Rapid communicationIn vivo P2X7 inhibition reduces amyloid plaques in Alzheimer's disease through GSK3β and secretases
Introduction
Two main neuropathological hallmarks have been described in Alzheimer's disease (AD): neurofibrillary tangles and amyloid plaques. Amyloid plaques are primarily composed of β-amyloid (Aβ) peptide, derived from sequential proteolysis of amyloid precursor protein (APP) by β- and γ-secretase (Selkoe, 2001). It has been postulated that the generation of Aβ peptides represents the initial event triggering neurological dysfunction (Hardy and Selkoe, 2002, Price et al., 1998). An alternative form of nonamyloidogenic APP processing by α- and γ-secretases has also been well described (Selkoe, 2001). While both APP processing pathways occur in the central nervous system (CNS) (Hardy and Selkoe, 2002), it is postulated that APP is cleaved preferentially via the nonamyloidogenic pathway in the healthy brain (Tyler et al., 2002). Early-onset familiar AD (FAD) studies indicate that increased levels of Aβ are linked to missense mutations or alternative splicing of APP and presenilin-1 and −2 (PS1 and PS2) genes (Ling et al., 2003, Price and Sisodia, 1998). Why APP processing shifts to the amyloidogenic pathway in sporadic AD however, remains unknown (Stockley and O'Neill, 2008).
It has been reported that Aβ peptides can interact with insulin or frizzled receptors (Magdesian et al., 2008, Townsend et al., 2007) and this would increase glycogen synthase kinase 3 (GSK-3) activity (Cross et al., 1995), and in turn enhance Aβ production (Phiel et al., 2003, Rockenstein et al., 2007, Su et al., 2004) possibly through phosphorylation of the intracellular domain of APP (Aplin et al., 1996). In this manner, antisense-GSK-3β oligonucleotides can reduce the neurotoxicity associated with Aβ production (Takashima et al., 1993). Furthermore, lithium administration in transgenic APP mice decreases Aβ deposition, pointing to GSK-3 as a potential therapeutic target in AD (Phiel et al., 2003, Rockenstein et al., 2007, Su et al., 2004). However, the potential tumorigenic effect (Polakis, 2000) and toxicity (Gómez-Sintes et al., 2007, Kaidanovich-Beilin et al., 2009) of sustained decrease GSK-3 questions the feasibility of a therapy based on GSK-3 inhibitors (Hu et al., 2009).
Adenosine triphosphate (ATP) acts as a fast neurotransmitter through activation of ionotropic P2X receptors (Evans et al., 1992). These receptors are widely distributed in the central nervous system, and regulate calcium homeostasis, neurotransmitter release, and vital intracellular pathways (Burnstock, 2007). Aged APP transgenic mice exhibit aberrant regulation of calcium homeostasis (Kuchibhotla et al., 2008), alterations in neurotransmitter release (Wang et al., 2000), and upregulation of P2X7R in microglial cells (Parvathenani et al., 2003). Moreover, it has been reported that neuroinflammatory markers induced by exogenous administration of Aβ are partially inhibited by the P2X7R antagonist Brilliant Blue-G (BBG) (Ryu and McLarnon, 2008). We have previously described the modulation of GSK-3 activity in the hippocampal neurons by P2X7R (Díaz-Hernandez et al., 2008). Recent studies have also reported that P2X7R activity affects APP processing (Delarasse et al., 2011, Hernandez et al., 2010). In the current study, we investigated the potential of P2X7R as a therapeutic target in AD using a well-established transgenic mouse model of FAD that expresses mutant\human APP protein.
Section snippets
Chemicals and antibodies
ATP, 2′,3′-O-(4-benzoyl)-benzoyl ATP (BzATP), BBG, bovine serum albumin (BSA), Sudan Black, and Thioflavin-T were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fura 2-AM was from Molecular Probes (Leiden, the Netherlands). A438079 was obtained from Tocris BioScience (Bristol, UK). TAPI-1 was purchased from Calbiochem (Darmstadt, Germany). The commercial antibodies used in this study were P2X7 (intracellular epitope) receptor (P2X7R) from Alomone Labs (Jerusalem, Israel). GSK-3 α/β antibody
The P2X7R modifies GSK-3 phosphorylation state and α-secretase activity in HEK and N2a cells
In N2a cells, which possess native P2X7R (Gómez-Villafuertes et al., 2009), calcium responses elicited by 100 μM BzATP were blocked by both P2X7R antagonists, BBG (1 μM), and A438079 (1 μM). BBG acts as a slowly reversible antagonist, resulting in inhibition of the BzATP response for 25–30 minutes after washout (Fig. 1A). By contrast, the antagonistic effect of A438079 was rapidly reversed after washout (Fig. 1B). Blockade of P2X7R with either BBG or A438079 induced a significant increase in
Discussion
The present study describes P2X7R-mediated modulation of α-secretase activity, which in turn is implicated in nonamyloidogenic APP processing. Initial pharmacological approaches in N2a cells demonstrated that α-secretase activity was significantly increased following direct or indirect (via P2X7R antagonism) inhibition of GSK-3. P2X7R-mediated regulation of α-secretase activity via GSK-3 was corroborated in HEK cells transfected with this receptor.
J20 mice provide a valuable model for the study
Disclosure statement
There are no potential conflicts of interest in this study. The experimental protocol was approved by the Ethics Committee for Animal Research in Universidad Complutense de Madrid.
Acknowledgements
This work was supported by research grants from MICINN (BFU2008-02699, BFU2005-06034, and SAF2009-12249-CO2-02), CAM (S-SAL-0253-2006), the Spanish Ion Channel Initiative (SICI) (CSD2008-00005), UCM-Santander Central Hispano Bank (911585-670), and Fundación Marcelino Botín. R.G.-V. was supported by SICI. J.I.D.-H. was supported by Juan de la Cierva Program.
References (50)
- et al.
Age-dependent and -independent behavioral deficits in Tg2576 mice
Behav. Brain Res
(2008) - et al.
The purinergic receptor P2X7 triggers alpha-secretase-dependent processing of the amyloid precursor protein
J. Biol. Chem
(2011) - et al.
GSK3 inhibitors show benefits in an Alzheimer's disease (AD) model of neurodegeneration but adverse effects in control animals
Neurobiol. Dis
(2009) - et al.
Abeta plaques lead to aberrant regulation of calcium homeostasis in vivo resulting in structural and functional disruption of neuronal networks
Neuron
(2008) - et al.
Activation of Src/kinase/phospholipase C/mitogen-activated protein kinase and induction of neurite expression by ATP, independent of nerve growth factor
Neuroscience
(2006) - et al.
Amyloid precursor protein (APP) and the biology of proteolytic processing: relevance to Alzheimer's disease
Int. J. Biochem. Cell Biol
(2003) - et al.
Amyloid-beta binds to the extracellular cysteine-rich domain of Frizzled and inhibits Wnt/beta-catenin signaling
J. Biol. Chem
(2008) - et al.
P2X7 mediates superoxide production in primary microglia and is up-regulated in a transgenic mouse model of Alzheimer's disease
J. Biol. Chem
(2003) - et al.
Association of microglia with amyloid plaques in brains of APP23 transgenic mice
Am. J. Pathol
(1999) - et al.
Soluble Abeta inhibits specific signal transduction cascades common to the insulin receptor pathway
J. Biol. Chem
(2007)