Robert Levenson, M.D., Ph.D. - US grants

culture; emotions; face expression; paralinguistic behavior; gender difference; fear; angers; questionnaires;

The objective of this proposal is to apply the techniques of molecular genetics to the analysis of Na,K-ATPase structure, function and biogenesis. We have developed an expression system to test the biological activity of cloned rodent Na,K-ATPase genes. This system forms the framework for experiments designed to analyze structure function relationships for the ATPase and control mechanisms underlying ATPase biosynthesis and assembly. The specific aims of the proposal include: 1) Interaction of the Na,K- ATPase with Cardiac Glycosides. We will evaluate the relationship between enzyme structure and the binding of cardiac glycosides. Initial experiments will involve the construction of chimeric cDNA molecules between cDNA molecules encoding ouabain resistant and ouabain sensitive forms of the Na,K-ATPase in order to delineate regions of the alpha chain which interact with the drug. We will then use site directed mutagenesis to alter specific residues in a cDNA encoding a ouabain sensitive ATPase in an effort to convert the encoded enzyme to ouabain resistance. 2) Subcellular Localization of Na,K-ATPase Isoforms. The technique of in situ hybridization will be used to learn whether alternative forms of Na,K-ATPase mRNA are expressed within the same or different cells of a particular tissue. Peptide derived antibodies specific for each ATPase isoform will be developed and used to distinguish alternative ATPase isoforms in cells synthesizing more than one isoform and to identify sites of subcellular localization. The goal of these experiments will be to obtain basic information regarding the relationship between isoform expression and function. 3) Control Mechanisms Affecting Na,K-ATPase Biosynthesis. We will attempt to isolate and characterize DNA sequences which may be responsible for the tissue specific and developmentally regulated expression of Na,K-ATPase mRNAs. Initial interest will focus on identification of the promoter and other 5' control regions involved in the regulation of Na,K-ATPase mRNA expression. We will also attempt to identify regions within alpha and beta subunit mRNAs which may play a role in the postranscriptional regulation of subunit biosynthesis. 4) Relationship of the beta Subunit to Na,K-ATPase Function. We will attempt to develop an assay system which measures the biological activity of the 8 subunit by creating a system in which overexpression of the beta subunit is required for cell viability. This system will then be used to assess structure function relationships for the beta subunit.

The goal of the proposed project is to isolate, characterize and use as an analytical tool the DNA sequences coding for the Na,K- ATPase alpha and beta subunits from rat heart. By comparing rat heart cDNAs with the previously characterized alpha and beta subunits genes from rat brain we will determine whether Na,K- ATPase molecules of differing primary sequence are present in heart tissue. The availability of cDNA probes for the Na,K- ATPase will permit us to address basic issues with respect to control mechanisms which affect myocardial Na,K-ATPase. These include: a) Analysis of ATPase gene expression in specific cells of the heart. We will use in situ hybridization techniques to determine whether alternative forms of ATPase mRNA are expressed in specific cells of the developing and adult rat heart. b) Regulation of ATPase gene expression. By nucleic acid hybridization techniques, we will determine whether physiological stress can lead to regulation of the activity of the Na,K-ATPase in a cell via alteration in the expression of Na,K-ATPase mRNA. c) Organization of the ATPase Gene Family. In order to understand the molecular basis for Na,K-ATPase isoform diversity, we will attempt to determine the number of copies of the ATPase gene in rat and examine the organization of these genes. This approach could also lead to the identification of regulatory regions which may control cell and tissue-specific expression of the Na,K-ATPase gene family. d) Development of Na,K-ATPase antibodies. Identification of sites of primary sequence difference between ATPase isoforms will form the basis for the development of a panel of antibodies capable of recognizing specific isoforms of the Na,K-ATPase. These antibodies will be used to gain insight into the anatomical location of ATPase isoforms and the mechanisms underlying ATPase biogenesis and subunit assembly. Introduction of the genes coding for the alpha subunit of the ATPase into a mammalian cell will allow us to study many aspects of the relationship between the structure and function of the Na,K-ATPase. For example, construction of chimeric cDNA molecules between ouabain- resistant and ouabain sensitive forms of the ATPase should permit us to define the region of the ATPase alpha subunit responsible for differential ouabain sensitivity. This type of approach, coupled with site specific mutagenesis, should make it feasible to identify and study other functional domains in the Na,K-ATPase.

The objective of this proposal is to apply the techniques of molecular genetics to the analysis of structure function relationships for the Na,K-ATPase. We have developed an expression system to test the biological activity of cloned Na,K-ATPase genes. This system forms the framework for experiments designed to analyze the relationship between enzyme structure and function. Specific aims include: 1) Na,K-ATPase alpha/beta subunit interaction. We will attempt to determine which combinations of alpha and beta subunits can assemble to form holoenzyme. To address this issue, we will use epitope addition to tag a cDNA encoding a specific alpha and beta subunit isoform. Introduction of the construct into CV-1 cells and immunoprecipitation of the expressed fusion protein with an antibody against the epitope tag will allow us to determine which alpha/beta subunits are produced. This approach should also allow us to study isoenzyme function in transfected cells. 2) Structure-Function of the alpha subunit. We will attempt to identify sequences responsible for the variation in Na+ affinity between the alpha1 and alpha3 subunit isoforms. Construction and expression of chimeras between alpha1 and alpha3 subunit cDNAs should permit identification of sites within the alpha subunit that interact with Na+ and contribute to Na+ binding. A second approach will be designed to analyze the biochemical properties of the alpha2 isoform. These experiments should allow us to derive a clearer understanding of the functional relationships among the three alpha subunit isoforms. 3) Function of the beta subunit. To analyze the role of the beta subunit, we will take advantage of the fact that a cDNA encoding the beta subunit of the H,K-ATPase can confer ouabain resistance to primate cells by transfection. We will use epitope addition to determine if the H,K- ATPase beta subunit can assemble with the Na,K-ATPase alpha subunit. The construction and expression of chimeric H,K-/Na,K-ATPase beta subunit cDNAs should permit us to identify sites within the beta subunit that contribute to ouabain resistance. Expression of the beta subunit provides an opportunity to study structure function relationships by expressing beta subunits with alterations in primary amino acid sequence. These experiments also have practical significance because of the use of cardiac glycosides in the treatment of congestive heart failure.

The objective of this proposal is to apply the techniques of molecular genetics to the analysis of structure function relationships for the Na,K-ATPase. We have developed an expression system to test the biological activity of cloned Na,K-ATPase genes. This system forms the framework for experiments designed to analyze the relationship between enzyme structure and function. Specific aims include: 1) Na,K-ATPase alpha/beta subunit interaction. We will attempt to determine which combinations of alpha and beta subunits can assemble to form holoenzyme. To address this issue, we will use epitope addition to tag a cDNA encoding a specific alpha and beta subunit isoform. Introduction of the construct into CV-1 cells and immunoprecipitation of the expressed fusion protein with an antibody against the epitope tag will allow us to determine which alpha/beta subunits are produced. This approach should also allow us to study isoenzyme function in transfected cells. 2) Structure-Function of the alpha subunit. We will attempt to identify sequences responsible for the variation in Na+ affinity between the alpha1 and alpha3 subunit isoforms. Construction and expression of chimeras between alpha1 and alpha3 subunit cDNAs should permit identification of sites within the alpha subunit that interact with Na+ and contribute to Na+ binding. A second approach will be designed to analyze the biochemical properties of the alpha2 isoform. These experiments should allow us to derive a clearer understanding of the functional relationships among the three alpha subunit isoforms. 3) Function of the beta subunit. To analyze the role of the beta subunit, we will take advantage of the fact that a cDNA encoding the beta subunit of the H,K-ATPase can confer ouabain resistance to primate cells by transfection. We will use epitope addition to determine if the H,K- ATPase beta subunit can assemble with the Na,K-ATPase alpha subunit. The construction and expression of chimeric H,K-/Na,K-ATPase beta subunit cDNAs should permit us to identify sites within the beta subunit that contribute to ouabain resistance. Expression of the beta subunit provides an opportunity to study structure function relationships by expressing beta subunits with alterations in primary amino acid sequence. These experiments also have practical significance because of the use of cardiac glycosides in the treatment of congestive heart failure.

The objective of this proposal is the molecular analysis of the expression, structure, and function of Na,K-ATPase isoforms expressed in brain. Specific aims include: 1) Regulation of Na,K,ATPase gene expression. We will attempt to characterize the DNA sequences and cellular factors that promote cell-specific activation of the Na,K-ATPase alpha3 and beta2 subunit genes in the CNS. We will first use gene transfer methods to identify sequence elements within the alpha3 and beta2 genes that promote efficient and cell-specific expression of a reporter gene. We will also attempt to use retrovirus-mediated gene transfer to introduce reporter gene constructs directly into the postnatal rat retina. Because alpha3 and beta2 subunits are naturally produced in photoreceptor cells, it should be possible to identify regulatory elements within the alpha3 and beta2 genes that are required to promote photoreceptor cell-specific gene expression. 2) Structure- function analysis. We will attempt to identify sequences within the a subunit responsible for the wide variation in sodium affinity between the Hydra alpha subunit and rat alpha1 subunit. Construction and expression of chimeras between Hydra alpha and rat alpha1 subunit cDNAs should permit identification of sites that interact with Na+ and contribute to Na+ binding. In order to develop a detailed topographical map of the a subunit, we will use epitope addition to create a panel of alpha1 subunit cDNAs carrying insertional tags. Indirect immunofluorescence microscopy of transfectants with a monoclonal antibody reactive with the epitope tag should permit us to determine whether an epitope-tagged domain is located on the inside or outside of the cell. 3) Functional significance for isoform diversity. The identification of cell types expressing limited combinations of alpha and beta subunits makes it possible to compare the biochemical properties of distinct Na,K-ATPase isoenzymes. Initial interest will focus on a comparison of the substrate requirements of the pineal gland alpha3/beta2-containing isoenzyme and the neuronal enzyme composed of alpha3 and beta1 subunits. This approach should allow us to begin to understand the functional differences between Na,K-ATPase isoenzymes and determine whether the beta subunit contributes to the affinity of a specific alpha subunit for Na+ and/or K+.

DESCRIPTION (adapted from the applicant's abstract): The objective of this proposal is to analyze the relationship between the structure and function of Na,K-ATPase. The specific aims are: 1. Na,K-ATPase alpha/beta subunit interaction. The applicant will attempt to determine which combinations of alpha and beta subunit isoforms can associate by inserting epitope tags into cDNAs encoding alpha and beta subunit isoforms and expressing the constructs in mammalian cells. 2. Structure-function of the alpha subunit. The applicant will develop a refined topographical map of the alpha subunit polypeptide by creating a cysteine-less alpha subunit mutant. Cysteine residues will be sequential introduced into predicted extracellular or cytoplasmic loops of the cysteine-less mutant, and the topology of individual cysteine tags determined using biotin-labeled thiol- or sulfhydryl-reactive reagents. A second goal will be to identify sequences within the alpha subunit that form the sodium binding site. 3. Genetic analysis of Looptail mice:Function of the alpha2 subunit. The applicant will attempt to determine whether Looptail (Lp) mice exhibit alterations in the sequence or expression of the Na,K-ATPase alpha2 subunit (Atp1a2) gene. 4. Molecular analysis of the beta3 subunit. The applicant proposes to further characterize the recently discovered Na,K-ATPase beta3 subunit. The goal is to isolate and characterize cDNA and genomic DNA sequences for the murine beta3 subunit and develop antibody probes for cell localization studies.

PROJECT SUMMARY (See instructions): We will study the effects of frontotemporal lobar degeneration (FTLD), Alzheimer's disease (AD), and mild cognitive impairment (MCI) on socioemotional functioning using laboratory procedures that allow for precise assessment of a number of aspects of actual emotional functioning including: (a) emotional reactivity (the type and magnitude of responses to salient challenges and opportunities), (b) emotion regulation (the ability to adjust emotional responses to situational demands) and (c) empathy and prosocial behavior (recognizing others' emotions and responding to their distress). These laboratory-based assessments measure the major aspects of emotional responding, including: (a) subjective emotional experience, (b) emotional expressive behavior, (c) emotional language, and (d) autonomic and somatic nervous system physiology. This approach allows us to identify specific areas of lost and preserved socioemotional functioning in FTLD and AD, compared to each other and to MCI and controls. A particular focus of the proposed work is on understanding the basis of symptoms of FTLD that are very troublesome for caregivers, including a progressive loss of warmth and an increase in socially inappropropriate behavior. We will use our detailed analyses of emotional functioning to explore the particular deficits that contribute to these real world symptoms. Delineating specific deficits will enable us to make strong links to damage in specific brain circuits (explored via structural imaging) and particular kinds of neuropathology (explored at autopsy). In addition, we will determine the extent to which changes in particular aspects of socioemotional functioning are related to genetic risk factors for FTLD by studying unaffected family members with and without suspected genetic markers in families with a high incidence of FTLD.

Imbalances in cortical dopamine receptor (DR) mediated signaling are believed to underly both the cognitive and psychotic aspects of schizophrenia. Using yeast two-hybrid screens, we identified several DR-interacting proteins (DRIPs) that are prominently involved in intracellular calcium (Ca++) homeostasis (calcyon, NCS-1, TRPC-1, and CAPS). Disease-related increases in calcyon and NCS-1 protein levels were detected in dorsolateral prefrontal cortex (DLPFC) of patients with schizophrenia. We propose to extend our discovery based research on DR signaling complexes (DRSCs) guided by the hypothesis that abnormal dopamine-mediated Ca++ signaling is the basis of prefrontal dysfunction in schizophrenia. The possibility that this approach may also identify potential schizophrenia susceptibility genes (SSGs) is supported by a recent genetic association study which found evidence for an SSG at 10q26, the chromosomal position of the calcyon gene. We will utilize MALDI TOF-TOF mass spectrometry to identify the regional and receptor-specific composition of D1 and D2 DR-containing DRSCs immunoprecipitated from rat and monkey prefrontal cortex and striatum. It is likely that among the DRSC components, some proteins will interact directly with the receptor (DRIPs), while others (DRAPs or DR associated proteins) may interact with peripheral components of the complex. We will confirm interaction of DRSC proteins using a combination of biochemical and immunohistochemical methods. The effects of DRIP and DRAP interactions on DR function will be studied in transfected mammalian cells and native neural systems with a focus on Ca++ signaling and homeostasis. Expression of DRIPs and DRAPs will be analyzed in collections of schizophrenic brains. We will generate transgenic mice overexpressing NCS-1 or calcyon within the prefrontal cortex to learn whether these candidate schizophrenia-associated DRIPs are involved in the etiology of the disease. Transgenic mice will be subjected to a battery of behavioral, anatomical, and Physiological tests relevant to prefrontal deficits manifested in schizophrenia in collaboration withother subprojects. Identifying previously unknown proteins directly or indirectly associated with DRs should provide new insights into mechanisms of dopaminergic signaling in DLPFC, and clues as to the etiology of natomical and physiological defects manifested in schizophrenia.

The mu-opioid receptor (MOR) mediates most of the actions of morphine and other clinically relevant analgesics as well as drugs of abuse such as heroin. The proteomic and functional studies proposed in this application are designed to elucidate the protein components of MOR signaling complexes. Identification of MOR interacting proteins will allow us to test our hypothesis that protein-protein interactions play an important role in regulating the MOR signal transduction pathway. In Aim I, we will identify and characterize MOR interacting proteins. A split-ubiquitin screen we have performed identified several novel MORIPs including GPR177, the mammalian ortholog of Drosophila Wntless. To identify additional MORIPs, each of the intracellular domains of the human MOR will be used as bait to separately screen a human brain cDNA library. We also propose to utilize highly selective anti-MOR antibodies combined with proteomics and mass spectrometry to identify a spectrum of MORIPs from immunoprecipitated mouse brain lysates. The major goal in Aim 2 will be to validate the MOR-protein interactions identified in Aim I. Cellular colocalization studies will confirm whether or not the MOR and candidate interactors are expressed within the same cells and intracellular compartments. Pull-down and coimmunoprecipitation will substantiate the MOR-protein interaction, while deletion mapping will permit identification of sites within the proteins that are necessary for the interactions to occur. Preliminary studies we have so far performed indicate that GPR177 meets all inclusion criteria and appears to represent a bona-fide MORIP. The goal in Aim 3 is to understand the functional significance of MOR/GPR177 interaction. We will examine the requirement for MOR/GPR177 interaction in MOR trafficking, desensitization, and signal transduction. We will also examine the role of the MOR/GPR177 interaction in regulating Wnt2 secretion from cells. To accomplish this goal, we will determine whether disrupting the MOR/GPR177 interaction, by expressing dominant negative forms of GPR177 or by knocking down GPR177 expression, affects the functional properties of the MOR. Identification of MOR interacting proteins will provide new insights into the etiology of drug abuse and dependence. RELEVANCE (See instructions): This project seeks to identify and evaluate the function of proteins that interact with and regulate the muopioid receptor (MOR), the cell associated receptor that mediates most of the analgesic actions of morphine as well as drugs of abuse. Understanding the function of GPR177, a novel interacting protein we identified, is likely to provide new insight into the etiology of drug abuse and dependence. MOR-interacting proteins may also represent novel therapeutic targets for the treatment of these important public health problems.

DESCRIPTION (provided by applicant): Addiction to opioid drugs has become a substantial public health problem due to the large number of Americans who use opioids for chronic pain, and the increased use by teenagers and adults of opioid prescription painkillers for nonmedical purposes. In this application, we describe development of a behavioral paradigm that models 'addiction-like' behavior for opioid drugs, we link this behavior with the expression of key molecular components, and we outline a novel strategy to translate this knowledge into potential therapeutic interventions. Specifically, using this behavioral paradigm, we linked 'addiction-like' behavior for heroin in rats following a one month test period to expression of several key regulators of the mu-opioid receptor (MOR), the primary mediator of the analgesic and rewarding properties of opioid drugs. One of these regulatory proteins is Wntless (WLS), a transport protein necessary for Wnt protein secretion. Wnts are ubiquitous glycoproteins with trophic properties for neurons, including dendritic spine maintenance and increased hippocampal neurogenesis, properties that are normally inhibited by opioid agonist drugs such as morphine, heroin, and fentanyl. Binding of morphine to the MOR enhances the interaction between MOR and WLS, resulting in the inhibition of Wnt protein secretion. Changes in WLS expression were also found to occur in the prefrontal cortex of rats demonstrating addiction-like behaviors for heroin. These results lead to the hypothesis that the opioid-induced MOR/WLS interaction is a critical molecular component contributing to the development of opioid addiction. Experiments proposed in this application are designed to challenge the MOR/WLS hypothesis by testing whether blockade of the MOR/WLS interaction in the prefrontal cortex can prevent acquisition of heroin 'addiction-like' behaviors in rats and/or rescue heroin-experienced rats from 'relapse'. Specific Aim 1 will test whether the disruptive effects of morphine on Wnt secretion can be prevented in vitro via administration of MOR/WLS blocking peptides expressed in a lentiviral vector. Specific Aim 2 will use a viral vector to test whether blockade of the MOR/WLS interaction in the prefrontal cortex will disrupt the development of addiction-like behavior for heroin in drug naïve rats, while Specific Aim 3 will use a viral vector to test whether blockade of the MOR/WLS interaction in the prefrontal cortex will disrupt drug-induced reinstatement of heroin-seeking behavior in heroin-experienced rats. If successful, results from the experiments proposed in this application will reveal a novel avenue for the pharmacotherapy of opioid addiction in humans.

? DESCRIPTION (provided by applicant): Corrosive couple conflict (CCC) and coercive parent-child conflict constitute a ubiquitous, potent, and destructive (but modifiable) interpersonal poison to a wide range of adult and children's health outcomes. Such patterns are also linked with poor parent-child relationships and with more harsh punishment, which is associated with disturbed responses to environmental stresses (e.g., disruption in sympathetic nervous system and hypothalamic-pituitary-adrenocortical responses), a wide variety of adverse health outcomes in childhood, including dental caries, obesity, and diabetes related metabolic markers. This phase of NIH's Science of Behavior Change program emphasizes an experimental medicine approach to behavior change necessitating identification of central interpersonal/social targets for maximal impact on far-reaching panoply of health outcomes. This project will focus on factors associated diabetes and oral health (though the processes affect many other disease outcomes). Both are associated with pain, distress, expense, loss of productivity, and even mortality. They share overlapping medical regimens, are driven by overlapping proximal health behaviors, and affect a wide developmental span, from early childhood to late adulthood. As requested by the RFA, we will isolate three proximal health behaviors: (a) medical regimen adherence; (b) eating and drinking high sugar/calorie items; and (c) self-care behaviors. CCC/coercive parent-child conflicts are marked by an interrelated set of affective, behavioral, and physiological signatures. In the UH2 phase, we will identify/develop/validate assays. We will also identify/develop, and test interventions to reduce CCC/coercion targets. In the UH3 phase, we expect to conduct at least 2 studies to test whether reduction in targets results in improvement in adherence and other health behaviors of interest. One study will focus on parents and children, the other on adults in intimate relationships. Health behaviors related to diabetes and oral health problems will serve as dependent variables as will self-care behaviors in both diabetes and oral health. To place these health behaviors in the context of disease conditions and medical regimen adherence, we expect to focus one study on a sample of children with early childhood caries and the other study on an adult sample with diabetes.

? DESCRIPTION (provided by applicant): This application requests MSTP funding for the Penn State MD/PhD Program. Our program has a 20-year history of training outstanding students and providing them with the tools and experiences needed to become successful physician-scientists and leaders in academic medicine. Penn State has a talented and interactive faculty committed to biomedical research and MD/PhD career development. The students enrolled in our MD/PhD program are fully supported by the Dean of the College of Medicine during the medical school years and by faculty grants and individual fellowships during the graduate phase of the program. An MSTP award would leverage this institutional commitment, allowing us to increase the program from our current level of 6 students admitted per year to 8 students admitted per year. Over the past 10 years, Penn State has substantially improved the quality of the MD/PhD program through several targeted investments: (i) full autonomy of the MD/PhD program to select and admit qualified students; (ii) ~100% increase in institutional support, from less than $1 million to nearly $2 million per year; (iii) recruitment of >70 physician-scientist faculty members, including several departmental chairs and program directors, plus a recent initiative to recruit an additional 5-10 new physician-scientist faculty members; (iv) a 45% increase in the applicant pool size; (v) an increase in the number of matriculating students from 33 to 51; (vi) a doubling of the number of matriculated underrepresented minority students; and (vii) establishment of closer ties with our sister campus at University Park (UP) through the development of joint programs with Engineering Sciences & Mechanics and Molecular & Cellular Integrated Biosciences, as well as the appointment of an Associate Director of the MD/PhD program on the UP campus. The curriculum for the MD/PhD program is interdisciplinary and integrated. Students are exposed to both clinical and basic scientific activities longitudinally throughout their training, thus emphasizing the need to integrate biomedical research with patient care. MD/PhD-specific programmatic activities include a monthly seminar series where students present their own research and learn from visiting speakers; a bimonthly Clinical Research Conference where students learn to use clinical problems as a springboard for investigating the basic and translational sciences literature; a longitudinal Clinical Exposure Program, in which students hone their clinical skills by selecting a clinical preceptor and attending clinic at least one half day per month while pursuing their research; an annual retreat held on the UP campus; and several courses and seminars that specifically deal with translational biomedical research and MD/PhD career development. Most importantly, Program leaders use a very hands-on approach with our students. We take mentorship extremely seriously and we invest a significant amount of time meeting with and listening to our students to make their training experience at Penn State one that will not only be rigorous, but also provide a training path that fosters their career development, enabling them to excel in their future careers as physician-scientists.

ABSTRACT This project applies basic affective science methodology to assess socioemotional functioning in patients with frontotemporal dementia (FTD) and Alzheimer's disease (AD). By measuring multiple emotion processes (reactivity, regulation, and recognition), types (positive, negative, self-conscious emotions), and response systems (subjective experience, expressive behavior, peripheral physiology, emotional language), we obtain an unusually comprehensive and fine-grained assessment of socioemotional functioning. Using this approach, we have identified particular aspects of socioemotional functioning that distinguish among FTD-spectrum disorders, AD, and normal aging. This assessment has also been well-suited for establishing links with anatomical data derived from structural neuroimaging and post-mortem neuropathology. In the next project period, we will expand this research to include: (a) psychiatric patients with major depressive disorder (MDD) and bipolar affective disorder (BD), which also produce changes in socioemotional functioning and can be difficult to distinguish from dementia in late life; (b) additional measures of social/interpersonal functioning in the realms of emotional reactivity (pride, an emotion that arises from complex social comparisons), regulation (social down-regulation of response to stress associated with presence of caregiver), and recognition (continuous ratings of one's own emotions, recognizing comfortable interpersonal distance); (c) new measures of autonomic nervous system functioning (stomach activity) and visceral awareness (gastric filling); (d) longitudinal assessments of patients' real-world socioemotional functioning; and (e) additional imaging measures (PET assays of tau and amyloid accumulation and circuit-focused assessment of functional connectivity. We will pursue three specific aims: Aim 1. Social/Interpersonal functioning. We will utilize our expanded assessment of social/interpersonal functioning to characterize social/interpersonal functioning in FTD-spectrum disorders, AD, MDD, and BD; Aim 2. Difficult diagnoses. We will identify optimal groups of predictors from our laboratory-assessment of socioemotional functioning for distinguishing among patients with FTD, AD, MDD, and BD. Aim 3. Brain-behavior relationships. We will delineate brain circuitry associated with deficits in specific aspects of emotional reactivity, regulation, and recognition. Innovations of this project include: (a) applying basic affective science methodology to characterize social and emotional functioning in patients with dementia and mood disorders; (b) using classification analysis techniques with strong criterion measures (state-of-the-art clinical diagnoses, and, when possible, autopsy-confirmed diagnoses) to identify optimal groups of predictors for increasing diagnostic accuracy; and (c) studying brain-behavior relationships using fine-grained measures of emotional reactivity, regulation, and recognition linked with anatomical data derived from MRI and PET imaging, functional connectivity analyses, and autopsy-based neuropathology focused on selectively vulnerable brain regions.