Karen J. Parker - US grants

DESCRIPTION (provided by applicant): Primate stress research has focused on severe social stress and its pathogenic effects on brain and behavior development. Consequently, few researchers have examined the possibility that mild forms of early social stress promote adaptive outcomes in primate development. Recently, our lab reported that brief, intermittent maternal separations (IS) in squirrel monkey infants subsequently lead to diminished anxiety, prosocial tendencies, enhanced glucocorticoid negative-feedback sensitivity, better spatial memory, and enlarged prefrontal brain regions. Since submitting my original NRSA proposal, I have determined that IS alters mother-infant social behavior at reunions. Also, in an unfamiliar environment, IS infants spend less time clinging to their mothers and more time exploring novel objects than NS infants. Although these data provide compelling evidence that IS triggers adaptations in postnatal development, it is not yet known a) whether IS produces differences in stress hormone responses and behavioral performance on a prefrontal-dependent cognitive task, b) whether the prefrontal enlargement previously identified in adult IS monkeys emerges earlier in postnatal brain development, and c) whether IS produces differences in prefrontal white matter integrity. This proposal delineates a series of experiments designed to answer these questions. Results from these studies will advance our understanding of how mild stressful experiences promote cognitive, emotional, and postnatal brain development. This animal model provides valuable insights on a basic process in human development, insofar as early emotional challenges foster adaptations useful later in life.

DESCRIPTION (provided by applicant): Using a primate model, we build on our previous research to test the novel hypothesis that exposure to moderate levels of maternal rejection early in life "inoculates" the developing infant by permanently altering cognitive appraisal of, and neuroendocrine sensitivity to, subsequent stressors (i.e., stress resilience). In contrast, we posit that exposure to too little or too much rejection-related stress leads to stress vulnerability later in life. We also test the hypotheses that these maternal influences on the development of stress resilience and vulnerability 1) result from long-term alterations in the activity of the serotonergic system, the HPA axis, and other stress-sensitive neurobiological systems, and 2) are modulated by risk or protective factors such as polymorphisms in the serotonin transporter (SERT) gene and amount of social support. This 5-year project will be conducted with the free-ranging population of rhesus macaques on Cayo Santiago, PR. Two cohorts of 45 infants (n=90) will be followed longitudinally from birth through 3 years of age. Infants will be classified on the basis of the amount of maternal rejection they receive (low, moderate, high) in the first 2-3 months of life. All infants will be SERT genotyped and homozygous or heterozygous individuals for the long and the short allele (l/l, l/s, and s/s) will be identified. Data on dominance rank, maternal protectiveness, and social support (social network size;grooming received;aid during agonistic interactions) will be quantified. Stress vulnerability and resilience will be operationalized with behavioral and neuroendocrine measures. Behavioral measures will include: a) independence from the mother;b) social competence during interactions with conspecifics;and c) behavioral inhibition and anxiety in response to "challenge" tests involving exposure to novel objects, unfamiliar humans, and risky social situations. Detailed characterization of HPA axis physiology and other stress-related neurobiological systems will be obtained through: a) frequent fecal sample collection to determine basal and stress-induced cortisol levels;b) assessment of plasma concentrations of ACTH and cortisol in response to psychosocial stress (social separations) and pharmacological challenges (CRF challenge, dexamethasone suppression test, metyrapone test, ACTH challenge);and c) assessment of stress-induced CSF concentrations of CRF, oxytocin, and monoamine metabolites. By conducting experimental research with free-ranging primates we maximize the ecological validity of our findings. This research will provide original information on the neuroendocrine mechanisms through which exposure to variable parenting can affect the development of stress vulnerability and resilience in children, and how genetic and environmental factors may influence these development outcomes. This research will also enhance our understanding of normative interindividual variation in the development of emotion regulation and stress-related disorders. Ultimately, this research may provide important information on the efficacy and potential limitations of parenting interventions designed to foster resilience in children based on controlled exposure to, and mastery of, psychosocial adversity. PUBLIC HEALTH RELEVANCE: The proposed project will provide original information on the neuroendocrine mechanisms through which exposure to variable parenting can affect the development of stress vulnerability and resilience in children. It will also enhance our understanding of how parental influences on the development of stress reactivity are modulated by genetic and environmental factors. Findings of this research have important implications for understanding normative interindividual variation in the development of emotion regulation as well as stress-related disorders in children.

PROJECT SUMMARY. Autism spectrum disorders (ASD) are characterized by social impairments and are one of the most devastating childhood disorders, but remain poorly understood. Few robust biomarkers of ASD have been identified, hindering the understanding of basic biology; nor are there any pharmacotherapies that treat the social deficits of ASD. Progress has been impeded, in part, by the difficulty of obtaining relevant tissue samples from patients and their matched neurotypical controls. In mouse models, while tissue is available, there is not infrequently discordance between complex human behavior and laboratory-based mouse behavior, even with shared genetic etiologies. These two limitations underscore the tremendous value in developing an animal model of social impairments with more reliable behavioral and biological correlates to the human disease. Rhesus monkeys are an ideal model organism. Like humans, they are highly social, and both species display stable and pronounced individual differences in social functioning. At the behavioral extremes, low-sociable compared to high-sociable male rhesus monkeys initiate fewer affiliative interactions and display more inappropriate social behavior, suggesting both lower social motivation and poorer social skills. Naturally occurring low sociability in male rhesus monkeys therefore presents an exceptional opportunity to study the biology of social impairments. This research leverages a novel statistical classification model which predicts, from infant behavior, subjects that are subsequently found to be low-sociable or high-sociable. In this grant, we propose to collect quantitative social behavior data in a larger validation cohort of 1-5 year old male monkeys to confirm the discriminant power of this statistical model. If successful, this statistical model will serve as a robust high- throughput screening tool to rapidly identify phenotypic social extremes in a large population (n=5,200 monkeys), allowing these monkeys to serve as a model for early-life interventions, whether pharmacologic or behavioral. To assess the construct validity of this model, we will test whether in low-sociable vs. high-sociable male monkeys there is an excess of rare pathogenic variants in genes previously implicated in autism. We will also test whether candidate biomarkers of social functioning (e.g., oxytocin, arginine-vasopressin, extracellular regulated kinase and related kinases), as measured in the CSF and blood from these monkeys, are associated with sociability status, whether the degree of biomarker dysregulation co-varies with the degree of social deficits, and whether CSF and blood measures are equally informative. These findings will link abnormal social behavior with protein and gene based biological changes in a way that has not been previously achievable. We are optimistic that further development of this model will accelerate the discovery of autism biomarkers and novel drugable targets, provide new blood-based metrics for treatment response efficacy, and streamline the development of effective therapeutics that will benefit patients with presently intractable social impairments.

DESCRIPTION (provided by applicant): Autism is characterized by core social impairments which limit patients' ability to form and maintain meaningful social relationships. At present, antipsychotic medications are the only pharmacotherapeutic option approved to treat autism. However, these agents mainly target associated symptoms (e.g., irritability), have unfavorable side-effects (e.g., lethargy, weight gain), and remain ineffective in treating the social features f this disorder. Developing new medications that specifically target social functioning thus will address a critical unmet need. A large body of research has shown that the closely related neuropeptides arginine-vasopressin (AVP) and oxytocin (OT) play critical roles in adaptive social functioning. For instance, AVP or OT impairments induced pharmacologically or transgenically produce a variety of social deficits in animals. Importantly, single-doses of intranasally administered OT improve several complex social functions (e.g., processing of social information, emotion recognition) in people with autism. Although intranasally administered AVP improves social cognition and memory in neurotypical individuals, and has been used safely for years to treat diabetes insipidus and nocturnal enuresis, no studies have tested the effects of AVP treatment on social functioning in individuals with autism. However, preclinical research has shown that in an OT receptor null (Oxtr-/-)mouse model of autism, social behavior is modulated selectively through brain AVP V1a receptors and that AVP administration rescues social deficits in these animals. Moreover, in other preclinical models, AVP, acting via V1a receptors, more selectively enhances male social behavior than does OT. In contrast, OT, acting via OT receptors, more selectively enhances female social behavior. Given that autism is a predominantly male-biased disorder, that in animals AVP is more important than OT for male social behavior, and that even so, acute OT does help male autistic patients, we hypothesize that AVP will be particularly effective in treating social symptoms in autism. The first step is to test whether AVP ameliorates social impairments in autism and whether males respond more robustly to AVP than females. We will test the effects of single-dose (20 IU) and 4-week (20 IU BID) intranasal AVP administration on social deficits in 50 high functioning males and females with autism aged 6 to 12 years using a double-blind, randomized, placebo controlled, parallel design. Study outcome measures are improvements on parent ratings of the Social Responsiveness Scale (SRS) and laboratory-based assessments of social behavior and cognition (i.e., facial emotion recognition, eye gaze to social cues, social memory, and social perceptual abilities). Consistent with preclinical evidence, we predict that AVP will improve social functioning in individuals with autism. Data from this study will be leveraged to secure additional funding to initiate head-to-head comparisons of AVP and OT treatment in a larger autism cohort. This research has high potential to lead to the development of the first effective treatments and earlier interventions for social impairments in individuals wth autism.

? DESCRIPTION (provided by applicant): Autism spectrum disorder (ASD) is characterized by core social impairments and affects 1 in 68 US children, but remains poorly understood. ASD is currently diagnosed on the basis of behavioral criteria because no robust biomarkers have been identified, nor are there any pharmacotherapies that effectively treat the social features of ASD. Biomarkers improve our ability to make accurate diagnoses and provide biological targets for drug development and testing. Two promising biomarkers of ASD are the closely related neuropeptides oxytocin (OXT) and arginine-vasopressin (AVP). OXT and AVP are critical for mammalian social functioning, and experimental disruption of these signaling pathways produces social deficits in animal models with relevance to ASD. In parallel with these preclinical studies, diminished blood OXT and AVP concentrations have been associated with social impairments in children with ASD, suggesting that both neuropeptides hold therapeutic promise for treating this disorder. Furthermore, human genetic studies have shown an association between common genetic variants of both the OXT receptor (OXTR) and AVP receptor (AVPR) v1a genes and ASD. A complementary regulatory mechanism to direct alteration of the DNA sequence is epigenetic modification of DNA, which may have extremely important scientific implications for understanding ASD disease biology. One epigenetic process, DNA methylation, influences gene expression and, at the extreme, can greatly diminish or even silence gene expression. Our team is now conducting OXT and AVP treatment trials in children with ASD. Treatment response is promising, but variable, and this may be due to variation in the availability of OXTR and AVPR v1a on which the drugs can effectively act to enhance social functioning in each patient. Despite the obvious need, no prior research has tested whether variation in DNA methylation of the OXTR and/or AVPR v1a genes has social, diagnostic, and/or treatment implications for individuals with ASD. These critical gaps in knowledge will be addressed in this highly innovative R21 research project. Our initial project goals are: (1) To test whether OXTR and/or AVPR v1a gene methylation differs between, and predicts disorder classification of, children with and without ASD; (2) To test whether OXTR and/or AVPR v1a gene methylation predicts social functioning and diagnostic scores; and (3) To identify, on an exploratory basis, additional genes that may be epigenetically regulated in children with ASD using genome-wide discovery methods. We will next confirm promising findings from this first study in a validation cohort. We will also test in this cohort whether variation in DNA methylation and expression of previously identified genes predicts treatment response in children with ASD who have completed our OXT and AVP clinical trials. This research could lead to the implementation of earlier and more accurate diagnostic methods, discovery of novel biological targets, and development of the first personalized therapeutics to treat the social impairments in ASD.

? DESCRIPTION (provided by applicant): Autism spectrum disorder (ASD) is characterized by social impairments and affects 1 in 68 US children, but remains poorly understood. Few biomarkers of ASD have been identified, hindering the understanding of its basic biology; nor are there any medications that treat the social deficits of ASD. Progress has been impeded by: 1) the difficulty of obtaining relevant tissue samples from patients and matched controls, and 2) in mouse models, the discordance between complex human behavior and laboratory-based mouse behavior, even with shared genetic etiologies. These two limitations underscore the tremendous value in developing an animal model of social deficits with more reliable behavioral and biological correlates to the human disease. Rhesus monkeys are an ideal model organism. Like humans, they are highly social, and both species display stable and pronounced individual differences in social functioning. At the behavioral extremes, low-social compared to high-social male rhesus monkeys initiate fewer affiliative interactions and display more inappropriate social behavior, suggesting both lower social motivation and poorer social skills. Naturally occurring low-social behavior in male rhesus monkeys therefore presents an exceptional opportunity to study the biology of social impairments. This research leverages a novel statistical classification model which predicts, from infant behavior, subjects that are subsequently found to be low-social or high-social. On the basis of promising pilot data, we will collect quantitative social behavior data in a larger validation cohort of 1-5 year old male monkeys to confirm the discriminant power of this statistical model to be used as a robust high-throughput screening tool to rapidly identify social extremes in a large population. We will also test whether: our candidate biomarkers of social functioning (e.g., oxytocin and arginine-vasopressin; kinase signaling) correctly classify monkeys as low- social vs. high-social; the degree of biomarker dysregulation co-varies with the degree of social deficits; and cerebrospinal fluid and blood measures are equally informative. Because exclusion of females may impede identification of important disease mechanisms, we will also test, for the first time, whether our statistical classification model can be used to identify social extremes in female monkeys, and whether the same or different biomarkers predict social deficits in low-social females. Finally, we will create the first primate social behavior test battery to better characterize the impairments of low-social monkeys with direct relevance to core autism symptoms (e.g., deficits in joint attention, face recognition, social learning, social competence, theory of mind, peer preferences). Creation of this battery will also allow us to test which biomarker measurements most robustly predict low-social monkey test performance to identify the most promising targets for therapeutic intervention. We are optimistic that further development of this model will accelerate the discovery of autism biomarkers and novel druggable targets, provide non-behavioral biometrics for treatment response, and streamline the development of the first effective autism therapeutics.

PROJECT SUMMARY Autism spectrum disorder (ASD) is characterized by core social impairments which limit patients? ability to form and maintain meaningful relationships. At present, antipsychotics are the only medication approved to treat ASD, but they target associated symptoms, have unfavorable side-effects, and do not treat ASD?s core social deficits. Developing new medications that specifically target social functioning will thus address an important unmet need. A large body of research has shown that the neuropeptide arginine vasopressin (AVP) plays a critical role in promoting social behavior and that experimental dysregulation of the AVP signaling pathway produces social deficits in animal models. Although intranasal AVP administration improves social cognition and memory in neurotypical individuals, no published research has tested the effects of AVP treatment in ASD patients. Several lines of evidence underscore the necessity of such research. For example, we recently reported that blood AVP levels predict theory of mind ability in children with ASD, such that children with the lowest AVP levels have the most marked theory of mind deficits. This finding is consistent with our preclinical research showing that socially impaired monkeys have significantly diminished cerebrospinal fluid AVP levels compared to control monkeys. Similarly, data from the first neuropeptide receptor mapping study of postmortem primate brain tissue revealed that AVP V1a receptors are widely distributed throughout the extended neural amygdala pathway, suggesting that AVP administration can target directly neural pathways known to regulate social functioning. Interestingly, AVP?s pharmacological effects are especially evident in male animals, and given ASD?s male-biased prevalence, AVP deficits may be particularly relevant to understanding the risk for, and treatment of, ASD. We recently tested the effects of 4-week intranasal AVP treatment in children with ASD in a double-blind randomized placebo- controlled pilot trial (R21 MH100387; MPI: Parker & Hardan). AVP was overall well tolerated in this small sample, and importantly, AVP treatment improved social abilities in children with ASD as assessed by parent ratings on the Social Responsiveness Scale, 2nd Ed (SRS-2). This result was more pronounced when we accounted for pre-treatment blood AVP levels. Here we seek to extend these findings in a larger ASD study cohort (N=100), aged 6 to 17 years, in this double-blind randomized placebo-controlled 8-week trial. Our primary outcome measure is improvement in child social abilities as assessed by parent ratings on the SRS-2. We will also test the safety and tolerability of AVP treatment, and whether pre-treatment blood AVP levels are a personalized predictor of treatment efficacy. Finally, we will test whether AVP treatment improves ASD symptoms as assessed by clinician impression, additional parent report measures, and child performance on laboratory tests of social cognition and communication. We predict that AVP treatment will improve social abilities in children with ASD, and that AVP will be well tolerated, in keeping with our preliminary data. This research has high potential to lead to development of the first effective medication to treat ASD?s currently intractable social deficits.