Antonio Ferragud

Trace Amine-Associated Receptor 1 Partial Agonism Reveals Novel Paradigm for Neuropsychiatric Therapeutics

BACKGROUND Trace amines, compounds structurally related to classical biogenic amines, represent endogenous ligands of the trace amine-associated receptor 1 (TAAR1). Because trace amines also influence the activity of other targets, selective ligands are needed for the elucidation of TAAR1 function. Here we report on the identification and characterization of the first selective and potent TAAR1 partial agonist. METHODS The TAAR1 partial agonist RO5203648 was evaluated for its binding affinity and functional activity at rodent and primate TAAR1 receptors stably expressed in HEK293 cells, for its physicochemical and pharmacokinetic properties, for its effects on the firing frequency of monoaminergic neurons ex vivo, and for its properties in vivo with genetic and pharmacological models of central nervous system disorders. RESULTS RO5203648 showed high affinity and potency at TAAR1, high selectivity versus other targets, and favorable pharmacokinetic properties. In mouse brain slices, RO5203648 increased the firing frequency of dopaminergic and serotonergic neurons in the ventral tegmental area and the dorsal raphe nucleus, respectively. In various behavioral paradigms in rodents and monkeys, RO5203648 demonstrated clear antipsychotic- and antidepressant-like activities as well as potential anxiolytic-like properties. Furthermore, it attenuated drug-taking behavior and was highly effective in promoting attention, cognitive performance, and wakefulness. CONCLUSIONS With the first potent and selective TAAR1 partial agonist, RO5203648, we show that TAAR1 is implicated in a broad range of relevant physiological, behavioral, and cognitive neuropsychiatric dimensions. Collectively, these data uncover important neuromodulatory roles for TAAR1 and suggest that agonists at this receptor might have therapeutic potential in one or more neuropsychiatric domains.

The hippocampal dentate gyrus is essential for generating contextual memories of fear and drug-induced reward.

The hippocampus is believed to play a role in processing information relative to the context in which emotionally salient experiences occur but evidence on the specific contribution of the hippocampal dentate gyrus (DG) to these processes is limited. Here, we have used two classical behavioral paradigms to study the participation of the dorsal DG in context-conditioned reward and context-conditioned fear. Rats received intra-hippocampal vehicle or colchicine injections (4 microg/microl solution; 0.2 microl injections at 10 sites) that damaged the DG but spared other hippocampal subfields. In the first experiment, we used a place conditioning procedure pairing cocaine exposure (20 mg/kg, i.p.) with a specific context and vehicle treatment with another. While rats with sham lesions exhibited preference for the cocaine-paired context following conditioning, rats with lesions of the DG showed no evidence of cocaine-induced place preference. In the second experiment, rats with sham or colchicine lesions received a foot shock in a given context and conditioned freezing was measured upon reexposure to the shock-paired context (2, 24, 48 and 96 h after conditioning). Rats with sham lesions exhibited high levels of conditioned freezing when exposed to the conditioning context but rats with lesions of the DG showed impaired conditioning, behaving as controls that had experienced shock in a different context. These observations indicate that the integrity of the DG is essential for establishing a coherent representation of the context to which emotional experiences, either hedonic or aversive, are bound.

High Trait Impulsivity Predicts Food Addiction-Like Behavior in the Rat

Impulsivity is a behavioral trait frequently seen not only in drug-addicted individuals but also in individuals who pathologically overeat. However, whether impulsivity predates the development of uncontrollable feeding is unknown. In this study, we hypothesized that a high impulsivity trait precedes and confers vulnerability for food addiction-like behavior. For this purpose, we trained ad libitum-fed male Wistar rats in a differential reinforcement of low rates of responding (DRL) task to select Low- and High-impulsive rats. Then, we allowed Low- and High-impulsive rats to self-administer a highly palatable diet (Palatable group) or a regular chow diet (Chow group) in 1-h daily sessions, under fixed ratio (FR) 1, FR3, FR5, and under a progressive ratio (PR) schedules of reinforcement. In addition, we tested the compulsiveness for food in Low- and High-impulsive rats by measuring the food eaten in the aversive, open compartment of a light/dark conflict test. Finally, we measured the expression of the transcription factor ΔFosB in the shell and the core of the nucleus accumbens, which is a marker for neuroadaptive changes following addictive drug exposure. The data we obtained demonstrate that impulsivity is a trait that predicts the development of food addiction-like behaviors, including: (i) excessive intake, (ii) heightened motivation for food, and (iii) compulsive-like eating, when rats are given access to highly palatable food. In addition, we show that the food addiction phenotype in high impulsive subjects is characterized by an increased expression of the transcription factor ΔFosB in the nucleus accumbens shell. These results reveal that impulsivity confers an increased propensity to develop uncontrollable overeating of palatable food.

Neurotoxicity and persistent cognitive deficits induced by combined MDMA and alcohol exposure in adolescent rats

Recent trend assessments of drug consumption reveal an increase in the simultaneous use of several drugs at raves, clubs and college settings among youngsters and young adults. We studied in adolescent rats the effects of repeated exposure to cocaine and 3,4‐methylenedioxymethanphetamine (MDMA, ecstasy), given alone or in combination with alcohol, on memory performance, adult hippocampal neurogenesis and neurotoxicity. Rats were trained two weeks after the drug treatments in the radial arm maze. The results showed that only rats exposed to combinations of alcohol and MDMA exhibited significant memory deficits. Alcohol, MDMA and combinations thereof significantly decreased 5‐bromodeoxyuridine labeling in the dentate gyrus (DG), indicating reduced survival of neuronal precursors. None of the treatments altered the length of the dendritic arbors of doublecortin (DCX)‐positive neurons or the number and length of DCX‐negative gaps in the DG. Thus, changes in adult neurogenesis were not causally related to the cognitive alterations induced by the treatments. Only the combination of alcohol and MDMA significantly decreased the population of mature granule neurons in the DG and increased the presence of cluster of differentiation 11b+ reactive microglia in the bordering areas of the subgranular zone. Critically, memory impairment was correlated with granule cell depletion. These observations demonstrate that exposure to alcohol and MDMA during adolescence, at doses that do not provoke apparent cognitive impairment when given separately, causes neurotoxic alterations affecting the DG region as well as persistent memory deficits. The findings highlight the elevated risk associated with the concurrent recreational use of alcohol and MDMA.

The Dopamine Uptake Inhibitor 3α-[bis(4′-fluorophenyl)metoxy]-tropane Reduces Cocaine-Induced Early-Gene Expression, Locomotor Activity, and Conditioned Reward

Benztropine (BZT) analogs, a family of high-affinity dopamine transporter ligands, are molecules that exhibit pharmacological and behavioral characteristics predictive of significant therapeutic potential in cocaine addiction. Here, we examined in mice the effects of 3α-[bis(4′-fluorophenyl)metoxy]-tropane (AHN-1055) on motor activity, conditioned place preference (CPP) and c-Fos expression in the striatum. AHN-1055 produced mild attenuation of spontaneous locomotor activity at a low dose (1 mg/kg) and weak stimulation at a higher dose (10 mg/kg). In parallel, the BZT analog significantly increased c-Fos expression in the dorsolateral caudoputamen at the high dose, whereas producing marginal decreases at low and moderate doses (1, 3 mg/kg) in both dorsal and ventral striatum. Interaction assays showed that cocaines ability to stimulate locomotor activity was decreased by AHN-1055 treatment, but not by treatment with D-amphetamine. Such reduced ability did not result from an increase in stereotyped behavior. Another dopamine uptake inhibitor, nomifensine, decreased cocaine-induced locomotor activity but evoked by itself intense motor stereotypies. Remarkably, the BZT analog dose-dependently blocked cocaine-induced CPP without producing CPP when given alone, and blocked in conditioned mice cocaine-stimulated early-gene activation in the nucleus accumbens and dorsomedial striatum. These observations provide evidence that AHN-1055 does not behave as a classical psychomotor stimulant and that some of its properties, including attenuation of cocaine-induced striatal c-Fos expression, locomotor stimulation, and CPP, support its candidacy, and that of structurally related molecules, as possible pharmacotherapies in cocaine addiction.

Exposure to N-Ethyl-N-Nitrosourea in Adult Mice Alters Structural and Functional Integrity of Neurogenic Sites

Background Previous studies have shown that prenatal exposure to the mutagen N-ethyl-N-nitrosourea (ENU), a N-nitroso compound (NOC) found in the environment, disrupts developmental neurogenesis and alters memory formation. Previously, we showed that postnatal ENU treatment induced lasting deficits in proliferation of neural progenitors in the subventricular zone (SVZ), the main neurogenic region in the adult mouse brain. The present study is aimed to examine, in mice exposed to ENU, both the structural features of adult neurogenic sites, incorporating the dentate gyrus (DG), and the behavioral performance in tasks sensitive to manipulations of adult neurogenesis. Methodology/Principal Findings 2-month old mice received 5 doses of ENU and were sacrificed 45 days after treatment. Then, an ultrastructural analysis of the SVZ and DG was performed to determine cellular composition in these regions, confirming a significant alteration. After bromodeoxyuridine injections, an S-phase exogenous marker, the immunohistochemical analysis revealed a deficit in proliferation and a decreased recruitment of newly generated cells in neurogenic areas of ENU-treated animals. Behavioral effects were also detected after ENU-exposure, observing impairment in odor discrimination task (habituation-dishabituation test) and a deficit in spatial memory (Barnes maze performance), two functions primarily related to the SVZ and the DG regions, respectively. Conclusions/Significance The results demonstrate that postnatal exposure to ENU produces severe disruption of adult neurogenesis in the SVZ and DG, as well as strong behavioral impairments. These findings highlight the potential risk of environmental NOC-exposure for the development of neural and behavioral deficits.

Therapeutic-like properties of a dopamine uptake inhibitor in animal models of amphetamine addiction

N-substituted benztropine (BZT) analogs are molecules that display high affinity for the dopamine transporter (DAT), therapeutic-like effects in animal models of cocaine abuse, and psychopharmacological characteristics consistent with those of a substitute medication for cocaine addiction. Since amphetamine (Amph) and cocaine share mechanisms of action at the DAT, we evaluated the effectiveness of a BZT analog in animal models of Amph addiction. We tested in mice and rats the effects of the BZT derivative, 3α-[bis(4-fluorophenyl)methoxy]-tropane (AHN-1055), on Amph-induced conditioned place preference (CPP), locomotor activity, sensitization, self-administration and ΔFosB accumulation in the nucleus accumbens (NAc). The results showed that AHN-1055 did not produce rewarding, stimulant, or sensitized locomotor effects in mice when administered alone but it readily blocked the rewarding, stimulant, and sensitizing effects of repeated Amph exposure. Furthermore, in mice undergoing conditioning in the CPP paradigm, the BZT analog prevented the accumulation of ΔFosB protein induced in the NAc shell region by Amph treatment. Notably, treatment with AHN-1055 dose-dependently reduced Amph self-administration in rats with a steady history of voluntary Amph intake. These results provide a straightforward demonstration that a BZT derivative with binding affinity for DAT exhibits high efficacy in animal models of Amph abuse, suggesting that the novel generation of BZT analogs could have wider therapeutic applications in stimulant-spectrum disorders than those previously recognized.

The Trace Amine-Associated Receptor 1 Agonist RO5256390 Blocks Compulsive, Binge-Like Eating in Rats.

Compulsive, binge eating of highly palatable food constitutes a core feature of some forms of obesity and eating disorders, as well as of the recently proposed disorder of food addiction. Trace amine-associated receptor 1 (TAAR1) is a highly conserved G-protein-coupled receptor bound by endogenous trace amines. TAAR1 agonists have been shown to reduce multiple behavioral effects of drugs of abuse through their actions on the mesocorticolimbic system. In this study, we hypothesized that TAAR1 may have a role in compulsive, binge-like eating; we tested this hypothesis by assessing the effects of a TAAR1 agonist, RO5256390, in multiple excessive feeding-related behaviors induced by limiting access to a highly palatable diet in rats. Our results show that RO5256390 blocked binge-like eating in rats responding 1 h per day for a highly palatable sugary diet. Consistent with a palatability-selective effect, drug treatment selectively reduced the rate and regularity of palatable food responding, but it did not affect either baseline intake or food restriction-induced overeating of the standard chow diet. Furthermore, RO5256390 fully blocked compulsive-like eating when the palatable diet was offered in an aversive compartment of a light/dark conflict box, and blocked the conditioned rewarding properties of palatable food, as well as palatable food-seeking behavior in a second-order schedule of reinforcement. Drug treatment had no effect on either anxiety-like or depressive-like behavior, and it did not affect control performance in any of the tests. Importantly, rats exposed to palatable food showed decreased TAAR1 levels in the medial prefrontal cortex (mPFC), and RO5256390 microinfused into the infralimbic, but not prelimbic, subregion of the mPFC-reduced binge-like eating. Altogether, these results provide evidence for TAAR1 agonism as a novel pharmacological treatment for compulsive, binge eating.

Substituting a long-acting dopamine uptake inhibitor for cocaine prevents relapse to cocaine seeking

The treatment of cocaine addiction remains a challenge. The dopamine replacement approach in cocaine addiction involves the use of a competing dopaminergic agonist that might suppress withdrawal and drug craving in abstinent individuals. Although it has long been postulated that such an approach may be therapeutically successful, preclinical or clinical evidence showing its effectiveness to prevent relapse is scant. We used in rats a procedure that involved substitution of the N‐substituted benztropine analog 3α‐[bis(4′‐fluorophenyl)methoxy]‐tropane (AHN‐1055), a long‐acting dopamine uptake inhibitor (DUI), for cocaine. Maintenance treatment was self‐administered. After extinction, reinstatement of drug seeking was induced by cocaine priming. We measured the contents of brain‐derived neurotrophic factor (BDNF), c‐Fos and Fas‐associated death domain (FADD) proteins in the medial prefrontal cortex (mPFC) following reinstatement. DUI, but not amphetamine, substitution led to extinction of active lever presses, as did saline substitution. DUI substitution significantly reduced cocaine‐induced reinstatement of drug‐seeking behavior, which was strongly elicited after saline substitution. Rats passively yoked to DUI also showed reduced cocaine‐primed reinstatement. Reductions in drug seeking during reinstatement were matched by downward shifts in the contents of BDNF, c‐Fos and FADD proteins in the mPFC, which were elevated in relapsing rats. These data indicate that DUI substitution not only leads to extinction of self‐administration behavior but also prevents reinstatement of drug seeking induced by cocaine re‐exposure. Thus, DUI substitution therapy using compounds with low abuse potential, even if received passively in the context previously paired with drug taking, may provide an effective treatment for stimulant addiction.

Reducing the RNA binding protein TIA1 protects against tau-mediated neurodegeneration in vivo

Emerging studies suggest a role for tau in regulating the biology of RNA binding proteins (RBPs). We now show that reducing the RBP T-cell intracellular antigen 1 (TIA1) in vivo protects against neurodegeneration and prolongs survival in transgenic P301S Tau mice. Biochemical fractionation shows co-enrichment and co-localization of tau oligomers and RBPs in transgenic P301S Tau mice. Reducing TIA1 decreased the number and size of granules co-localizing with stress granule markers. Decreasing TIA1 also inhibited the accumulation of tau oligomers at the expense of increasing neurofibrillary tangles. Despite the increase in neurofibrillary tangles, TIA1 reduction increased neuronal survival and rescued behavioral deficits and lifespan. These data provide in vivo evidence that TIA1 plays a key role in mediating toxicity and further suggest that RBPs direct the pathway of tau aggregation and the resulting neurodegeneration. We propose a model in which dysfunction of the translational stress response leads to tau-mediated pathology.Apicco and colleagues show that reducing TIA1 inhibits tau-mediated neurodegeneration and improves survival in a mouse model of tauopathy. This rescue occurs with a transition in tau aggregation from oligomeric to fibrillar forms of tau. These findings suggest a key role for RNA binding proteins in the pathophysiology of tau.