Extracellular tau promotes intracellular calcium increase through M1 and M3 muscarinic receptors in neuronal cells
Introduction
Alzheimer disease (AD) is characterized by the presence in the brain of the patients of two structures, the senile plaques and neurofibrillary tangles (NFT). The main component of NFTs is the microtubule associated protein tau in hyperphosphorylated form. Tau pathology in AD follows a reproducible pattern, whereby hyperphosphorylated (and aggregated) tau first appears in the entorhinal cortex and hippocampus, and from there, the pathology spreads to the surrounding areas (Braak and Braak, 1991). In this pathological process there is neuron death and as a consequence of this, intracellular components like tau in soluble form or forming NFT could be found at the extracellular space, becoming ghost tangles.
It has been proposed that extracellular tau could be toxic for neurons (Gomez-Ramos et al., 2006), playing a role in the spreading pathology of AD. The possibility exists that tau toxicity could be dependent on the interaction of this protein with cellular receptors able to promote an increase in intracellular calcium levels. It has been previously indicated that not only tau protein but others related with neurodegenerative disorders such as α-synuclein (Danzer et al., 2007), β-amyloid peptide or prion protein (Demuro et al., 2005) may induce a disruption of calcium homeostasis when added to cultured neuronal cells. However, the mechanisms for that disruption of calcium homeostasis, differ with the type of assayed protein (Adamczyk and Strosznajder, 2006, Danzer et al., 2007, Gomez-Ramos et al., 2006). In the case of tau protein it has been suggested that changes in the level of intracellular calcium could be mediated by the interaction of tau with muscarinic receptors, that induce calcium release from intracellular stores (Gomez-Ramos et al., 2006).
There are five cholinergic muscarinic receptor subtypes (M1 to M5), all of them being expressed in the Central Nervous System (CNS), but at different levels and in different locations, for example, M1 and M3 appear to be the most abundant muscarinic receptors expressed in hippocampus and entorhinal cortex in adult mouse, whereas M5 is poorly expressed (data from Allen Brain Atlas; http://www.brain-map.org/). The M1–M5 receptors can be subdivided into two major functional classes according to their G-protein coupling preference. The M1, M3 and M5 receptors, selectively couple to G-proteins of the Gq/G11 family, whereas the M2 and M4 receptors preferentially activate Gi/Go-type G-proteins (Wess et al., 2007). Coupling through the first group (M1, M3 and M5), but not through the second group, results in an increase in intracellular calcium (Lanzafame et al., 2003).
In this work, we tried to identify the muscarinic receptor or receptors involved in the response of neuronal cells to extracellular tau. Using pharmacological tools and transfected cells with cDNA of muscarinic receptors, we have determined that M1 and M3 muscarinic receptors are involved in this cellular response to tau, whereas M2 did not play any role in tau-induced increase in intracellular calcium. In addition, we have also found that tau induced a sustained intracellular calcium increase in primary cultures of hippocampal and cortical neurons.
Section snippets
Exposure to tau deregulates calcium homeostasis in SH-SY5Y neuroblastoma cells, but not in the presence of atropine
The SH-SY5Y human neuroblastoma cell line is a well characterized model to study muscarinic cholinergic function (Lambert and Nahorski, 1990, Murphy et al., 1991) and we decided to use this cellular model to establish the effect that extracellular tau induces on cholinergic neurotransmission system.
In order to check the functional status of these cells, FURA-2 AM loaded SH-SY5Y cells were exposed to 50 μM ACh (Fig. 1A a). The calcium increase induced by ACh was totally abolished when SH-SY5Y
Discussion
Thirty years ago, it was described that Alzheimer disease was associated with a severe loss of cholinergic markers in the brain (Bowen et al., 1976, Davies and Maloney, 1976). This observation raised the hypothesis suggesting that cholinergic loss could be related to the severity of the dementia (Perry et al., 1981) and settled the basis for the cholinergic deficit hypothesis in Alzheimer disease (Coyle et al., 1983). However, studies of other pathological features of the disease, such as the
Materials
The muscarinic receptor antagonists: atropine, Pirenzepine, Gallamine and 4-DAMP were purchased from Sigma (St. Louis, MO, USA). Monoclonal mouse anti-HA antibody (clone 12CA5) was obtained from the “Optic and Confocal Microscopy Service” (S.M.O.C., Centro de Biología Molecular “Severo Ochoa”, Madrid, Spain). Monoclonal mouse anti-β-actin antibody was obtained from Sigma (St. Louis, MO, USA). Polyclonal rabbit anti-muscarinic acetylcholine receptor M1 was purchased from Santa Cruz Biotechnology
Acknowledgments
This work was supported by grants from the Spanish Plan Nacional, from the Spanish Ministry of Health, Neuropharma, Fundación M. Botín, Comunidad de Madrid and an institutional grant to CBMSO from the “Fundación R. Areces”. We thank Drs J. Lucas and F. Hernandez for the critical reading of the manuscript.
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