Extracellular tau promotes intracellular calcium increase through M1 and M3 muscarinic receptors in neuronal cells

Abstract

Extracellular tau promotes an increase in the level of intracellular calcium in cultured neuronal cells. We have found that such increase is impaired in the presence of antagonists of muscarinic receptors. In order to identify the nature of those receptors, we have tested the effect of different specific muscarinic receptor antagonists on tau promoted calcium increase. Our results indicate that the increase does not take place in the presence of antagonists of muscarinic (mainly M1 and M3) receptors. A similar increase in intracellular calcium was found in non-neuronal cells transfected with cDNA of M1 and M3 muscarinic receptors when tau was added. These results suggest that observed effect of tau protein on neuronal (neuroblastoma and primary cultures of hippocampal and cortical neurons) cells is through M1 and M3 muscarinic receptors. Therefore blocking M1 and for M3 receptors, by using specific receptor antagonists, can prevent that tau toxic effect that could take place in tauopathies.

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 G_q/G₁₁ family, whereas the M2 and M4 receptors preferentially activate G_i/G_o-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.