Noboru Hiroi - US grants

DESCRIPTION(Adapted from applicant's abstract): The molecular basis of drug addiction has recently been explored using knock-out mice. These studies have revealed that a number of molecules contribute to distinct aspects of addiction to stimulants, opiates, and ethanol. However, the molecular basis of nicotine addiction remains unclear despite the fact that addiction to nicotine and other drugs shares a common neuroanatomical basis. The transcription factor FosB and the protein phosphatase inhibitor DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa) have been shown to be critical determinants for different aspects of behavioral responsiveness to cocaine in mice. These two intracellular molecules are unique in that their disruption makes animals more vulnerable to cocaine's behavioral effects. The present application is designed to test the hypothesis that these two molecules are also critical for nicotine addiction and that, if so, they play distinct roles in specific aspects of nicotine dependence. The experimental design is unique in three aspects. First, a number of behavioral models will be used to assess different aspects of nicotine addiction. They include tolerance, sensitization, conditioned place preference, withdrawal-associated conditioned place aversion, and self-administration. Second, an attempt will be made to assess the influence of genetic backgrounds on behavioral phenotypes. The dissimilar genetic backgrounds of knock-out and wild-type mice littermates have confounded the behavioral phenotypes of knock-out mice. Heterozygous mice will be repeatedly back-crossed to C57BL/6J mice to achieve a higher degree of similarity in the genetic backgrounds of knock-out mice and wild-type littermates (i.e., congenic mice). Third, anatomical analysis will determine the involvement of neuroanatomical adaptations in behavioral phenotypes. Based on the outcome of similar approaches to cocaine addiction, it is expected that these two genes contribute to specific aspects of nicotine addiction. If these molecules turn out to be important for vulnerability to nicotine addiction, this mouse study will provide a solid basis for genetic analysis of human addiction vulnerability.

DESCRIPTION (provided by applicant): The long-term objective of the principal investigator is to ascertain the molecular mechanisms underlying nicotine addiction/dependence. One salient aspect of nicotine addiction is that cues that are usually associated with nicotine exert a powerful control over craving and relapse in smokers. Although some attempts have been made to implement cue extinction to aid smoking cessation, lack of knowledge regarding the mechanisms of cue control of smoking has hampered progress. The proposed studies are designed to elucidate the molecular mechanisms underlying extinction of cue control of nicotine addiction in the place conditioning paradigm in mice. This R01 application tests the overall hypothesis that cGMP-dependent protein kinase subtype II (PKG-II), in regions along the mesocorticolimbic dopamine system, is a determinant of the rate of extinction of nicotine-induced conditioned place preference (CPP) in mice. Our published and preliminary studies show that a) nicotine elevates the concentration of cGMP in the striatum (nucleus accumbens and caudate-putamen), b) nicotine can up-regulate PKG activity in the nucleus accumbens and the VTA, which represent a target and the origin of the mesocorticolimbic dopamine pathway, respectively, c) nicotine causes phosphorylation of a PKG substrate in the striatum (including the nucleus accumbens), d) mice exhibit intensified nicotine CPP on the first few drug-free test days and extinction thereafter, and e) extinction of nicotine CPP is accelerated in PKG-II knockout (KO) mice. The proposed studies rely on genetic manipulation of PKG levels in the mouse brain. Specific Aim 1 will test the hypothesis that the level of PKG-II, but not PKG-I, determines the rate of extinction of nicotine CPP. PKG-I and PKG-II wild-type (WT), heterozygous (HT), and KO mice will be employed. Moreover, we will determine the exact behavioral processes in which PKG subtypes play a role during extinction of nicotine CPP and the general role played by PKG subtypes in extinction of cue control by other addictive substances. Specific Aim 2 will identify the specific brain region(s) in which PKG may act to regulate extinction of nicotine CPP. We will use a lentiviral vector to locally restore PKG-II (or PKG-I) in PKG-II (or PKG-I) KO mice. Moreover, we will correlate maintenance of nicotine CPP during extinction with PKG activities in selected brain regions along the mesocorticolimbic dopamine pathway. The outcome of the proposed studies is significant, as it will provide evidence that can be used to devise therapeutic options for smokers. Identification of a PKG subtype in specific brain loci, in connection with cue control of nicotine addiction, would be an important step toward enhancing our ability to fine-tune molecular targets to prevent cue-triggered smoking relapse and accelerate extinction. If this kinase turns out to be important for cue control of other addictive substances during extinction, the outcome of these studies will have a much broader implication for treatment of cue-triggered relapses in other forms of addiction. Recent estimates indicate that there are approximately 1.3 billion smokers world-wide and 5 million deaths are attributable to tobacco use annually. Currently available therapies are not effective in aiding cessation, partly because the exact neuronal mechanisms underlying extinction of nicotine addiction are still poorly understood. The proposed studies are designed to fill in this knowledge gap.

[unreadable] DESCRIPTION (provided by applicant): DiGeorge syndrome (DGS)/velo-cardio-facial syndrome (VCFS) is one of the common genetic disorders and affects approximately one in 4000 livebirths. Hemizygosity of a 1.5-3.0 Mb region of the human 22q11 underlies various neuropsychiatric, behavioral and physical abnormalities in DGS/VCFS. They include behavioral excitation, impaired prepulse inhibition, social interaction problems, as well as cardiovascular defects. Although efforts have been made in identifying individual 22q11 genes responsible for the behavioral abnormalities of DGS/VCFS, little is known about how individual 22q11 genes interact in increasing the susceptibility to these behavioral abnormalities. We and others have shown that deletion of either the T-box transcription factor Tbx1 or Cdcrel (cell division control related protein, also called Sept5) induces some, but not all DGS/VCFS symptoms in mice. To study the interactive role of Tbx1 and Cdcrel, the Principal Investigator has developed a double Tbx1/Cdcrel heterozygous mouse. Using this mouse line together with Tbx1 heterozygous mice and Cdcrel heterozygous/knockout mice, we have further shown that mice with combined heterozygosity of Tbx1 and Cdcrel, but not mice with deletion of either Tbx1 or Cdcrel alone, have sensitized hyperactivity. This suggests that Tbx1 and Cdcrel synergistically increase the susceptibility to the behavioral abnormalities of DGS/VCFS. Our long-term goal is to ascertain the nature of interaction among 22q11 genes as one of the underlying mechanisms for the behavioral abnormalities of DGS/VCFS. The specific hypothesis to be tested in this R21 proposal is that Tbx1 and Cdcrel interactively contribute to distinct behavioral abnormalities in mice. The specific aims to test this hypothesis are: Specific Aim 1: To determine whether heterozygosity of Tbx1, Cdcrel, or their combination causes abnormalities in locomotor activity/habituation, prepulse inhibition and social behaviors in mice (Experiments 1- 3). We will use Tbx1 heterozygous mice, Cdcrel heterozygous and knockout mice, double Tbx1/Cdcrel heterozygous mice, and their wild-type littermates. Specific Aim 2: To determine whether behavioral abnormalities seen in double heterozygous mice are attenuated by restoration of Cdcrel by a viral vector in the brain (Experiment 4). The present R21 proposal will provide a mouse model to further ascertain the nature of interaction between Tbx1 and Cdcrel in the brain in relation to behavioral abnormalities. The proposal will form a solid basis to further study the genetic basis of this common developmental disorder. Because deletion of 22q11 is also associated with high rates of schizophrenia, obsessive compulsive disorder, and attention deficit hyperactivity disorder, the outcome of this project will have significant implications for a better understanding of the genetic mechanisms of these neuropsychiatric disorders as well. The proposed project will ascertain the role of two 22q11 genes in behavioral abnormalities in a double heterozygous mouse model. Because hemizygosity of this chromosomal region is associated with many neuropsychiatric disorders and behavioral abnormalities, the outcome of the proposed studies will contribute to a better understanding of the genetic mechanisms underlying neuropsychiatric disorders, including schizophrenia. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Memory deficits are one of the disabling impairments associated with autism, mental retardation, and schizophrenia. Because the underlying genetic and neuronal mechanisms of memory impairments are still poorly understood, mechanism-based therapeutic options do not exist to ameliorate such impairment, hindering the effective integration of patients into society. Interestingly, a high activity allele of catechol-O-methyl-transferase (COMT) is associated with lower levels of working memory performance after, but not before, puberty in children and adolescents. Our published mouse work shows that elevated activity of human COMT impairs working memory of mice at 2 months but not 1 month of age, where 1 month of age corresponds to puberty in the mouse. While this correlation in humans and mice suggests that there is a developmental timetable along which COMT levels begin to exert an influence on working memory, these studies did not elevate COMT levels at specific brain loci and developmental time points. The precise anatomical and cellular substrates and their developmental programming through which high COMT activity affects working memory capacity are still poorly understood. The objective of the proposed project is to test the overarching hypothesis that working memory and associated synaptic plasticity become increasingly dependent on an endogenous dopamine tone in the medial prefrontal cortex or hippocampus during postnatal development. To test this hypothesis, the PI has developed a lentiviral vector that temporally and spatially up-regulates COMT in the mouse brain. A team of investigators will achieve the following Specific Aims: Specific Aim 1: To ascertain the impact of elevated COMT expression in the medial prefrontal cortex or hippocampus on working memory capacity at specific developmental time points. Specific Aim 2: To determine the impact of spatially and temporally targeted reductions in an endogenous dopamine tone on synaptic plasticity in the prefrontal cortex and hippocampus. Upon completion of the proposed studies, these technically innovative experiments will, for the first time, identify the anatomical and cellular mechanisms through which an endogenous dopamine tone determines the developmental maturation of working memory capacity and synaptic plasticity. Identifying the developmental time window, anatomical region(s), and cellular substrate(s) necessary for an endogenous dopamine tone to induce behavioral and cellular working memory phenotypes will have a major impact on our understanding of working memory. Because working memory deficits have been observed in individuals with mental retardation, autism, and schizophrenia, this proposal could lead to a better understanding of the neurobiological substrates for one aspect of developmental neuropsychiatric disorders.

Cognitive deficits are major disabling impairments associated with autism and schizophrenia. Because the underlying genetic and cellular mechanisms of such deficits are still poorly understood, mechanism-based therapeutic options do not exist, limiting the effective integration of patients into society. Previous clinical work has shown that executive functions such as working memory and cognitive flexibility start to lag behind from adolescence to adulthood in individuals with autism and schizophrenia. However, the precise genetic, anatomical and cellular substrates through which this occurs are still poorly understood. We have identified two genes encoded in copy number variants (CNVs) at human chromosome 22q11.2, a high-risk factor for autism and schizophrenia, for which dose alterations impair the developmental maturation of working memory. Our published work shows that mice developmentally expand working memory capacity from adolescence to adulthood and that constitutively elevated activity of catechol-O-methyl-transferase (COMT) impairs the working memory of mice during adulthood, but not adolescence. During the previous funding period, we have further found that over-expression of COMT and the transcription factor TBX1, another 22q11.2 gene, in adult neural progenitor cells of the hippocampus recapitulates this age-dependent deficit in working memory capacity. The objective of the proposed project is to test our overarching hypothesis that dose alterations of CNV-encoded genes impair the developmental maturation of executive function through defective adult neurogenesis in the hippocampus. To test this hypothesis, we developed experimental tools to regulate CNV- encoded genes in adult neural progenitor cells in the hippocampus at specific developmental time points. Moreover, we have established a screening system to identify other autism- and schizophrenia-associated CNV genes whose dose alterations affect adult neurogenesis and executive function. Our experimental readouts include executive function and adult neurogenesis. Upon completion of the proposed studies, these technically innovative experiments will, for the first time, establish a common cellular mechanism through which altered doses of autism- and schizophrenia-associated genes impair the developmental maturation of executive function. Identification of the developmental time window, neuroanatomical region(s), and cellular subtypes necessary for maturation of executive function will have a major impact on our understanding of the developmental mechanisms of executive function and its derailed trajectories. This proposal could lead to a better understanding of the neurobiological substrates for an important dimensional aspect of developmental neuropsychiatric disorders.

A critical step to improving our understanding of autism spectrum disorder (ASD) is to identify underlying genetic and environmental risk factors. A number of rare copy number variants (CNVs) have emerged as robust genetic risk factors for ASD. However, not all individuals exhibit ASD and the severity of ASD symptoms varies among carriers of a CNV. Given that early therapeutic intervention attenuates symptoms, it is reasonable to assume that early environmental factors also influence ASD symptoms later. However, manipulation of the interplay between genetic and early environmental factors to identify mechanisms is difficult in humans. Our team is uniquely positioned to experimentally address this issue. First, we have identified atypical pup vocal call sequences of a genetic mouse model of ASD. To do so, our team applied a set of sophisticated statistical tools. Second, our team developed innovative experimental tools and demonstrated that such atypical pup call sequences induce less maternal approach. This observation shows that neonatal vocalization is a means of social communication with mothers and suggests that it influences the level of maternal care. Third, we found that enriched housing, an environmental manipulation known to reverse the detrimental behavioral effects of maternal separation in mice, alters the expression and methylation of a CNV-encoded gene in mouse brains. Capitalizing on these innovative methods and observations, we propose to test our central hypothesis that CNVs disturb neonatal social communication with the mother and this early experience exacerbates ASD-like behaviors via epigenetically modified expression of CNV- encoded genes. We will use mouse models of paternal 15q11-13 duplication, 15q13.3 hemizygosity and 22q11.2 hemizygosity, as they represent three robust genetic risk factors for ASD. Use of multiple models will allow us to determine common, as well as distinct roles of neonatal social communication in CNV-associated ASD. We propose to achieve the following three Aims: Aim 1: Determine if CNVs result in atypical vocalization structure during the neonatal period and if it is correlated with ASD-like behaviors at 2 months of age; Aim 2: Determine the impact of atypical neonatal vocalization on maternal care; Aim 3: Measure the effect of altered maternal care on the severity of ASD-like behaviors and CNV gene expression and epigenetic modification. The outcomes of this project will reveal the interplay between genetic factors and neonatal social communication with mothers resulting in the ultimate severity of ASD-like behaviors through epigenetic mechanisms. The project has high translational value because it could provide a novel mechanistic base to understand the gene-environment interaction underlying ASD.

Summary One in every 10 babies is born prematurely in the US, and this contributes to high rates of long-term negative health consequences. An even higher rate of preterm births is expected in the near future due to maternal coronavirus disease 2019 (COVID-19) infection. While premature birth does not necessarily result in developmental neuropsychiatric disorders, it is associated with elevated rates of autism spectrum disorder (ASD)1-9, intellectual disability10, 11, attention-deficit/hyperactivity disorder (ADHD)2, 12, learning disabilities13, cerebral palsy13, and delays in cognitive, social, and motor development2, 4, 8, 10, 14-16. The developmental risks and uncertainty associated with premature birth may overwhelm caregivers with confusion, stress, and anxiety about the future. Moreover, the heterogeneity of cognitive, social, and motor developmental trajectories among preterm infants confounds the accurate, early detection of developmental issues and the early implementation of therapeutic interventions17-21. As some forms of early intervention improve the prognoses of infants with (or at high risk of) developmental neuropsychiatric disorders22-28, an objective, quantitative method of predicting (or improving the prediction of) the developmental trajectories of preterm infants is urgently needed. A number of research groups have attempted to use computational models to differentiate the cries of preterm and term infants29-32. However, computational models that can predict the heterogeneous cognitive, social, and motor developmental trajectories among preterm infants do not exist. Moreover, the neural basis of how preterm birth alters the cognitive, social, and motor developmental trajectories is poorly understood. Based on our preliminary data, which showed that the volume of the amygdala is selectively impacted by a gene variant that is linked to social deficits in mice, we hypothesize that preterm birth also alters the cognitive, social, and motor developmental trajectories from the neonatal to early adult periods via variable alterations of brain structures relevant to cognitive, social, and motor capacities. To test this hypothesis, we will leverage our expertise in computational feature selection, using variables of neonatal vocalization and volumes of various mouse brain regions that best account for variances in cognitive, social, and motor capacities. A positive outcome will provide much-needed predictive models to further explore the mechanistic bases of cognitive, social, and motor development in mouse models. This should enable investigators to further evaluate the mechanistic, structural bases for heterogeneous cognitive, social, and motor trajectories in preclinical models of environmental and genetic risk factors.