Marco Bortolato, MD, PhD - US grants

DESCRIPTION (provided by applicant): Pathological aggression has a devastating socio-economic impact, and is a leading mortality in young male Americans. The development of preventative and therapeutic tools for the management of this disorder is greatly limited by our partial understanding of its pathophysiology. The objective of this exploratory R21 proposal is to understand the mechanisms of interaction between the three best-characterized vulnerability factors for pathological aggression: early psychosocial stress;low activity of brain monoamine oxidase (MAO) A, the major enzyme for the degradation of brain serotonin (5-HT), norepinephrine (NE) and dopamine (DA);male gender, in relation to the high levels of androgen testosterone and its metabolites. In fact, recent data show that the high risk to develop post-pubertal aggression in maltreated boys is mainly observed in carriers of genetic variants associated with low MAO A activity. We found that 5-1 reductase (5AR), the enzyme that converts testosterone into its potent androgenic metabolite dihydrotestosterone (DHT), is affected by early stress and MAO-A deficiency;furthermore, its inhibition by finasteride reduces the high aggression of patients and MAO A knockout mice. The leading hypothesis of this proposal is that the interaction of low MAO A activity and early psychosocial stress results in long-term changes in monoamine levels and steroidogenic pathways in the brain. In males, the increase in testosterone levels at puberty interacts with these changes, resulting in imbalances in steroids and monoamines in prefrontal cortex and other key brain regions for emotional regulation. These neurochemical perturbations lead to aggression and antisocial behavior. We will address this hypothesis using male WT, MAO A KO mice and a newly-developed line of MAO A hypomorphic (MAO Aneo) mice, with very low brain MAO A activity. These mice show lower levels of aggression than MAO A KO mice and are an excellent model for carriers of low MAO A- activity genetic variants. In Aim 1, we will study the effects of the interaction between MAO-A genetic variants and early stress in the developmental trajectory of aggression in male mice. To this end, we will subject male MAO Aneo and KO pups (and their WT littermates) to maternal separation (a highly isomorphic model of child neglect) for the first three weeks of postnatal life;aggression-related behaviors before, during and after puberty will be correlated with the levels of MAO A and 5AR, as well as 5-HT, NE, DA, testosterone and their metabolites in key brain regions for the regulation of aggression. In Aim 2, we will study the role of testosterone and 5AR in the aggression of MAO A-deficient mice, by assessing the behavioral and molecular changes induced by castration and finasteride treatment. The proposed project will help establish the neurobiological bases of pathological aggression. The translational application of these findings will be critical to define new biomarkers, endophenotypes and molecular targets for early prevention, diagnosis and treatment of this disorder. PUBLIC HEALTH RELEVANCE: This application proposes to use a highly innovative approach to identify the mechanisms of interactions between the three best-characterized vulnerability factors for pathological aggression: early psychosocial stress, low activity of monoamine oxidase and male gender (in relation to testosterone and its metabolism). This knowledge will help establish the neurobiological bases of pathological aggression, which will be translated into novel biomarkers, endophenotypes and molecular targets for early prevention, diagnosis and treatment of this disorder.

DESCRIPTION (provided by applicant): Pathological reactive aggression is a condition characterized by frequent outbursts of impulsive violence and anger. The devastating socio-economic impact of this problem underscores the urgent need to identify effective strategies for its prevention and treatment; unfortunately, current efforts in this direction are thwarted by our poor knowledge of the pathophysiology of reactive aggression. The long-term goal of our research is to elucidate the neurobiological bases of aggression, and identify novel molecular targets for the prevention and therapy of this complex condition. Animal models are indispensable tools to understand the mechanisms of aggression and test novel therapies, but their translational and predictive validity is often compromised by their failure to accurately reproduce the mechanisms of pathological aggression in humans. Thanks to our previous NIH R21 grant, we began to address this problem by developing the first mouse model of the best-characterized interaction of genetic and environmental vulnerability factors for pathological reactive aggression. This interaction involves: i) low activity of monoamine oxidase (MAO) A, the major enzyme for the metabolism of serotonin (5HT); ii) child neglect or abuse. To simulate this interaction, we generated a novel line of mice with low MAOA activity (MAOANeo), and subjected them to maternal separation (MS, simulating child neglect) during the first week of life. Similarly to the clinical scenario, MS induced high levels of aggression in adolescent and adult MAOANeo mice, but not in their wild-type littermates. Our preliminary data suggest that MS predisposes MAOANeo mice to develop aggression through the interplay of age-specific processes: 1) the activation of 5HT2A receptors during the first week of life; 2) progressive, age- dependent deficits of N-methyl-D-aspartate glutamate receptors (NMDARs) in the prefrontal cortex (PFC) throughout adolescence; and 3) the disinhibition of glutamatergic subcortical connections in adulthood. Based on these data, we hypothesize that the interaction of low MAOA activity and MS produces 5HT2A receptor overstimulation and age-dependent NMDAR alterations in the PFC. In turn, these PFC deficits lead to aggression through the disinhibition of downstream glutamate pathways across subcortical regions. We will test this hypothesis in three specific aims: in Aim 1, we will determine how the interaction of low MAO A activity and MS leads to early 5HT 2A receptor overstimulation; in Aim 2, we will examine how early 5HT2A receptor overstimulation leads to age-specific deficits of NMDAR signaling pathways in the PFC; in Aim 3, we will identify the subcortical regions that mediate the role of glutamate in the aggressive responses of MAOANeo mice subjected to MS. These aims will be accomplished with a unique combination of cutting-edge behavioral, neurochemical and proteomic technologies. The results of these translational studies will help us understand the biological bases of reactive aggression, identify new potential biomarkers and therapeutic targets for this condition, and eventually reduce its staggering socio-economic burden.

ABSTRACT Tourette syndrome (TS) is a neurodevelopmental disorder, affecting 0.5-1% of the pediatric population, with a marked male predominance. TS is characterized by multiple, recurring tics, which are a source of significant disability. The clinical management of TS poses considerable challenges, partially due to the limited efficacy and numerous side effects of available pharmacotherapies. Tics are triggered and exacerbated by stress, but the underpinnings of this association remain unclear. This proposal seeks to test a novel candidate mechanism whereby stress exacerbates tics, which may represent a new therapeutic target. Over the past few years, the Bortolato lab has shown that neurosteroids, a class of brain-produced steroids that play a central role in the orchestration of stress response, are involved in the neurobiological mechanisms of tic modulation. In particular, our preliminary data in animal models strongly suggest that the adverse effects of stress on tic severity are mediated by the neurosteroid allopregnanolone (AP). The implication of neurosteroids in TS is further suggested by our findings on finasteride, an inhibitor of the key steroidogenic enzymes 5?-reductase 1 (5?R1) and 2 (5?R2). In preliminary clinical trials, this drug led to a significant reduction of tic severity in adult TS patients refractory to standard treatments; furthermore, in animal models of TS, this drug reduced both stereotyped movements and other TS-related behavioral deficits. Although these data are promising, finasteride is contraindicated in childhood due to its demasculinizing effects. Nevertheless, preliminary data in animals suggest that the therapeutic effects of finasteride may be due to 5?R1 inhibition, while the antiandrogenic effects of this drug are primarily due to 5?R2 inactivation. Based on this background, our central hypothesis is that acute stress triggers tics by increasing 5?R1- mediated synthesis of AP. To test this hypothesis, we will use a mouse model of TS firmly grounded in tic pathophysiology, recently developed by the Pittenger group. This model, which reproduces the depletion of striatal cholinergic interneurons observed in post-mortem samples of subjects with severe TS, is particularly well-suited to test our hypothesis, since it exhibits tic-like behaviors in response to stress. The central hypothesis will be tested in two aims, to study how AP (Aim 1) and 5?R1 (Aim 2) contribute to the tic-like responses induced by stress in this model. The proposed research will be the first to test a specific mechanistic hypothesis explaining how acute stress exacerbates tics in TS. If our hypotheses are confirmed, these experiments may lead to a novel therapeutic approach for tics, since highly specific 5?R1 inhibitors have already been developed for clinical use and have excellent tolerability. Furthermore, because neurosteroids modulate the neuroactive functions of sex hormones, a better understanding of their role in tic genesis may help explain the male predominance of TS.

PROJECT SUMMARY Opioid misuse and dependence have become one of the largest public health crises in the US, with devastating socio-economic repercussions. In the attempt to curb this epidemic, NIH has recently issued the HEAL initiative to promote the development of novel and better strategies for OUD prevention and therapy. The goal of our team is to develop novel non-opioid drugs that may prevent or counter the abuse liability of opioids. To this end, the Peterson lab has recently developed a high-throughput zebrafish assay to screen for drugs that may reduce opioid self-administration. Drug screening with this paradigm revealed that one of the most effective molecules to suppress opioid seeking was finasteride (FIN), a drug approved for clinical use with good safety and tolerability. FIN inhibits 5?-reductase (5?R), the enzyme catalyzing the rate-limiting step in the synthesis of several neurosteroids (including allopregnanolone and other allosteric modulators of GABA-A receptors), as well as in the metabolism of testosterone and corticosterone. However, the specific changes in steroid profiles whereby FIN reduces opioid self-administration remain unclear. Over the past few years, the Bortolato group has studied the behavioral effects of FIN in rodent models of psychiatric disorders and found that this drug reduces the severity of impulsive behaviors and self-administration of other drugs of abuse. These studies have also shown that FIN?s effects are related to dopamine signaling, but do not affect the function of opioid receptors. Accordingly, our preliminary data show that FIN does not interfere with the analgesic properties of opioids in rat models. The exploratory collaborative studies proposed in this application are aimed at obtaining critical data on the translational power of our discoveries on the zebrafish model and ascertain the mechanisms whereby FIN reduces opioid self-administration. The two aims of this proposal will: 1) use a well-validated rat model to test whether opioid self-administration is reversed or prevented by FIN, and study potential sex differences in relation to these effects; and 2) use the high-throughput zebrafish model to identify which steroids (substrates and products of 5?R) are responsible for the effects of FIN on opioid self-administration. The results of the proposed studies will lead to future studies to validate the effects of FIN on multiple stages of opioid self- administration (also in rat models of chronic and neuropathic pain) and the steroid-based mechanisms whereby FIN modifies the rewarding effects of opioids. Given that FIN has been used in patients for more than 25 years and has a very good tolerability and safety profile, these studies may rapidly lead to clinical trials on its use as an adjunct treatment to opioid pain-killers that may reduce opioid abuse liability without interfering with the analgesic properties of these drugs.