Activation of M4 muscarinic receptors in the striatum reduces tic-like behaviours in two distinct murine models of Tourette syndrome.

BACKGROUND AND PURPOSE: Current pharmacotherapies for Tourette syndrome (TS) are often unsatisfactory and poorly tolerated, underscoring the need for novel treatments. Insufficient striatal acetylcholine has been suggested to contribute to tic ontogeny. Thus, we tested whether activating M1 and/or M4 receptors-the two most abundant muscarinic receptors in the striatum-reduced tic-related behaviours in mouse models of TS. EXPERIMENTAL APPROACH: Studies were conducted using CIN-d and D1CT-7 mice, two TS models characterized by early-life depletion of striatal cholinergic interneurons and cortical neuropotentiation, respectively. First, we tested the effects of systemic and intrastriatal xanomeline, a selective M1/M4 receptor agonist, on tic-like and other TS-related responses. Then, we examined whether xanomeline effects were reduced by either M1 or M4 antagonists or mimicked by the M1/M3 agonist cevimeline or the M4 positive allosteric modulator (PAM) VU0467154. Finally, we measured striatal levels of M1 and M4 receptors and assessed the impact of VU0461754 on the striatal expression of the neural marker activity c-Fos. KEY RESULTS: Systemic and intrastriatal xanomeline reduced TS-related behaviours in CIN-d and D1CT-7 mice. Most effects were blocked by M4, but not M1, receptor antagonists. VU0467154, but not cevimeline, elicited xanomeline-like ameliorative effects in both models. M4, but not M1, receptors were down-regulated in the striatum of CIN-d mice. Additionally, VU0467154 reduced striatal c-Fos levels in these animals. CONCLUSION AND IMPLICATIONS: Activation of striatal M4, but not M1, receptors reduced tic-like manifestations in mouse models, pointing to xanomeline and M4 PAMs as novel putative therapeutic strategies for TS.

Systemic administration of ivabradine, a hyperpolarization-activated cyclic nucleotide-gated channel inhibitor, blocks spontaneous absence seizures.

OBJECTIVE: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are known to be involved in the generation of absence seizures (ASs), and there is evidence that cortical and thalamic HCN channel dysfunctions may have a proabsence role. Many HCN channel blockers are available, but their role in ASs has been investigated only by localized brain injection or in in vitro model systems due to their limited brain availability. Here, we investigated the effect on ASs of orally administered ivabradine (an HCN channel blocker approved for the treatment of heart failure in humans) following injection of the P-glycoprotein inhibitor elacridar, which is known to increase penetration into the brain of drug substrates for this efflux transporter. The action of ivabradine was also tested following in vivo microinjection into the cortical initiation network (CIN) of the somatosensory cortex and in the thalamic ventrobasal nucleus (VB) as well as on cortical and thalamocortical neurons in brain slices. METHODS: We used electroencephalographic recordings in freely moving Genetic Absence Epilepsy Rats From Strasbourg (GAERSs) to assess the action of oral administration of ivabradine, with and without elacridar, on ASs. Ivabradine was also microinjected into the CIN and VB of GAERSs in vivo and applied to Wistar CIN and GAERS VB slices while recording patch-clamped cortical Layer 5/6 and thalamocortical neurons, respectively. RESULTS: Oral administration of ivabradine markedly and dose-dependently reduced ASs. Ivabradine injection into CIN abolished ASs and elicited small-amplitude 4-7-Hz waves (without spikes), whereas in the VB it was less potent. Moreover, ivabradine applied to GAERS VB and Wistar CIN slices selectively decreased HCN channel-dependent properties of cortical Layer 5/6 pyramidal and thalamocortical neurons, respectively. SIGNIFICANCE: These results provide the first demonstration of the antiabsence action of a systemically administered HCN channel blocker, indicating the potential of this class of drugs as a novel therapeutic avenue for ASs.

Functional characterization of α7 nicotinic acetylcholine and NMDA receptor signaling in SH-SY5Y neuroblastoma cells in an ERK phosphorylation assay.

In the present study, the functional properties of α7 nicotinic acetylcholine receptors (α7 nAChRs) and N-methyl-D-aspartate receptors (NMDARs) endogenously expressed in SH-SY5Y human neuroblastoma cells were characterized in an extracellular-signal regulated kinase (ERK) phosphorylation assay. Both choline and N-methyl-D-aspartate (NMDA) mediated robust concentration-dependent increases in ERK phosphorylation in the SH-SY5Y cells, exhibiting EC50 values in good agreement with those reported for the agonists at recombinant α7 nAChRs and NMDARs, respectively. Importantly, the responses evoked by choline (10 mM) and by NMDA (50 µM) were significantly inhibited by the α7-selective antagonist α-bungarotoxin (100 nM) and by the NMDAR-selective antagonist MK-801 (50 µM), respectively. The increased ERK phosphorylation levels observed upon co-application of choline (1, 3, 10 mM) and NMDA (50 µM) compared to those produced by the two agonists on their own were fully reconcilable with additive effects and did not reveal substantial synergy between α7 nAChR and NMDAR signaling. Interestingly, however, the responses evoked by the "choline (10 mM) - NMDA (50 µM)" combination were almost completely inhibited by α-bungarotoxin (100 nM) as well as by MK-801 (50 µM), suggesting some sort of a link between α7 nAChR- and NMDAR-mediated ERK phosphorylation. Finally, oligomeric amyloid-ß1-42 peptide (1000 nM) mediated robust inhibition of the ERK phosphorylation induced by choline (10 mM), NMDA (50 µM) and the "choline (10 mM) - NMDA (50 µM)" combination. In conclusion, ERK phosphorylation measurements in SH-SY5Y cells provides a robust assay for studies of α7 nAChR- and NMDAR-mediating signaling and putative functional interactions between the receptors.

Probing the putative α7 nAChR/NMDAR complex in human and murine cortex and hippocampus: Different degrees of complex formation in healthy and Alzheimer brain tissue.

α7 nicotinic acetylcholine receptors (nAChRs) and N-methyl-D-aspartate receptors (NMDARs) are key mediators of central cholinergic and glutamatergic neurotransmission, respectively. In addition to numerous well-established functional interactions between α7 nAChRs and NMDARs, the two receptors have been proposed to form a multimeric complex, and in the present study we have investigated this putative α7 nAChR/NMDAR assembly in human and murine brain tissues. By α-bungarotoxin (BGT) affinity purification, α7 and NMDAR subunits were co-purified from human and murine cortical and hippocampal homogenates, substantiating the notion that the receptors are parts of a multimeric complex in the human and rodent brain. Interestingly, the ratios between GluN1 and α7 levels in BGT pull-downs from cortical homogenates from Alzheimer's disease (AD) brains were significantly lower than those in pull-downs from non-AD controls, indicating a reduced degree of α7 nAChR/NMDAR complex formation in the diseased tissue. A similar difference in GluN1/α7 ratios was observed between pull-downs from cortical homogenates from adult 3xTg-AD and age-matched wild type (WT) mice, whereas the GluN1/α7 ratios determined in pull-downs from young 3xTg-AD and age-matched WT mice did not differ significantly. The observation that pretreatment with oligomeric amyloid-ß1-42 reduced GluN1/α7 ratios in BGT pull-downs from human cortical homogenate in a concentration-dependent manner provided a plausible molecular mechanism for this observed reduction. In conclusion, while it will be important to further challenge the existence of the putative α7 nAChR/NMDAR complex in future studies applying other methodologies than biochemical assays and to investigate the functional implications of this complex for cholinergic and glutamatergic neurotransmission, this work supports the formation of the complex and presents new insights into its regulation in healthy and diseased brain tissue.

Nicotinic Acetylcholine Receptors in the Pathophysiology of Al zheimer's Disease: The Role of Protein-Protein Interactions in Current and Future Treatment.

Nicotinic acetylcholine receptors (nAChRs) have been pursued for decades as potential molecular targets to treat cognitive dysfunction in Alzheimer's disease (AD) due to their positioning within regions of the brain critical in learning and memory, such as the prefrontal cortex and hippocampus, and their demonstrated role in processes underlying cognition such as synaptic facilitation, and theta and gamma wave activity. Historically, activity at these receptors is facilitated in AD by use of drugs that increase the levels of their endogenous agonist acetylcholine, and more recently nAChR selective ligands have undergone clinical trials. Here we discuss recent findings suggesting that the expression and function of nAChRs in AD may be regulated by direct interactions with specific proteins, including Lynx proteins, NMDA-receptors and the Wnt/ß-catenin pathway, as well as ß-amyloid. The ability of protein interactions to modify nAChR function adds a new level of complexity to cholinergic signaling in the brain that may be specifically altered in AD. It is currently not known to what degree current nAChR ligands affect these interactions, and it is possible that the difference in the clinical effect of nAChR ligands in AD is related to differences in their ability to modulate nAChR protein interactions, rather than their effects on ion flow through the receptors. Drugs designed to target these interactions may thus provide a new avenue for drug development to ameliorate cognitive symptoms in AD. Notably, the development of experimental drugs that specifically modulate these interactions may provide the opportunity to selectively affect those aspects of nAChR function that are affected in AD.

α7 and ß2 Nicotinic Acetylcholine Receptor Subunits Form Heteromeric Receptor Complexes that Are Expressed in the Human Cortex and Display Distinct Pharmacological Properties.

The existence of α7ß2 nicotinic acetylcholine receptors (nAChRs) has recently been demonstrated in both the rodent and human brain. Since α7-containing nAChRs are promising drug targets for schizophrenia and Alzheimer's disease, it is critical to determine whether α7ß2 nAChRs are present in the human brain, in which brain areas, and whether they differ functionally from α7 nAChR homomers. We used α-bungarotoxin to affinity purify α7-containing nAChRs from surgically excised human temporal cortex, and found that α7 subunits co-purify with ß2 subunits, indicating the presence of α7ß2 nAChRs in the human brain. We validated these results by demonstrating co-purification of ß2 from wild-type, but not α7 or ß2 knock-out mice. The pharmacology and kinetics of human α7ß2 nAChRs differed significantly from that of α7 homomers in response to nAChR agonists when expressed in Xenopus oocytes and HEK293 cells. Notably, α7ß2 heteromers expressed in HEK293 cells display markedly slower rise and decay phases. These results demonstrate that α7 subunits in the human brain form heteromeric complexes with ß2 subunits, and that human α7ß2 nAChR heteromers respond to nAChR agonists with a unique pharmacology and kinetic profile. α7ß2 nAChRs thus represent an alternative mechanism for the reported clinical efficacy of α7 nAChR ligands.

Expression of the Ly-6 family proteins Lynx1 and Ly6H in the rat brain is compartmentalized, cell-type specific, and developmentally regulated.

The Ly-6 superfamily of proteins, which affects diverse processes in the immune system, has attracted renewed attention due to the ability of some Ly-6 proteins to bind to and modulate the function of neuronal nicotinic acetylcholine receptors (nAChRs). However, there is a scarcity of knowledge regarding the distribution and developmental regulation of these proteins in the brain. We use protein cross-linking and synaptosomal fractions to demonstrate that the Ly-6 proteins Lynx1 and Ly6H are membrane-bound proteins in the brain, which are present on the cell surface and localize to synaptic compartments. We further estimate the amount of Lynx1 in the rat cortex using known amounts of a heterologously expressed soluble Lynx1 variant (ws-Lynx1) to be approximately 8.6 ng/µg total protein, which is in line with the concentrations of ws-Lynx1 required to affect nAChR function. In addition, we demonstrate that Lynx1 and Ly6H are expressed in cultured neurons, but not cultured micro- or astroglial cultures. In addition, Lynx1, but not Ly6H was detected in the CSF. Finally, we show that the Ly-6 proteins Lynx1, Lynx2, Ly6H, and PSCA, display distinct expression patterns during postnatal development in the rat frontal cortex and hippocampus at the mRNA and protein level, and that this is paralleled to some degree by the expression of the nAChR subunits α2, α4, α7 and ß2. Our results demonstrate a developmental pattern, localization, and concentration of Ly-6 proteins in the brain, which support a role for these proteins in the modulation of signaling at synaptic membranes.