Parkin Reverses Intracellular β-Amyloid Accumulation and Its Negative Effects on Proteasome Function

Parkin is a 465-amino-acid protein containing an N-terminal ubiquitin-like (Ubl) domain linked to a C-terminal RING box (Shimura et al., 2000). Parkin functions as an E3 ubiquitin-protein ligase (Imai et al., 2000; Shimura et al., 2000; Zhang et al., 2000), facilitating the proteasomal degradation of misfolded proteins. Several Parkin gene mutations have been linked to autosomal-recessive Parkinsonism with juvenile onset (Kitada et al., 1998; Lucking et al., 2000). In cell culture systems, Parkin fusion proteins have been shown to interact with several proteins, including the α-synuclein-binding protein synphilin-1 (Chung et al., 2001), actin filaments (Huynh et al., 2000), and α/β tubulin (Ren et al., 2003). Parkin has also been found to be up-regulated during the integrated cellular response to misfolded protein-induced ER stress (Imai et al., 2001). Specific targets of Parkin activity having intrinsic toxic and aggregative properties include Pael-R, the Parkin-associated endothelin-like receptor (Imai et al., 2001), and possibly an O-glycosylated form of α-synuclein (Shimura et al., 2001). Thus, Parkin has been shown to suppress the toxicity of PAEL-R (Imai et al., 2001), mutated α-synuclein A30P (Petrucelli et al., 2002; Lo Bianco et al., 2004), and a poly(Q)-expanded mutant of ataxin-3 (Tsai et al., 2003). Deletions in the Parkin gene result in the accumulation of nonubiquitinated forms of α-synuclein and Pael-R in the brain (Imai et al., 2001; Shimura et al., 2001). The accumulation of neuronal β-amyloid (Aβ) is increasingly recognized as a critical factor in Alzheimer's disease (AD) and related pathologies (Hartmann, 1999; Wilson et al., 1999; Gouras et al., 2005). Soluble fractions of AD brain Aβ are better correlated with disease severity than the larger insoluble pool (McLean et al., 1999), pointing to a possible contribution from the cellular compartment. There is also evidence to suggest that extracellular, parenchymal Aβ plaques in the brain are derived from degenerating Aβ42-laden neuronal cell bodies (Gouras et al., 2000; D'Andrea et al., 2001). Expanding data have shown the importance of Aβ degradation or clearance mechanisms. Among these, the endopeptidases insulin-degrading enzyme (IDE) and neprilysin (NEP) have received the most attention (Selkoe, 2001). IDE engages extracellular secreted monomeric and plaque Aβ and, through a cytosolic pool of enzyme, the amyloid intracellular domain (AICD; Qiu et al., 1998; Edbauer et al., 2002; Farris et al., 2003; Leissring et al., 2003). NEP is a major extracellular Aβ-degrading enzyme; it decreases plaque formation and is active against mono- and oligomeric Aβ (Kanemitsu et al., 2003; Marr et al., 2003). Less is known about the clearance of intracellularly generated Aβ. Both IDE and a proteasome-dependent pathway have been shown to degrade endoplasmic reticulum (ER)-localized Aβ in transfected HeLa cells. However, only 30% of expressed Aβ was sensitive to the inhibitor MG132, suggesting limited proteasome involvement in that system (Schmitz et al., 2004). The mechanism behind the proteasome contribution was not further explored. Nevertheless, synthetic Aβ and purified 20S proteasome preparations interact to form complexes (Gregori et al., 1997). Studies of cells and tissues derived from Parkin knockout mice and flies have indicated an increased sensitivity to cellular stressors of various types (Palacino et al., 2004; Pesah et al., 2004). Our earlier study with Parkin knockout skeletal muscle (Rosen et al., 2006) found enhanced accumulation of fragments of the endogenous β-amyloid precursor protein, including intracellular β-amyloid, and a heightened sensitivity to oxidative stress. Here we examine whether additional manipulations of Parkin expression can influence the accumulation and toxic effects of intracellular β-amyloid in neuronal cells. From its action as a ubiquitin ligase, but likely having other functions, we predicted that Parkin confers cytoprotection against Aβ42 when over-expressed and that this property would be proteasome dependent. Indeed, we found that proteasome inhibition interferes with Parkin's ability to reverse intracellular β-amyloid-induced toxicity and accumulation. These findings were extended to uncover Parkin and β-amyloid associations in the AD brain.