published_or_final_version / Physiology / Master / Master of Medical Sciences

Myriad small molecule compounds targeting metabotropic glutamate receptors (mGluRs) have been investigated for the treatment of various neuropsychiatric diseases and displayed promise in preclinical studies. At the clinical level, many of these compounds have been well tolerated by human subjects but have eluded success as promising therapeutics. There are eight subtypes of mGluRs, which express as constitutive dimers. This dimerization can occur between identical (homodimerization) or different (heterodimerization) mGluR protomer subtypes, which are subject to pairing-specific signaling mechanisms. Subtype expression of mGluRs is heterogenous between brain regions and cell types, yielding probable cell-specific homo- and heterodimer combinations that respond differently to certain drugs. While G protein recruitment to active mGluR dimers has been studied extensively, little is known about arrestin recruitment to these receptors. I used bioluminescence resonance energy transfer (BRET) assays, which provide a quantitative measure of protein-protein proximity, to observe and quantify arrestin recruitment to specific mGluR subtype pairings upon ligand administration in heterologous cells. I studied how select allosteric ligands affect communication between protomers to enhance arrestin recruitment to dimers. My findings indicate that arrestin recruitment occurs only at select mGluR homodimers upon orthosteric stimulation but is frequently stimulated or enhanced by administration of activating allosteric ligands. Additionally, I found that trans-protomer communication is highly specific to mGluR protomer subtype pairings, the ligand administered,a nd inter-protomer signal direction. Lastly, my findings reveal a cooperative effect of mGluR2 and 3 heterodimerization on arrestin recruitment that is dependent on the functional ability of each protomer to bind orthosteric agonist and responds distinctively from homodimers to stimulation by certain allosteric ligands. Taken together, this work shows that mGluR signaling can be tuned using strategic pharmacology and energizes hope for future clinical success of mGluR-targeting ligands.

The purpose of the study was to investigate the role of metabotropic glutamate receptors (mGluR) in the ontogeny of amphetamine-induced behavioral sensitization. Eleven-day-old rat pups were given five daily bilateral infusions of the mGluR antagonist, (RS)-methyl-4-carboxyphenylglycine (MCPG) followed by a systemic injection of amphetamine and locomotor activity was measured. It was hypothesized that rats receving amphetamine pretreatment and an amphetamine challenge would exhibit a significant increase in activity, indicating short-term behavioral sensitization. As predicted, repeated amphetamine administration during the pretreatment phase produced progressively enhanced locomotor activity, indicating the development of behavioral sensitization. The effect of MCPG on locomotor activity appears to be independent from the effects of amphetamine-induced locomotor activity and MCPG pretreatment failed to consistently block the expression of behavioral sensitization in rats pretreated with amphetamine and challenged with amphetamine. This study demonstrated that contrary to previous studies on adult rats, the mGluR system does not appear to consistently mediate the development of amphetamine-induced sensitization in neonatal rats.

The present study examined the role of group I metabotropic glutamate receptors (mGluRs) in mammalian spinal motor networks and investigated the potential role of mGluRs in the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Group I mGluR activation was found to modulate locomotor-related activity recorded from ventral roots of in vitro mouse spinal cord preparations. Activation of group I mGluRs led to an increase in the frequency of locomotor-related bursts and a decrease in their amplitude. The cellular mechanisms underlying group I mGluR-mediated modulation were investigated using whole-cell patch-clamp recordings from spinal neurons. Recordings from motoneurons revealed a wide range of effects, some of which were expected to increase motoneuron excitability, such as membrane depolarisation and hyperpolarisation of action potential thresholds. However, the net modulatory effect of group I mGluR activation was a reduction in motoneuron excitability, likely reflecting a reduction in the density of fast inactivating Na+ currents. The activation of group I mGluRs also reduced excitatory synaptic input to motoneurons, suggesting that modulation of motoneuron properties and synaptic transmission both contribute to group I mGluR-mediated reductions in locomotor motoneuron output. Recordings from spinal interneurons revealed a smaller range of modulatory effects for group I mGluRs. The clearest effect on interneurons, membrane depolarisation, may underlie group I mGluR-mediated increases in the frequency of locomotor activity. Finally, the potential role of group I mGluRs in the pathogenesis of ALS was investigated using a mouse model of the disease. Although no major perturbations in group I mGluR-mediated modulation were demonstrated in ALS affected spinal cords, there appeared to be a difference in the intrinsic excitability of spinal interneurons between wild type and ALS affected animals. Together these data highlight group I mGluRs as important sources of neuromodulation within the spinal cord and potential targets for the treatment of ALS.

Indiana University-Purdue University Indianapolis (IUPUI) / Background: Group II metabotropic glutamate receptors (mGluR2/3) are predominately presynaptically located Gi/o coupled receptors that are highly expressed in the cortex, nucleus accumbens, amygdala, and hippocampus. Previous studies suggest that group II mGluRs are involved in regulating ethanol (EtOH) consumption and seeking following extinction (Backstrom and Hyytia, 2005; Kufahl, et al., 2011). The sipper tube model, which allows for procedural separation of seeking and consumption, was used to further clarify the role of mGluR2/3 in EtOH-seeking and consumption. The non-selective group II mGluR agonist LY379268 (LY37) and selective mGluR2 positive allosteric modulator (PAM) BINA were used to determine the relative contribution of mGlu2 and mGlu3 receptors on EtOH seeking and consumption. Following characterization of the agonist and PAM on EtOH reinforcement, a microinjection study was performed examining the effect of blockade of nucleus accumbens core mGluR2/3 on systemic agonist induced suppression of EtOH-seeking. Methods: For the systemic agonist/PAM experiments, separate groups of male Wistar rats [n=8-9 group; LY37 (0-2.0 mg/kg) and BINA (0-20 mg/kg)] were trained to complete a response requirement (RR) of 10 lever presses that resulted in access to 10% EtOH or 2% sucrose (in separate groups) for a 20-minute drinking period. For consummatory testing, animals received weekly drug injections with a RR1. The RR was then increased over sessions to a RR20. For appetitive testing, animals received weekly drug injections followed by a non-reinforced extinction session. To determine effects of blockade of NAc core mGluR2/3 receptors on agonist-induced suppression of EtOH-seeking, a separate group of male Wistar rats (n=15) was trained to complete a RR10 for access to 10% EtOH. Animals were surgically implanted with bilateral guide cannulae terminating 1mm above the NAc core. Following recovery, animals received four sets of microinjections in a balanced design (systemic vehicle + core vehicle, systemic LY37 + core vehicle, systemic LY37 + core LY34, and systemic vehicle + core LY34). A final non-balanced microinjection of LY37 was then performed. Results and Conclusions: Systemic administration of the mGluR2/3 agonist LY37 significantly reduced EtOH- and sucrose- seeking with no systematic effect on locomotion. Systemic administration of the selective mGluR2 PAM BINA had no significant effect on either seeking or consumption. These findings suggest that modulation of glutamatergic neurotransmission by a systemic mGluR2/3 agonist, but not allosteric modulation of mGluR2, significantly reduces reinforcer seeking. Intra- accumbens core administration of LY37 significantly reduced EtOH-seeking, suggesting a role of NAc core mGluR2/3 modulation in EtOH-seeking during maintenance drinking. Systemic administration of LY37 was also found to significantly reduce sucrose consumption and body weight 24-hours following systemic administration, meriting further examination of the role of mGluR2/3 receptors on feeding behavior.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: During glutamate excitotoxicity, overstimulation of glutamate receptors leads to sustained elevation in cytosolic Ca2+ ([Ca2+]c), or delayed Ca2+ dysregulation (DCD), which is causally linked to cell death. There are two major hypothetical mechanisms for DCD: the continuous activation of N-methyl-D-aspartate-subtype of the ionotropic glutamate receptors (NMDAR) and the reversal of the plasmalemmal Na+/Ca2+ exchanger. However, the contribution of each of these mechanisms in DCD is not completely established. Major results: Neurons exposed to excitotoxic glutamate produced DCD, an increase in cytosolic Na+ ([Na+]c), and plasma membrane depolarization. MK801 and memantine, noncompetitive NMDAR inhibitors, added after glutamate, completely prevented DCD; however AP-5, a competitive NMDAR inhibitor, failed to do so. The NMDAR inhibitors had no effect on lowering elevated [Na+]c or on restoring plasma membrane potential, which are conditions suggesting NCXrev could be involved. In experiments inducing NCXrev, MK801 and memantine completely inhibited Ca2+ dysregulation after glutamate while AP-5 did not. Inhibition of NCXrev, either with KB-R7943 or by preventing the increase in [Na+]c, failed to avert DCD. However, NCXrev inhibition combined with NMDAR blocked by AP-5 completely prevented DCD. Overall, these data suggested that both NMDAR and NCXrev are essential for glutamate-induced DCD, and inhibition of only one mechanism is insufficient to prevent collapse of calcium homeostasis. Based on the data above, we investigated a NMDA receptor antagonist currently in clinical trials for reducing the effects of glutamate excitotoxicity, ifenprodil. Ifenprodil is an activity-dependent, NMDAR inhibitor selective for the NR2B subunit. We found that ifenprodil not only inhibited the NR2B-specific NMDAR, but also inhibited NCXrev. If ifenprodil is combined with PEAQX, a NMDAR inhibitor selective for the NR2A subunit, low concentrations of both inhibitors completely prevent DCD. Conclusion: The inhibition of a single Ca2+ influx mechanism is insufficient in preventing DCD, which requires simultaneous inhibition of both the NMDAR and NCXrev. These findings are critical for the correct interpretation of the experimental results obtained with these inhibitors and for better understanding of their neuroprotective actions.

Indiana University-Purdue University Indianapolis (IUPUI) / Glutamate clearance by astrocytes is an essential part of physiological excitatory neurotransmission. Failure to adapt or maintain low levels of glutamate in the central nervous system is associated with multiple acute and chronic neurodegenerative diseases. The primary excitatory amino acid transporters (EAATs) in human astrocytes are EAAT1 and EAAT2 (GLAST and GLT-1 respectively in rodents). While the inhibition of a ubiquitously-expressed serine/threonine protein kinase, the calcium/calmodulindependent kinase (CaMKII) results in diminished glutamate uptake in cultured primary rodent astrocytes, the molecular mechanism underlying this regulation is unknown. In order to delineate this mechanism, we use a heterologous expression model to explore CaMKII regulation of EAAT1 and EAAT2. In transiently transfected HEK293T cells, pharmacological inhibition of CaMKII and overexpression of a dominant-negative version of CaMKII (Asp136Asn) reduces [3H]-glutamate uptake by EAAT1, without altering EAAT2 mediated glutamate uptake. Surprisingly, overexpression of a constitutively active autophosphorylation mutant (Thr287Asp) to increase autonomous CaMKII activity and a mutant incapable of autophosphorylation (Thr287Val) had no effect on either EAAT1 or EAAT2 mediated glutamate uptake. Pulldown of FLAGtagged glutamate transporters suggests CaMKII does not interact with EAAT1 or EAAT2. SPOTS peptide arrays and recombinant GST-fusion proteins of the intracellular N- and C-termini of EAAT1 identified two potential phosphorylation sites at residues Thr26 and Thr37 in the N-terminus. Introducing an Ala (a non-phospho mimetic) but not an Asp (phosphomimetic) at Thr37 diminished EAAT1-mediated glutamate uptake, suggesting that the phosphorylation state of this residue is important for constitutive EAAT1 function. In sum, this is the first report of a glutamate transporter being identified as a direct CaMKII substrate. These findings indicate that CaMKII signaling is a critical driver of homeostatic glutamate uptake by EAAT1. Aberrations in basal CaMKII activity disrupt glutamate uptake, which can perpetuate glutamate-mediated excitotoxicity and result in cellular death.