicant): The goal of this application is to identify plasma peptide signatures that reflect neuroplasticity in regions of the brain associated with multiple stages of the addictive process, modeled initially using the simple conditioned place preference (CPP) procedure in rats. A focused approach is proposed in which the initial search will begin in the extracellular compartment of brain regions implicated in addictive processes, using microdialysis linked to mass spectrometry (high resolution nanobore LC-MS and data-dependent LC-MS2). We will then search for, and quantify, such identified candidate peptides in plasma in a targeted fashion, using LC-MS3 and/or LC-MRM. The discovery phase of the project will identify arrays of candidate peptides that, in brain regions considered central to addictive behavior, change extracellularly in response to chronic exposure to, and abstinence from, two highly addictive drugs of different pharmacological classes;namely morphine and cocaine. A parallel strategy will focus entirely on readily releasable peptides by constructing a subtractive screen of brain dialysates under potassium depolarization versus basal conditions. In both of these approaches blood plasma samples will be collected simultaneously. Peptides identified in either of these dialysate screens will subsequently be searched for in plasma in a targeted fashion. Peptides that are (a) detectable in both dialysate and plasma, and (b) change in concentration in tandem as a product of drug history or depolarization will be selected for the validation phase. In the validation phase, candidates identified in the discovery phase will be studied in longitudinal experiments using the place preference paradigm to correlate changes in plasma content with multiple phases of the conditioning experiment: drug conditioning, initial testing, incubation (withdrawal), re-testing, extinction and reinstatement. We expect to identify individual peptide signatures in plasma that reflect different stages of the addiction process to the extent that they are modeled by CPP. These experiments will focus on cocaine and morphine, but will be extended to other abused drugs and to other behavioral models (e.g. self-administration) in future experiments beyond the scope of this proposal. PUBLIC HEALTH RELEVANCE: These studies will provide plasma peptide signatures unique to different stages of one simple model of addictive behavior. Once this proof of principle is established the approach could be extended to other more complex addiction models, and ultimately to humans, thereby providing markers of underlying addictive processes. This is a key issue for the development of effective medications to treat addiction to multiple classes of abused substances.

The ability of reward-predictive cues to trigger reward seeking is a major facet of addiction. This ability develops through the process of Pavlovian incentive learning. Importantly, such Pavlovian incentive learning or motivation is distinct from the emotional/hedonic experience of rewards. This distinction is often referred to in the literature as wanting versus liking. Endogenous opioid peptides have long been considered mediators of the hedonic aspects of reward. However, recent evidence also implicates endogenous opioid peptides in the motivational aspects of reward processing, including Pavlovian incentive motivation. We predict that hedonia and incentive motivation are underpinned by distinct opioid-containing elements of striatal output circuitry. To test this prediction, we will combine a measure of licking architecture with the Pavlovian-instrumental transfer task to assess both the hedonic impact of sucrose reward and the influence of conditioned cues on sucrose seeking in mice with genetic manipulations of striatal opioid-containing circuitry. Optogenetics will be employed to selectively modulate the activity of direct and indirect striatal outputs while monitoring both behavioral output and dopamine release with fast scan cyclic voltammetry or microdialysis. We will test two major hypotheses. Firstly, that the hedonic effects of opiates are mediated through mu opioid receptors in the indirect striatal output pathway while opiate facilitation of cue-induced reward seeking is mediated through mu opioid receptors in the direct pathway in a dopamine-dependent fashion. This will be addressed using genetic manipulations that either ablate mu opioid receptors from specific elements of striatal circuitry or isolate mu opioid receptor expression to those elements. The second hypothesis tested will be that enkephalin in the indirect pathway mediates palatability in the absence of exogenous drug by acting in the ventral pallidum. We speculate that this same source of enkephalin may influence cue-induced incentive motivation via collaterals to direct pathway neurons. This hypothesis will be tested using selective genetic manipulations of pro-enkephalin expression. Alongside the Pavlovian-instrumental transfer task, we will also apply advanced methodologies such as optogenetics, microdialysis and fast cyclic voltammetry to test our hypotheses.

DESCRIPTION (provided by applicant): Despite evidence linking mesolimbic dopamine sensitization to hypersensitivity to reward-paired cues during protracted cocaine withdrawal; the molecular mechanisms underlying this phenomenon are poorly understood. Here, we test the novel hypothesis that changes in energy utilization within dopamine neurons brought about by repeated cocaine exposure result in disruption of insulin signaling, which negatively impacts synaptic dopamine clearance via the dopamine transporter (DAT). This then results in heightened dopamine transmission in response to cocaine-paired cues, thereby precipitating cocaine-seeking behaviors. The synergistic influence of dietary changes associated with cocaine use on these processes will be assessed using a high-fructose diet that we have previously shown to disrupt insulin signaling in the brain. Markers of mitochondrial function and insulin signaling will be assessed in ventral tegmental area dopamine neurons, post-mortem, collected after varying durations of cocaine self-administration followed by withdrawal, with and without access to a high-fructose diet. In parallel studies, the impact of such exposures on dopamine clearance will be assessed by fast-scan-cyclic voltammetry while monitoring the impact of cocaine-paired cues on cocaine seeking actions. Finally, a causal link between insulin signaling dysregulation and these neurochemical and behavioral phenomena will be assessed by blocking development of insulin resistance with pioglitazone, a drug currently approved for the treatment of Type 2 diabetes. These studies will provide fundamental information regarding the role of cocaine-induced metabolic aberrations in lowering the threshold for relapse and may uncover new targets for therapeutic intervention.

DESCRIPTION (provided by applicant): The decision to engage in an action leading to a rewarding outcome is governed by several factors that may vary with age. Primary among these is the value placed on the reward at the time of the decision, which is continually updated on the basis of previous emotional experience with the reward. Cues associated with reward experience also impact decision-making through a Pavlovian incentive motivation process. Endogenous opioids play a major role in mediating emotional responsiveness to palatable food rewards through actions in the nucleus accumbens/ventral pallidum, and independently facilitate instrumental incentive learning via mu opioid receptors in the basolateral amygdala. Dopamine transmission in the nucleus accumbens is essential for the invigorating effects of reward-associated cues. The goal of this proposal is to examine whether developmental transitions in endogenous opioid and dopamine transmission in these brain regions manifest as shifts in the manner in which adolescent, middle-aged, and aged rodents react to changes in experienced reward value and to environmental cues predictive of reward. We will use licking architecture during consumption of sweet solutions to assess how emotional experience of reward (palatability) changes over the lifespan and an instrumental incentive learning task to parse the effects of aging on how the emotional impact of a reward is translated into a goal value used in the decision-making process. The Pavlovian-to- instrumental transfer test will assess the ability of reward-paired cues to initiate and invigorate reward-seeking actions and how this process changes over the lifespan. The role of developmental shifts in opioid and dopamine transmission in the shifting influence of these factors across ages will be determined using central pharmacological manipulations, viral-mediated gene transfer, in vivo measures of enkephalin and dopamine release, and mice with targeted conditional manipulation of pro-enkephalin expression. Understanding how aging impacts cue-evoked incentive motivation, reward palatability, and incentive learning may inform development of targeted cognitive-behavioral and pharmacological therapies for age-dependent decision- making impairments and behavioral control disorders.

DESCRIPTION (provided by applicant): Efforts to understand how neurons within networks interact to control behavior will be greatly facilitated by a means with which to measure multiple neuroactive molecules in the brain simultaneously and in near-real time. Existing methods either offer rapid measurements of a single analyte (e.g., fast-scan cyclic voltammetry) or provide multiple analyte measurement with insufficient temporal resolution (microdialysis). Here, we will develop an implantable microprobe capable of simultaneous rapid monitoring of 3 ubiquitous neurotransmitters/neuromodulators: dopamine (DA), glutamate (Glut), and acetylcholine (ACh). Further, we will harness the power of optogenetics by incorporating an optical waveguide into the microprobe providing directional illumination of tissue juxtaposed to each sensing electrode on the microelectrode array. This will be achieved using an innovative silica-based microprobe design with embedded waveguides and light splitters, and with a micromirror embedded underneath each microelectrode for uniform directional light illumination. The resultant microprobe will combine local modulation of genetically isolated neuron terminals with measurement of transmitter release from those terminals, together with resultant changes in the release of other transmitters from neurons within the network, all in the context of a freel behaving animal. These sensors will be transformative in a wide range of neuroscience applications due to the involvement of DA, Glut and ACh as inter-neuronal signaling molecules in multiple brain regions controlling a plethora of functions from learning and memory to motor control. Our evaluation of their performance in vivo will focus on the striatum, a brain region involved in motor control and motivated behavior. Precise coupling of multiple transmitter release events to behavioral actions will provide valuable information pertinent to the search for therapeutic interventions for multiple neurological and psychiatric disorders.

DESCRIPTION (provided by applicant): Obesity has reached a crisis point in the Western world, especially in the US where one third of the population is obese, and many more are classified as overweight. A major cause of obesity is overeating, the majority of which is targeted to energy-dense, highly palatable foods. Feeding is the product of a complex interaction between humoral, metabolic, physiological and psychological processes. Regarding the latter, advances in the neurobiology of motivated behavior and addiction demonstrate that endogenous opioid peptides and dopamine are central to affective processing of rewarding stimuli, especially food, and to learning about rewards and the relevance of reward-related stimuli, making them logical suspects for focused investigation in the etiology of obesity. In the first part of this project, we will test the hypothesis that chronic consumption of energy-dense highly palatable foods disrupts opioid-dependent instrumental incentive learning processes, resulting in discordance between the pleasurable experience of palatable food consumption and the incentive value assigned to the food that guides food-seeking behavior, leading ultimately to compulsive food-seeking. The hypothesized role of disruption in endogenous opioid transmission in the basolateral amygdala will be tested using a combination of pharmacological and genetic interventions and by measurement of enkephalin release. As environmental stimuli, such as carefully engineered advertising, packaging and product placement can potently influence feeding, the second part of the project will test the hypothesis that chronic consumption of highly palatable foods accentuates cue-induced food-seeking, and that this process involves plasticity in central dopamine and/or opioid transmission. Thus, we will combine a preclinical model of diet-induced obesity with a combination of advanced behavioral, neurochemical and genetic methodologies to yield data that promise to have important implications for guiding public policy and treating overeating and obesity.

Summary Apathy, defined as ?lack of motivation? or a ?quantitative reduction in goal-directed behavior? is a major and prevalent (up to 72%) debilitating symptom of Alzheimer's disease and related dementias. It is also associated with poor outcome, and is the primary cause of caregiver distress. Remarkably, however, very few studies have probed for disrupted reward processing and goal-directed decision-making underlying the apathy domain using rodent models of Alzheimer's disease. Apathy may result from dysfunction of one or more of the behavioral processes necessary to achieve goal-directed behavior, including internal and external determinants that motivate behavior, selection of goals, elaboration of a plan of action, initiation, execution, evaluation of goals achieved and feedback control of the behavioral response. We will employ a battery of behavioral tasks, used routinely in our labs to probe reward and decision making deficits in rats related to normal aging and addiction, to examine such processes in a double- transgenic rat model of Alzheimer's disease (TgF344-AD). This model expresses a single `Swedish mutation' human amyloid precursor protein (APPsw) gene plus a delta exon 9 mutant human presenilin-1 (PS1deltaE9) gene and manifests age-dependent cerebral amyloidosis, including an abundance of soluble amyloid-beta, that precedes tauopathy, gliosis, apoptopic loss of neurons in the cortex and hippocampus, and cognitive deficits. We will perform in-depth microstructural analysis of licking behavior during reward consumption as a measure of experienced hedonia; instrumental incentive learning to determine how experienced reward value is used to guide reward-seeking actions; Pavlovian-to-instrumental transfer to evaluate the motivational impact of reward-paired cues; and progressive ratio instrumental testing as a catch-all motivational assay of willingness to work for rewards, to test the hypothesis that these core emotional and motivational processes are disrupted in the TgF344-AD rat. In addition to probing for an apathy phenotype at a late stage of development when neuropathology is profound (18 months), we will also probe prior to presentation of overt neuropathology (3 months) and at an early stage of progression (6 months), to test the hypothesis that such deficits precede cognitive impairments in this model and that individual variability in motivation at early stages predicts severity of cognitive deficits and neuropathology assessed later in the animals' life. Apathy is a core behavioral symptom of Alzheimer's disease. Identifying the neurobehavioral basis for apathy in an animal model of the disease will provide targets for behavioral and pharmacological therapeutic intervention. Further, demonstration that emotional and/or motivational deficits precede and predict cognitive decline and overt neuropathology in an animal model will inform the development of tools for using measures of apathy (and their neural correlates) as biobehavioral markers of disease risk and onset in humans, allowing for the implementation of interventions that can be tailored to the unique needs of individual patients.

Demographic considerations predict an overwhelming burden on the U.S. health care system within the next few decades due to an influx of elderly patients with neurodegenerative disease. The most prevalent of these, Alzheimer's and Parkinson's diseases (AD and PD), are predicted to affect tens of millions of Americans by 2040. Both of these diseases involve progressively more aberrant brain proteostasis, associated with massive neuronal cell death. A variety of cytosolic and secreted brain chaperones contribute to maintenance of neuronal proteostasis in these and other neurodegenerative proteinopathies; of these, the secretory chaperone proSAAS has many compelling features. ProSAAS is expressed only in neurons and endocrine cells; because it traffics through the regulated secretory pathway, it becomes concentrated within dense core synaptic granules, and is released during neuronal activity. ProSAAS has been identified by five proteomics groups as a potential biomarker in neurodegenerative disease, and is found associated with aggregated proteins in the substantia nigra of PD patients as well as with amyloid plaques in AD?affected individuals. ProSAAS blocks the aggregation of both Abeta and alpha synuclein at highly substoichiometric ratios, and both endogenous overexpression as well as exogenous application reduce Abeta? and alpha synuclein?mediated neurotoxicity in primary neurons and cell lines. Most recently, we have shown that proSAAS overexpression is also functionally protective in vivo in a rat model of alpha?synuclein overexpression. In the proposed work, we will investigate the likely common mechanisms by which proSAAS protects neurons from neurotoxic aggregating proteins and peptides such as alpha synuclein and Abeta 1?42. We hypothesize that secreted proSAAS sequesters cytotoxic oligomers and fibrils extracellularly, reducing their concentrations at the synapse. Secondly, we hypothesize that endocytosed proSAAS acts intracellularly to similarly sequester cytotoxic proteins, speeding their degradation. Using cultured primary hippocampal and nigral neurons, we will determine whether proSAAS is involved in intracellular and extracellular Abeta and alpha synuclein sequestration. We will also determine whether intracellular expression of proSAAS confers a cytoprotective advantage compared to extracellular addition. Lastly we will assess whether endocytosed proSAAS accelerates the intracellular degradation of Abeta and alpha synuclein. In parallel, we will expand our exciting in vivo results to include the alpha?synuclein preformed fibril model to tease apart the potential sites of action of proSAAS in substantia nigra and striatum. Pre?degenerative changes in dopamine homeostasis, assessed using fast?scan?cyclic?voltammetry, will be correlated with proSAAS?mediated neuroprotection. Similarly, a mouse model of AD will be used to test the effects of proSAAS AAV?mediated over? and underexpression on the development of amyloid pathology. Collectively, these experiments will provide insight into biochemical mechanisms underlying the potent cytoprotective effects of the proSAAS chaperone protein.

PROJECT SUMMARY Decision-making operates over vastly different temporal scales. Although perceiving a sound requires information gathered over a time span of just milliseconds, deciding whom to marry may require years. Decision- makers also change over time. As we age, our perceptual and motor processing abilities change but also our knowledge base, our motivations and our comfort with risk change, all factors that contribute to our decisions. Recent work indicates that older adults may rely more on heuristics to inform their decisions, compared to younger adults. Conversely, we recently discovered that people with Parkinson?s disease, one of several related neurodegenerative proteinopathies falling along a continuum of alpha-synuclein and tauopathy that includes Alzheimer?s disease and variants of these two diseases, are impaired in the use of previously learned information (priors or heuristics) to guide perceptual decisions. Together these results indicate that the ability to use prior information for decisions changes over the lifespan and with diseases associated with aging. It is unknown how the use of priors or heuristics for perceptual decision-making develops or changes over the lifespan. It is equally unknown how the ability to use heuristics for decision-making is impaired with aging or neurodegenerative disease. Therefore, we propose to develop a novel animal model of decision-making behavior over the lifespan to test the hypothesis that the use of heuristics for decision-making differs between young and old and those with age-related proteinopathy and neurodegeneration. The results of our proposed experiments will provide a timeline of decision-making changes in a novel animal model of aging in health and disease. The results will provide the groundwork for future experiments designed to unravel the neural circuitry and neuropharmacology underlying decision-making changes over the lifespan and with age-related proteinopathies along the synuclein- tauopahy continuum, including Alzheimer?s disease, Parkinson?s disease and variants of these diseases, and thus facilitate the development of novel treatments for cognitive impairment associated with these diseases.

SUMMARY In our original grant, we proposed to investigate the likely common mechanisms by which the proSAAS chaperone protects neurons from neurotoxic aggregating proteins and peptides, such as alpha synuclein and Abeta. We hypothesized that secreted proSAAS sequesters cytotoxic oligomers and fibrils extracellularly, reducing their concentrations at the synapse, and that endocytosed proSAAS might act intracellularly to similarly sequester cytotoxic species. Using cultured primary hippocampal and nigral neurons, we are currently investigating whether secretory proSAAS is involved in intracellular and extracellular Abeta and alpha synuclein sequestration, and whether secretory proSAAS also accelerates the intracellular degradation of Abeta and alpha synuclein. However, three major findings were made recently in carrying out the above project which take the work in an unexpected but important new direction. The first is the discovery that delivery of proSAAS to the cytoplasm, by expression of signal-less proSAAS (?cyto-proSAAS?), results in the liquid-liquid phase separation and formation of large (2 - 4 µm) symmetric proSAAS spheres, formed by dynamic fusion of smaller spheres. The second major finding is that these cyto-proSAAS spheres specifically interact with TDP-43216-414 aggregates, and efficiently sequester these aggregates within the sphere cores. Thirdly, and most importantly, a collaboration with the Shorter laboratory provided important information that the interaction between proSAAS and TDP-43 is cytoprotective in a yeast model cell system. The proposed supplement to our existing ?Common Mechanisms? grant is designed to determine whether cytoplasmic proSAAS should also be studied, not only in proteostatic mechanisms in Alzheimer's disease, but also in the context of TDP-43 aggregation in another neurodegenerative disease, fronto-temporal dementia. Obtaining a one-year supplement to investigate the functional properties of cyto-proSAAS will provide us with the opportunity to exploit our exciting findings regarding the highly unusual physical properties of cyto-proSAAS in forming ?aggregate sequestration? spheres. This supplement will also permit us to determine whether cyto-proSAAS expression is relevant to blocking Abeta and TDP-43 cytotoxicity in human cells (indeed, cyto-proSAAS expression may represent an improved avenue to achieve our original specific aim of ameliorating Abeta cytotoxicity -original Proposal Aims 1 and 3). Ultimately, this research will provide insight into whether proSAAS-mediated cytoplasmic aggregate sequestration should be further explored as a possible therapeutic approach in neurodegenerative disease. Lastly, it should be mentioned that given the unpredicted costs of the Covid-19 research shutdown, we clearly require additional funding to work on cyto-proSAAS, as we will otherwise be solely dedicated to recovering lost time in completing our original Aims. 1