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Subthreshold Concentrations of Melatonin and Galantamine Improves Pathological AD-Hallmarks in Hippocampal Organotypic Cultures

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Abstract

Melatonin is a neurohormone whose levels are significantly reduced or absent in Alzheimer’s disease (AD) patients. In these patients, acetylcholinesterase inhibitors (AChEI) are the major drug class used for their treatment; however, they present unwanted cholinergic side effects and have provided limited efficacy in clinic. Because combination therapy is being extensively used to treat different pathological diseases such as cancer or acquired immune deficiency syndrome, we posed this study to evaluate if melatonin in combination with an AChEI, galantamine, could provide beneficial properties in a novel in vitro model of AD. Thus, we subjected organotypic hippocampal cultures (OHCs) to subtoxic concentrations of β-amyloid (0.5 μM βA) plus okadaic acid (1 nM OA), for 4 days. This treatment increased by 95 % cell death, which was mainly apoptotic as shown by positive TUNEL staining. In addition, the combination of βA/OA increased Thioflavin S aggregates, hyperphosphorylation of Tau, oxidative stress (increased DCFDA fluorescence), and neuroinflammation (increased IL-1β and TNFα). Under these experimental conditions, melatonin (1–1000 nM) and galantamine (10–1000 nM), co-incubated with the toxic stimuli, caused a concentration-dependent neuroprotection; maximal neuroprotective effect was achieved at 1 μM of melatonin and galantamine. Most effective was the finding that combination of sub-effective concentrations of melatonin (1 nM) and galantamine (10 nM) provided a synergic anti-apoptotic effect and reduction of most of the AD-related pathological hallmarks observed in the βA/OA model. Therefore, we suggest that supplementation of melatonin in combination with lower doses of AChEIs could be an interesting strategy for AD patients.

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Acknowledgments

This work was supported by the Spanish Ministry of Economy and Competence Ref. SAF2012-32223 to MGL. IB has a predoctoral FPU fellowship from the Spanish Ministry of Economy and Competence (AP2010/1219), EP has a predoctoral FPI fellowship from the Spanish Ministry of Economy and Competence, and EN (FPI-UAM2012) from Universidad Autónoma de Madrid. IS Carlos III, Programa Miguel Servet (CP11/00165) and FIS proyect (grant PI14/00372) and European Commission, Marie Curie Actions FP7 (FP7-People-2012-CIG-322156) to RL. JE has a grant from IS Carlos III, Programa Miguel Servet (CP14/00008) and IS Carlos III research contract under Miguel Servet Program. We would like to thank Vanessa Gómez Rangel for technical support, Mª Dolores Morales García and Ana Isabel de las Heras Núñez from the Confocal Service of de Universidad Autónoma de Madrid. We also thank the continuous support of Fundación Teófilo Hernando.

Compliance with Ethical Standards

All animal assays were carried out following the Guide for the Care and Use of Laboratory Animals and were previously approved by the Institutional Ethics Committee of the Autonomous University of Madrid, Spain, according to the European guidelines for the use and care of animals for research in accordance with the European Union Directive of 22 September 2010 (2010/63/UE) and with the Spanish Royal Decree of 1 February 2013 (53/2013). All efforts were made to minimize the number of animals used and their suffering.

Conflict of Interest

All authors have no conflict of interest.

Author Contributions

Izaskun Buendia has contributed to acquisition of data, data analysis/interpretation, writing, and critical revision of the manuscript. Esther Parada has contributed to acquisition of data and data analysis/interpretation. Elisa Navarro has contributed to acquisition of data and data analysis/interpretation. Rafael León has contributed to critical revision of the manuscript. Pilar Negredo has contributed to critical revision of the manuscript. Javier Egea has contributed to concept/design, acquisition of data, data analysis/interpretation, drafting of the manuscript, critical revision of the manuscript, and approval of the article. Manuela García López has contributed to concept/design, drafting of the manuscript, critical revision of the manuscript, and approval of the article.

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    Authors and Affiliations

    1. Instituto Teófilo Hernando (ITH), Departamento de Farmacología y Terapéutica. Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo, 4, Madrid, 28029, Spain

      I. Buendia, E. Parada, E. Navarro, R. León, J. Egea & M. G. López

    2. Instituto de Investigación Sanitaria, Hospital Universitario de la Princesa, C/ Diego de León 62, Madrid, 28006, Spain

      I. Buendia, E. Parada, E. Navarro, R. León, J. Egea & M. G. López

    3. Department of Anatomy, Histology and Neuroscience, School of Medicine, Universidad Autónoma de Madrid, Madrid, Spain

      P. Negredo

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    Correspondence to J. Egea or M. G. López.

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    I. Buendia and E. Parada contributed equally to this work.

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    Buendia, I., Parada, E., Navarro, E. et al. Subthreshold Concentrations of Melatonin and Galantamine Improves Pathological AD-Hallmarks in Hippocampal Organotypic Cultures. Mol Neurobiol 53, 3338–3348 (2016). https://doi.org/10.1007/s12035-015-9272-5

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