α-Synuclein enhances secretion and toxicity of amyloid beta peptides in PC12 cells

Abstract

α-Synuclein is the fundamental component of Lewy bodies which occur in the brain of 60% of sporadic and familial Alzheimer’s disease patients. Moreover, a proteolytic fragment of α-synuclein, the so-called non-amyloid component of Alzheimer’s disease amyloid, was found to be an integral part of Alzheimer’s dementia related plaques. However, the role of α-synuclein in pathomechanism of Alzheimer’s disease remains elusive. In particular, the relationship between α-synuclein and amyloid beta is unknown. In the present study we showed the involvement of α-synuclein in amyloid beta secretion and in the mechanism of amyloid beta evoked mitochondria dysfunction and cell death. Rat pheochromocytoma PC12 cells transfected with amyloid beta precursor protein bearing Swedish double mutation (APPsw) and control PC12 cells transfected with empty vector were used in this study. α-Synuclein (10 μM) was found to increase by twofold amyloid beta secretion from control and APPsw PC12 cells. Moreover, α-synuclein decreased the viability of PC12 cells by about 50% and potentiated amyloid beta toxicity leading to mitochondrial dysfunction and caspase-dependent programmed cell death. Inhibitor of caspase-3 (Z-DEVD-FMK, 100 μM), and a mitochondrial permeability transition pore blocker, cyclosporine A (2 μM) protected PC12 cells against α-synuclein or amyloid beta evoked cell death. In contrast Z-DEVD-FMK and cyclosporine A were ineffective in APPsw cells containing elevated amount of amyloid beta treated with α-synuclein. It was found that the inhibition of neuronal and inducible nitric oxide synthase reversed the toxic effect of α-synuclein in control but not in APPsw cells. Our results indicate that α-synuclein enhances the release and toxicity of amyloid beta leading to nitric oxide mediated irreversible mitochondria dysfunction and caspase-dependent programmed cell death.

Introduction

The pathogenesis of Alzheimer’s disease (AD), a progressive neurodegenerative disorder of the elderly and the most common form of dementia, is till now not fully elucidated and there is no efficient method for its treatment. The most frequent sporadic forms of AD are associated with an oligomerisation and abnormal accumulation of amyloid beta (Aβ) peptides into senile plaques (Selkoe, 2000, Marks and Berg, 2008, Meyer-Luehmann et al., 2008, Ye et al., 2008). Aβ is formed by proteolytic processing of amyloid precursor protein (APP) by β- and γ-secretase (Marks and Berg, 2008). These enzymes generate the Aβ peptide and carboxyl terminal fragments (CTF) of APP, which have been implicated in the pathogenesis of AD (Suh and Checler, 2002, Walsh et al., 2005, Cacquevel et al., 2008). Extracellular Aβ peptides are highly cytotoxic to neuronal cells by activating a variety of cell signaling pathways, such as mitogen-activated protein (MAP) kinase cascades (Dineley et al., 2001), by inducing the dephosphorylation/inactivation of Akt (Nassif et al., 2007) and by the activation of NMDA receptors and disruption in calcium homeostasis (Domingues et al., 2007). Besides the presence of amyloid β-peptides, the major component of senile plaques corresponds to a component now referred to as NAC (non-amyloid component of Alzheimer’s disease plaques; Tanaka et al., 2002). The molecular cloning of NAC indicated that it derives from the proteolytic cleavage of a precursor protein (NACP) of 19 kDa now known as α-synuclein (ASN; Ueda et al., 1993). ASN is a small protein recognized in various cell types of the central nervous system, and is especially abundant in presynaptic terminals of neocortex, hippocampus, dentate gyrus, olfactory bulb, thalamus, cerebellum and striatum (Sidhu et al., 2004, Adamczyk et al., 2005).

It is postulated that in physiological conditions, in nanomolar concentration, ASN plays an important role in synaptic plasticity, regulation of vesicle transport, dopaminergic neurotransmission and also acts as a chaperon protein (Sidhu et al., 2004). It can also protect neurons against oxidative stress and inhibit apoptosis. Its protective effect is suggested to be mediated by inactivation of JNK signaling pathway (Hashimoto et al., 2002), by activation of PI3/Akt kinase pathway (Seo et al., 2002) and or by inhibition of p53 mediated apoptosis (Alves Da Costa et al., 2002). However, under some pathogenic conditions, oxidative and genotoxic stress, which lead to overexpression and mutations, ASN changes its native conformation and tends to form oligomers, which are highly toxic and induce cell death (Amer et al., 2006). The progressive aggregation of this protein leads to the formation of insoluble inclusions called Lewy bodies that can mechanically damage the cell structure. Nowadays it is obvious that over 60% of AD cases is accompanied by Lewy body formation (Mikolaenko et al., 2005, Jellinger, 2004). Lewy bodies and associated Lewy neurites are also pathological hallmarks of other neurodegenerative diseases such as Parkinson’s disease (PD) and dementia with Lewy bodies (Braak et al., 2003, Pletnikova et al., 2005). Abnormal ASN aggregates were also found in other neuronal and glial inclusions, such as Lewy body-like glial cytoplasmic inclusions of multiple system atrophy (MSA; Parkkinen et al., 2007, Ishizawa et al., 2008).

While the role of ASN in the pathogenesis of AD remains unclear, indirect evidence suggests that this protein may interact with Aβ and increase its toxicity. Full-length ASN is present in blood and CSF of normal subjects suggesting that this protein is released by neurons in the extracellular space as a part of its normal cellular processing (El-Agnaf et al., 2006). A recent study showed that membrane-bound ASN interacts with extracellular Aβ and that the NAC fragment is liberated from ASN (Mandal et al., 2006). Other data suggest that ASN liberated from presynaptic terminals during the process of neurodegeneration is cleaved to NAC by extracellular metaloproteinases (Sung et al., 2005).

The aim of this study was to investigate the role of extracellular ASN in Aβ secretion and in molecular mechanisms of its toxicity. Moreover, the protective effect of selected specific inhibitors against cytotoxicity of ASN and Aβ was evaluated.