Alzheimer’s disease (AD) is a neurodegenerative disorder causing progressive memory loss and cognitive dysfunction. Anti-AD strategies targeting cell receptors consider them as isolated units. However, many cell surface receptors cooperate and physically contact each other forming complexes having different biochemical properties than individual receptors. We here report the discovery of dopamine D1, histamine H3, and N-methyl-D-aspartate (NMDA) glutamate receptor heteromers in heterologous systems and in rodent brain cortex. Heteromers were detected by co-immunoprecipitation and in situ proximity ligation assays (PLA) in the rat cortex where H3 receptor agonists, via negative cross-talk, and H3 receptor antagonists, via cross-antagonism, decreased D1 receptor agonist signaling determined by ERK1/2 or Akt phosphorylation, and counteracted D1 receptor-mediated excitotoxic cell death. Both D1 and H3 receptor antagonists also counteracted NMDA toxicity suggesting a complex interaction between NMDA receptors and D1-H3 receptor heteromer function. Likely due to heteromerization, H3 receptors act as allosteric regulator for D1 and NMDA receptors. By bioluminescence resonance energy transfer (BRET), we demonstrated that D1 or H3 receptors form heteromers with NR1A/NR2B NMDA receptor subunits. D1-H3-NMDA receptor complexes were confirmed by BRET combined with fluorescence complementation. The endogenous expression of complexes in mouse cortex was determined by PLA and similar expression was observed in wild-type and APP/PS1 mice. Consistent with allosteric receptor-receptor interactions within the complex, H3 receptor antagonists reduced NMDA or D1 receptor-mediated excitotoxic cell death in cortical organotypic cultures. Moreover, H3 receptor antagonists reverted the toxicity induced by ß1–42-amyloid peptide. Thus, histamine H3 receptors in D1-H3-NMDA heteroreceptor complexes arise as promising targets to prevent neurodegeneration.
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We would like to thank Prof. Isidre Ferrer and Dr. Ester Aso for kindly providing the APP/PS1 transgenic animals used in this work and Dr. Julie Perroy for kindly providing constructs encoding NMDA receptor subunits and fusion proteins containing NMDA receptor subunits.
None of the authors have received compensation for professional services.
MRR had and has a predoctoral contract from the University of Barcelona. DMD had postdoctoral contracts from the Spanish Government. EM and GN had and have research fellow contracts from CIBERNED (Instituto Carlos III, Ministry of Health, Spanish Government). AC, JM, CL, EIC, VC, and RF had and have academic positions linked to the University of Barcelona.
Compliance with Ethical Standards
Animal procedures were conducted according to ethical guidelines (European Communities Council Directive 2010/63/EU) and approved by the animal experimentation ethics committee of the Catalan Government (CEEA-DAAM 6419 and CEEA/DMAH 4049 and 5664).
Conflict of Interest
This work was supported by grants SAF2009-07276 (RF) from Spanish Ministry of Economy and Innovation (MINECO), 2014-SGR-1236 (EIC) from Generalitat de Catalunya and 2140610 (EIC) from the Fundació La Marató de TV3. Some grants may include FEDER funds. PJM was supported by projects RYC-2009-05522, SAF2010-18472 and RG140118. Authors declare no conflict of interests.
ESM 1Fig. S1 Bimolecular fluorescence complementation optimization. Different ratios of plasmids encoding fusion proteins constituted by H3 or D1 receptors and either half of the YFP were assayed to optimize fluorescence emission after complementation. HEK293T cells were co-transfected with the indicated amounts of cDNAs and 48 h post-transfection, fluorescence was determined at 530 nm. The optimal combination of cDNAs was obtained using D1R-cYFP and H3R-nYFP (a), where the ratio 1.5 μg and 4 μg, respectively, showed the highest percentage of fluorescence emission respect to non-transfected cells. The inverse combination of cDNAs D1R-nYFP and H3R-cYFP (b) showed no significant differences compared to non-transfected cells in all the tested ratios. As negative controls, other non-interacting pairs of receptors were assayed: serotonin 5HT2B and H3 receptors fused to, respectively, the C-terminal and N-terminal hemi-domains of YFP (c), and cannabinoid CB1 and D1 fused to, respectively, the N-terminal and C-terminal hemi-domains of YFP (d). . All negative controls showed no significant differences compared to non-transfected cells. Values are means ± SEM of 3–5 different experiments. One-way ANOVA followed by Dunnett’s post-hoc test showed significant (*p<0.05, ***p< 0.001,) differences compared to non-transfected cells. (TIFF 1241 kb)
Borchelt DR, Thinakaran G, Eckman CB et al (1997) Cellular and molecular mechanisms involved in the neurotoxicity of opioid and psychostimulant drugs. J Neurosci 1762:10090–10101. doi:10.1523/JNEUROSCI.4147-13.2014Google Scholar
Borchelt DR, Thinakaran G, Eckman CB et al (1996) Familial Alzheimer’s disease-linked presenilin 1 variants elevate Abeta1-42/1-40 ratio in vitro and in vivo. Neuron 17:1005–13CrossRefPubMedGoogle Scholar
Cowburn RF, Wiehager B, Trief E et al (1997) Effects of beta-amyloid-(25–35) peptides on radioligand binding to excitatory amino acid receptors and voltage-dependent calcium channels: evidence for a selective affinity for the glutamate and glycine recognition sites of the NMDA receptor. Neurochem Res 22:1437–42CrossRefPubMedGoogle Scholar
Hersi AI, Richard JW, Gaudreau P, Quirion R (1995) Local modulation of hippocampal acetylcholine release by dopamine D1 receptors: a combined receptor autoradiography and in vivo dialysis study. J Neurosci 15:7150–7PubMedGoogle Scholar
Cacabelos R, Yamatodani A, Niigawa H et al (1989) Brain histamine in Alzheimer’s disease. Methods Find Exp Clin Pharmacol 11:353–60PubMedGoogle Scholar
Panula P, Rinne J, Kuokkanen K et al (1998) Neuronal histamine deficit in Alzheimer’s disease. Neuroscience 82:993–7CrossRefPubMedGoogle Scholar
Mazurkiewicz-Kwilecki IM, Nsonwah S (1989) Changes in the regional brain histamine and histidine levels in postmortem brains of Alzheimer patients. Can J Physiol Pharmacol 67:75–8CrossRefPubMedGoogle Scholar
Franco R, Martínez-Pinilla E, Lanciego JLJL, Navarro G (2016) Basic pharmacological and structural evidence for class A G-protein-coupled receptor heteromerization. Front Pharmacol In press:76. doi: 10.3389/fphar.2016.00076
Moreno E, Hoffmann H, Gonzalez-Sepúlveda M et al (2011) Dopamine D1-histamine H3 receptor heteromers provide a selective link to MAPK signaling in GABAergic neurons of the direct striatal pathway. J Biol Chem 286:5846–54. doi:10.1074/jbc.M110.161489CrossRefPubMedGoogle Scholar
Fiorentini C, Gardoni F, Spano P et al (2003) Regulation of dopamine D1 receptor trafficking and desensitization by oligomerization with glutamate N-methyl-D-aspartate receptors. J Biol Chem 278:20196–20202. doi:10.1074/jbc.M213140200CrossRefPubMedGoogle Scholar
Lee FJS, Xue S, Pei L et al (2002) Dual regulation of NMDA receptor functions by direct protein-protein interactions with the dopamine D1 receptor. Cell 111:219–30CrossRefPubMedGoogle Scholar
Wilkinson D, Wirth Y, Goebel C (2014) Memantine in patients with moderate to severe Alzheimer’s disease: meta-analyses using realistic definitions of response. Dement Geriatr Cogn Disord 37:71–85. doi:10.1159/000353801CrossRefPubMedGoogle Scholar
Navarro G, Carriba P, Gandía J et al (2008) Detection of heteromers formed by cannabinoid CB1, dopamine D2, and adenosine A2A G-protein-coupled receptors by combining bimolecular fluorescence complementation and bioluminescence energy transfer. ScientificWorldJournal 8:1088–97. doi:10.1100/tsw.2008.136CrossRefPubMedGoogle Scholar
Navarro G, Moreno E, Aymerich M, et al. (2010) Direct involvement of sigma-1 receptors in the dopamine D1 receptor-mediated effects of cocaine. Proc Natl Acad Sci U S A. doi: 10.1073/pnas.1008911107
Koh JY, Yang LL, Cotman CW (1990) Beta-amyloid protein increases the vulnerability of cultured cortical neurons to excitotoxic damage. Brain Res 533:315–20CrossRefPubMedGoogle Scholar
Le WD, Colom LV, Xie WJ et al (1995) Cell death induced by beta-amyloid 1–40 in MES 23.5 hybrid clone: the role of nitric oxide and NMDA-gated channel activation leading to apoptosis. Brain Res 686:49–60CrossRefPubMedGoogle Scholar
Wu J, Anwyl R, Rowan MJ (1995) Beta-amyloid selectively augments NMDA receptor-mediated synaptic transmission in rat hippocampus. Neuroreport 6:2409–13CrossRefPubMedGoogle Scholar
Passani MB, Blandina P (1998) Cognitive implications for H3 and 5-HT3 receptor modulation of cortical cholinergic function: a parallel story. Methods Find Exp Clin Pharmacol 20:725–33CrossRefPubMedGoogle Scholar
Grove RA, Harrington CM, Mahler A et al (2014) A randomized, double-blind, placebo-controlled, 16-week study of the H3 receptor antagonist, GSK239512 as a monotherapy in subjects with mild-to-moderate Alzheimer’s disease. Curr Alzheimer Res 11:47–58CrossRefPubMedGoogle Scholar