ATP leakage induces P2XR activation and contributes to acute synaptic excitotoxicity induced by soluble oligomers of β-amyloid peptide in hippocampal neurons

Highlights

• Aβ increases extracellular ATP causing activation of P2X receptors.

• ATP leakage contributes to acute synaptic excitotoxicity.

• Aβ induces excitotoxicity by binding preferentially to excitatory neuron.

• The effects were blocked by a non-specific P2XR antagonist.

• The neurotoxicity of Aβ is mediated by P2XR activation.

Abstract

Recent studies suggest that the toxic effects of Aβ can be attributed to its capability to insert in membranes and form pore-like structures, which are permeable to cations and molecules such as ATP. Our working hypothesis is that Aβ increases extracellular ATP causing activation of P2X receptors and potentiating excitatory synaptic activity. We found that soluble oligomers of β-amyloid peptide increased cytosolic Ca²⁺ 4-fold above control (415 ± 28% of control). Also, ATP leakage (157 ± 10% of control) was independent of extracellular Ca²⁺, suggesting that ATP traveled from the cytosol through an Aβ pore-mediated efflux and not from exocytotic mechanisms. The subsequent activation of P2XR by ATP can contribute to the cytosolic Ca²⁺ increase observed with Aβ. Additionally, we found that β-amyloid oligomers bind preferentially to excitatory neurons inducing an increase in excitatory synaptic current frequency (248.1 ± 32.7%) that was blocked by the use of P2XR antagonists such as PPADS (Aβ + PPADS: 110.9 ± 18.35%) or Apyrase plus DPCPX (Aβ + inhibitors: 98.97 ± 17.4%). Taken together, we suggest that Aβ induces excitotoxicity by binding preferentially to excitatory neuron membranes forming a non-selective pore and by increasing intracellular calcium by itself and through P2XR activation by extracellular ATP leading to an augmention in mEPSC activity. All these effects were blocked with a non-specific P2XR antagonist, indicating that part of the neurotoxicity of Aβ is mediated by P2XR activation and facilitation of excitatory neurotransmitter release. These findings suggest that P2XR can be considered as a potential new target for the development of drugs or pharmacological tools to treat Alzheimer's disease.

This article is part of the Special Issue entitled ‘Synaptopathy – from Biology to Therapy’.

Introduction

Alzheimer's disease (AD) is a neurodegenerative pathology (Castellani et al., 2010) characterized by a progressive loss of synapses and neurons (Dalvi, 2012) leading to memory impairment and cognitive dysfunction (Hardy and Selkoe, 2002). It is highly relevant to our society to study and obtain a better understanding of the processes involved in the onset and progression of AD mainly due to the aging of the population and the costs associated to the care of elderly people (Thies and Bleiler, 2012, Thies and Bleiler, 2013).

The etiology of AD remains elusive, but it is currently accepted that small, soluble oligomers of β-Amyloid peptide (SOAβ) are involved in the onset of the disease (Lesné et al., 2006, Carlo, 2010, DaRocha-Souto et al., 2011). The exact mechanism of action of the oligomers is not currently well defined, but there is growing evidence suggesting that SOAβ is able to disrupt the membrane by its insertion and formation of a non-specific pore-like structure (Arispe et al., 1993, Parodi et al., 2010, Sepúlveda et al., 2010). The perforation of the membrane is associated to an increase in [Ca²⁺]_i (Arispe et al., 1996, Demuro et al., 2010) and more recently it has been proposed that the pore has a diameter sufficient to allow the passage of molecules of up to 900 Da of weight (Sepúlveda et al., 2010, Sepúlveda et al., 2013). Thus, this diameter is large enough for molecules to enter the neurons, but more interestingly, to permit the leakage of some key molecules from the neurons. Previous studies showed that SOAβ increased the extracellular level of glutamate (Brito-Moreira et al., 2011) and also ATP from cell cultures or slices (Kim et al., 2007, Fuentealba et al., 2011, Orellana et al., 2011).

The leakage of ATP could mediate activation of purinergic receptors, which are classified according to their ligand and mechanism of signal transduction into 3 families of receptors: P1 (or A) activated by adenosine (ADO) and coupled to G-protein (Burnstock, 2007), P2Y activated by ATP, ADP or UDP and also coupled to G-protein (Burnstock, 2008), and P2X receptors (P2XRs) which are gated by ATP and belong to the ligand gated ionic channels (LGIC) super family (North, 2002). From a sequential point of view, the activation of the receptors is as follows: first P2XRs are activated (Abbracchio et al., 2009) and then ectonucleotidases hydrolyze ATP to ADP allowing the activation of P2Y (Benarroch, 2010) and finally ADP is dephosphorylated to ADO and activation of P1 receptors (Burnstock, 2007). The signaling is ended by the conversion of ADO to inosine and its intake to the cell (Zebisch and Sträter, 2008).

We are interested in investigating the physiological and pathophysiological role of P2XRs on synaptic function, since they were described as neuromodulators (Bowser and Khakh, 2004, Choi et al., 2009, Khakh Baljit and North, 2012). These receptors can facilitate the release of other neurotransmitters due to their permeability to Ca²⁺ and their location on presynaptic terminals (Rubio and Soto, 2001, Rodrigues et al., 2005). In addition, we were interested in studying P2X receptors on neurodegenerative conditions like AD where their participation in AD is not clear, but the focus has been on neuroinflammation (Apolloni et al., 2009). In this context, the expression of P2X7, for example on microglia, has been described as necessary for its activation (Choi et al., 2007), and more interestingly is found to be up-regulated in the brain of AD patients and the hippocampus of rats injected with SOAβ (McLarnon et al., 2006). However, few studies are available on the involvement of neuronal purinergic receptors. For example, Varma published a study in 2009 (Varma et al., 2009) where they described that P2X4 can be processed by caspase-3 and this modified the properties of the receptor, delaying the closure of the channel and preventing its internalization. But other authors have published results where they suggest that P2XRs could be acting to prevent cell death mediated by SOAβ (Delarasse et al., 2011, Jung et al., 2012). Therefore, more studies are needed to clarify the role of P2XRs in AD.

Therefore, the aim of this study was to examine the role of purinergic transmission and that of P2XRs in a neuronal model of AD, induced by acute exposure to SOAβ, and the changes to its properties as a neuromodulator and facilitator of neurotransmitter release.