Molecular modulation of dopamine transmission by TAAR1
Raul R. Gainetdinov Italian Institute of Technology, Genova, Italy St. Petersburg State University, St. Petersburg, Russia; Skolkovo Institute of Science and Technology, Moscow, Russia
ECNP Congress-2014, Berlin, Germany TAAR1 knockout (TAAR1-KO) mice
• Supersensitivity to behavioral and neurochemical effects of amphetamine (Wolinsky et al., Genes, Brain and Behavior, 2007)
• Deficit in prepulse inhibition • Enhaced behavioral and neurochemical effects of amphetamine • DA D2 receptors are supersensitive • Indicated as a model related to schizophrenia
Similarly, supersensitivity to psychostimulants was observed in other strains of TAAR1-KO mice developed independently (Linderman et al., 2008, Di Cara et al., 2011) Enhanced stimulatory effects of amphetamines and b-PEA in TAAR1-KO and heterozygous mice
d-Amphetamine (3 mg/kg, i.p.) 2000 L-Amphetamine (10 mg/kg, i.p.) 1500 TA1-WT (n=8) TA1-HET (n=8) TA1-WT TA1-KO (n=8) 1500 TA1-KO 1000
1000 (cm/5min)
(cm/5min) 500
Total distance Total 500 Total distance Total
0 0 0 30 60 90 120 150 0 30 60 90 120 Time (min) Time (min)
b-PEA (50 mg/kg, i.p.) 1500 TA1-WT (n=12) TA1-HET (n=12) TA1-KO (n=10 ) 1000
(cm/5min) 500 Total distanceTotal
0 0 30 60 90 Time (min) Suggests inhibitory action of TAAR1 on DA-dependent hyperactivity Selective TAAR1 ligands
• First selective TAAR1 antagonist EPPTB, selective for mouse TAAR1, less active for rat and human form (Bradaia et al., PNAS, 2009)
• Regulates the firing frequency of DA neurons in VTA • Blocks the TAAR1-mediated activation of an inwardly rectifying K(+) current.. • Increases the potency of DA at D2 receptors
Unfortunately, poor solubility and BBB permeability complicates the use of EPPTB in studies in vivo
First selective full TAAR1 agonist RO5166017, with high potency and efficacy at mouse, rat, monkey and human TAAR1 (Revel et al., PNAS, 2011)
• Regulates firing rate of both DA and 5-HT neurons potentially via interference with autoreceptor regulation • Potential anxiolytic effect in stress-induced hyperthermia test in mice • Reduces cocaine and NMDA antagonist induced hyperactivity in mice • Reduces hyperactivity of DAT-KO mice Dopamine transporter knockout (DAT-KO) Mice
Normal Lack of Neurotransmission Dopamine Transporter
DAT
DA DA
TH TH
Extracellular DA is elevated 5-fold Gainetdinov and Caron, Annu. Rev. Pharmacol. 2003 DA storage is reduced 20-fold Hyperactivity of DAT-KO mice Validation of selectivity of newly developed TAAR1 agonist (in collaboration with F. Hoffmann-La Roche Ltd )
• TAAR1 agonist inhibits hyperactive DAT-KO mice • This effect is absent in double KO mice lacking DAT and TAAR1 • Indicates an inhibitory action of TAAR1 on DA- dependent hyperactivity The full agonist The partial agonist
Potential antipsychotic-like activity: • Block cocaine and NMDA antagonists (PCP, L-687,414) - induced hyperactivity • Potentiate effects of Olanzapine on psychostimulant-induced hyperacitivity • Brain activation pattern reminiscent of the antipsychotic drug Olanzapine Potential antidepressant-like activity: • Porsolt forced-swim test in rats • The differential reinforcement of low-rate behavior paradigm in monkeys Potential pro-cognitive activity: • The object retrieval paradigm in Cynomolgus macaques • Attentional set-shifting in rats TAAR1 partial agonist RO5263397 increases wakefulness in rats
Potential mechanisms involved:
1. Role of TAAR1 in presynaptic regulation of DA transmission
2. Role of TAAR1 in the regulation of postsynaptivc D2Rs
3. Modulation of glutamate NMDA receptors in the prefrontal cortex by TAAR1
Role of TAAR1 in presynaptic regulation of DA transmission
• TAAR1-KO mice have altered firing rate of VTA dopaminergic neurons in slices
• p-tyramine decrease firing rate only in WT
• TAAR1 antagonist EPPTB reverse tyramine effect
• TAAR1 antagonist EPPTB increase firing rate in WT but not TAAR1-KO mice
• D2 DA receptor binding is increased in TAAR1-KO mice (Seeman et al, Synapse, 2007)
• Suggest an interaction of TAAR1 and D2 dopamine receptor signaling or regulation (Bradaia, et al., PNAS , 2009)
Role of TAAR1 in presynaptic regulation of DA transmission HPLC Analysis of total tissue content
Leo et al., Neuropharmacology, 2014
HVA, but not DA and DOPAC levels are increased in the striatum of TAAR1-KO mice Increased extracellular levels of DA in the Nucleus Accumbens but not Striatum of TAAR1-KO mice
(Quantitative Low Perfusion Rate Microdialysis in Awake Mice)
Leo et al., Neuropharmacology, 2014 Fast Scan Cyclic Voltammetry (FSCV) FSCV is a real-time electrochemical technique that detects electrochemically active neurotransmitters by their redox properties.
Applied waveform Applied waveform DA :-400 mV to 1V to -400 mV; scan rate 300V/s
Background current Ammine Oxidation pick Reduction pick (mV vs Ag/AgCl) (mV vs Ag/AgCl) Dopamine +500 -400
Adrenaline +500 -100 and -400 Voltamogram
Noradrenaline +500 -100
Serotonin +500 -100 and -800 An increased evoked DA release in the Nucleus Accumbens but not Striatum slices from TAAR1-KO mice
*
No alterations in measures of DA uptake (half-life; tau) are found in TAAR1 KO mice TAAR1 agonist reduces DA evoked overflow in Dorsal STR and NAc of control animals but does not affect TAAR1 KO mice
TAAR1 agonist RO5166017
N= 10, one way ANOVA But no alteration in DA half-life and Tau (indications of DA uptake) TAAR1 antagonist influences DA release in the NAc but not in the Dorsal STR
TAAR1 antagonist N-(3-Ethoxy-phenyl)-4-pyrrolidin-1- yl-3-trifluoromethyl-benzamide (EPPTB)
N= 10, one way ANOVA
But no alteration in DA half-life and Tau (indications of DA uptake) TAAR1 agonist potentiates effect of Quinpirole on evoked DA release in the NAc of WT but not TAAR1-KO mice
WT Quinpirole Wash 0.1 mM * * *
Two-pulse assessment of autoreceptor activity suggests impaired D2R autoreceptor function in TAAR1-Ko mice
Demonstrate that TAAR1 regulates sensitivity of D2R autoreceptos Role of TAAR1 in the regulation of postsynaptic D2R receptors D2R-related AKT3/GSK3 signaling pathway D2R binding and D2R-related signaling are altered in the striatum of TAAR1-KO mice
AKT/GSK3 signaling pathway is activated No alterations in the D2R but not D1R number is altered phosphorylation of pERK1/2, pCREB, pDARPP32 Haloperidol-induced catalepsy is reduced in TAAR1-KO mice
Bar test TAAR1 and D2R form a heterodimer in HEK-293 cells
Bioluminescence Resonance Energy Transfer (BRET) assay
TAAR1 and D2R form an heterodimer in cells that may be disrupted by haloperidol Role of TAAR1 in prefrontal cortex-related processes
Prefrontal cortex is implicated in cognitive behavior and in general in executive functions
Dysfunction of this areas are linked to psychiatric disorders such as schizophrenia, ADHD and bipolar disorder
VTA neurons project in different part of prefrontal cortex
TAAR1 is expressed and modulate DA VTA neurons and is also expressed in the PFC phMRI shows anypsychotic -like pattern of brain activation induced by TAAR1 agonists mPFC Pre-Limbic Area
Bregma 1.94
VI V II I III
Stimulation Recording Layer III Layer V
Patch-clump recordings in pyramidal neurons Evoked post-synaptic currents are altered
Evoked Post-Synaptic Currents (EPSCs)
Alterations in amplitude and and kinetics of EPSCs suggest changes in composition of postsynaptic glutamate receptors NMDA-dependent currents are reduced
NMDA/AMPA ratio is changed
NMDA mEPSCs are reduced
Indicates NMDA receptor-dependent synaptic deficits in PFC neurons of TAAR1-KO mice Glutamate NMDA receptor composition and phosphorylation is altered in the prefrontal cortex of TAAR1-KO mice
Taken together, these data indicate glutamate NMDA receptor hypofunction n the prefrontal cortex of TAAR1-KO mice Conclusions
• Stimulation of TAAR1 exerts an inhibitory action on dopamine-dependent locomotor activity • TAAR1 ligands can modulate dopamine release • TAAR1 can form a heterodimer with D2R and this interaction may be important for downstream signaling and behavior both at the level of presynaptic autoreceptors and postsynaptic receptors • TAAR1 modulates glutamate NMDA receptor function in the prefrontal cortex and related functions ACKNOWLEDGEMENTS
Ali Salahpour Stefano Espinoza University of Toronto Damiana Leo Bernard Masri Ilya Sukhanov INSERM, Tolouse Ludmilla Mus Larry Barak Tatyana Sotnikova Duke University, Durham Marc Caron Italian Institute of Technology, Genova, Italy Duke University, Durham
Fabio Fumagalli, University of Milan, Milan, Italy Marius Hoener, F. Hoffman La-Roche, Basel, Switzerland
Supported in part by F. Hoffman La-Roche, Basel, Switzerland and San Paolo Foundation, Torino, Italy