Molecular Neurobiology

, Volume 54, Issue 6, pp 4537–4550 | Cite as

Heteroreceptor Complexes Formed by Dopamine D1, Histamine H3, and N-Methyl-D-Aspartate Glutamate Receptors as Targets to Prevent Neuronal Death in Alzheimer’s Disease

  • Mar Rodríguez-Ruiz
  • Estefanía Moreno
  • David Moreno-Delgado
  • Gemma Navarro
  • Josefa Mallol
  • Antonio Cortés
  • Carme Lluís
  • Enric I. Canela
  • Vicent Casadó
  • Peter J. McCormick
  • Rafael FrancoEmail author


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.


Alzheimers disease Dementia G-protein-coupled receptors Heteroreceptor complexes Ionotropic receptor Neuroprotection Receptor heteromers Parkinson’s disease Neurodegeneration Transgenic animals 



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.

Supplementary material

12035_2016_9995_MOESM1_ESM.tiff (1.2 mb)
ESM 1 Fig. 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)


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Mar Rodríguez-Ruiz
    • 1
    • 2
  • Estefanía Moreno
    • 1
    • 2
  • David Moreno-Delgado
    • 1
    • 2
  • Gemma Navarro
    • 1
    • 2
  • Josefa Mallol
    • 1
    • 2
  • Antonio Cortés
    • 1
    • 2
  • Carme Lluís
    • 1
    • 2
  • Enric I. Canela
    • 1
    • 2
  • Vicent Casadó
    • 1
    • 2
  • Peter J. McCormick
    • 1
    • 2
    • 3
  • Rafael Franco
    • 1
    • 2
    • 4
    Email author
  1. 1.Molecular Neurobiology laboratory, Department of Biochemistry and Molecular BiologyUniversity of BarcelonaBarcelonaSpain
  2. 2.Centro de Investigación en Red, Enfermedades Neurodegenerativas (CIBERNED)Instituto de Salud Carlos IIIMadridSpain
  3. 3.School of PharmacyUniversity of East AngliaNorwichUK
  4. 4.Departament de Bioquímica i Biologia Molecular, Facultat de BiologiaUniversitat de BarcelonaBarcelonaSpain

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