Extracellular ATP induces intracellular alpha-synuclein accumulation via P2X1 receptor-mediated lysosomal dysfunction

Abstract

The pathologic hallmark of Parkinson's disease (PD) is the accumulation of alpha-synuclein (αsyn) in susceptible neurons in the form of Lewy bodies and Lewy neurites. The etiology of PD remains unclear. Because brain injury has been suggested to facilitate αsyn aggregation, we investigated whether cellular breakdown products from damaged cells can act on neighboring healthy cells and cause intracellular αsyn accumulation and/or aggregation. Using 2 neuronal cell models, we found that extracellular adenosine triphosphate (ATP) induced a significant increase in intracellular αsyn levels between 24 and 48 hours after treatment. Further investigation revealed that the observed αsyn accumulation is a result of lysosome dysfunction caused by extracellular ATP-induced elevation of lysosomal pH. Interestingly, P2X1 receptor appears to mediate the cells' response to extracellular ATP. Although Ca²⁺ influx via P2X1 receptor is necessary for αsyn accumulation, Ca²⁺ influx per se is not sufficient for increased αsyn accumulation. These findings provide new insight into our knowledge of the role of P2X receptors in PD pathogenesis and may be helpful in identifying new therapeutic targets for PD.

Introduction

Parkinson's disease (PD) is the second most common neurodegenerative disorder, pathologically characterized by the formation of filamentous alpha-synuclein (αsyn) neuronal inclusions and the loss of dopaminergic neurons in substantia nigra and dopaminergic terminals in the striatum (Dauer and Przedborski, 2003). The etiology of PD remains unclear and no disease-modifying treatment currently exists. Misfolding and aggregation of αsyn plays a central role in the pathogenesis of PD (Jellinger, 2012). Among the variety of aggregate species present, low-molecular weight oligomers, ranging from 2 to 30 mers are thought to be the primary neurotoxic species responsible for the impairment of neuronal function in PD (Planchard et al., 2014). Traumatic brain injury (TBI) has been considered a causative factor for PD based on case-control studies (Lee et al., 2012), and brain injury has been suggested to facilitate αsyn aggregation and PD development (Bramlett and Dietrich, 2003, Jafari et al., 2013). This notion is further supported in an experimental study showing that αsyn expression and aggregation increased transiently following TBI in aged mouse brain (Uryu et al., 2003). Focal cerebral ischemia was also found to induce both the aggregation of wild type and transgenic human mutant (A30P) αsyn in mouse brain (Unal-Cevik et al., 2011), and a recent study further linked ischemia to PD by showing early development of brain injury caused neuronal loss in substantia nigra in a mouse focal cerebral ischemia model (Rodriguez-Grande et al., 2013). Despite these intriguing data, the exact mechanism(s) underlying brain injury-induced aggregation of αsyn remain elusive. Considering that the aforementioned αsyn aggregation only appears approximate to the injured area and αsyn aggregates form in a relatively short time (from 3 days to 1 week) after brain injury (Uryu et al., 2003), we hypothesized that cellular breakdown products from damaged cells may influence neighboring healthy neuronal cells leading to intracellular αsyn accumulation and/or aggregation. Herein, we tested whether extracellular adenosine triphosphate (ATP) might be a key factor, inducing αsyn accumulation in the previously scenario because ATP acts as a potent stimulus for inflammation and can be released from damaged cells. Extracellular ATP plays an important signaling role by activating broadly distributed cell surface purinergic receptors (P2 receptors) (Seminario-Vidal et al., 2009). P2 receptors are divided into 2 subclasses, ATP-selective cation channels P2X and G-protein-coupled P2Y families. Functional P2X receptors are expressed on both neurons and glial cells (Volonte et al., 2003). So far, 7 members of P2X subfamily have been identified, P2X1-7 (Falzoni et al., 2013). In the present study, we confirm that extracellular ATP plays an important role in intracellular αsyn aggregation in 2 different neuronal cell models and further demonstrate that lysosomal dysfunction is linked to extracellular ATP-induced αsyn accumulation in neuronal cells. Importantly, we found that P2X1 receptors are key to mediating the observed effects.