The beneficial effect of a prolyl oligopeptidase inhibitor, KYP-2047, on alpha-synuclein clearance and autophagy in A30P transgenic mouse

Highlights

• PREP inhibition reduced the amount of aSyn oligomers on aSyn transgenic mice.

• PREP inhibition increased autophagy in vitro and in vivo.

• PREP inhibition enhances autophagy by increasing beclin 1 protein levels.

• Dopamine levels increased in the mouse striatum after chronic PREP inhibition.

Abstract

The misfolding and aggregation of α-synuclein (aSyn) eventually lead to an accumulation of toxic forms that disturb normal neuronal function and result in cell death. aSyn rich inclusions are seen in Parkinson's disease, dementia with Lewy bodies and other synucleinopathies. Prolyl oligopeptidase (PREP) can accelerate the aggregation process of aSyn and the inhibition of PREP leads to a decreased amount of aggregated aSyn in cell models and in aSyn transgenic mice. In this study, we investigated the effect of 5- and 28-day PREP inhibitor (KYP-2047) treatments on a mouse strain carrying a point mutation in the aSyn coding gene. Following PREP inhibition, we found a decrease in high molecular-weight oligomeric aSyn and a concomitant increase in the amount of the autophagosome marker, LC3BII, suggesting enhanced macroautophagy (autophagy) and aSyn clearance by KYP-2047. Moreover, 28-day treatment with KYP-2047 caused significant increases in striatal dopamine levels. In cell culture, overexpression of PREP reduced the autophagy. Furthermore, the inhibition of PREP normalized the changes on autophagy markers (LC3BII and p62) caused by an autophagy inhibition or aSyn overexpression, and induced the expression of beclin 1, a positive regulator of autophagy. Taken together, our results suggest that PREP inhibition accelerates the clearance of protein aggregates via increased autophagy and thus normalizes the cell functions in vivo and in vitro. Therefore, PREP inhibition may have future potential in the treatment of synucleinopathies.

Introduction

Synucleinopathies including Lewy body dementia, multiple system atrophy and the most common neurodegenerative movement disorder, Parkinson's disease (PD), all share the hallmark pathologic feature of α-synuclein (aSyn) protein accumulation in the brain (Spillantini and Goedert, 2000). Lewy bodies and Lewy neurites are neuropathological hallmarks of PD and Lewy body dementia and also multiple system atrophy is characterized by filamentous inclusions of insoluble form of aSyn (Baba et al., 1998, Spillantini et al., 1998, Ueda et al., 1993). aSyn consists of 140 amino acids (Jakes et al., 1994) and in its native forms it is a soluble, cytosolic protein that mainly localizes to presynaptic terminals (Iwai et al., 1995). aSyn is suggested to play a role in synaptic transmission, axonal transport and the regulation of dopamine (DA) homeostasis (Surguchov, 2008). This protein interacts with and regulates the function and distribution of the dopamine transporter (DAT), which is responsible for DA recycling (Bellucci et al., 2011, Wersinger and Sidhu, 2003). aSyn also localizes to synaptic vesicles where it can control vesicle recycling by modulating phospholipase D2 function (Jenco et al., 1998). Therefore, aSyn is purported to affect DA packaging and vesicle pools in DAergic nerve terminals (Murphy et al., 2000).

Genetic studies have revealed several forms of early-onset autosomal dominant familial Parkinson's disease that are associated with point mutations in the aSyn coding gene, namely A30P, A53T and E46K (Conway et al., 2000, Kruger et al., 1998, Polymeropoulos et al., 1997, Zarranz et al., 2004). Furthermore, duplications and triplications in the aSyn wildtype gene also increase the incidence of this disease (Chartier-Harlin et al., 2004, Singleton et al., 2003).

Wildtype aSyn (Wt aSyn) and its mutated forms are initially natively unfolded proteins (Conway et al., 1998). In the aSyn aggregation process, protein misfolding leads to the formation of insoluble fibrils and higher order aggregates. Many factors enhance aSyn nucleation including mutations, oxidative stress, low pH, metal ions and impairments in protein trafficking and processing (Cuervo et al., 2004, Lee et al., 2013, Uversky, 2007, Uversky et al., 2001). Interestingly, the aggregation of Wt aSyn is accelerated by the serine protease enzyme, prolyl oligopeptidase (PREP; POP; PO; EC 3.4.21.26), even under cell-free conditions, which implies a direct interaction between these proteins (Brandt et al., 2008, Lambeir, 2011). Furthermore, this action of PREP can be blocked by PREP inhibitors or by mutation of the active site (Brandt et al., 2008).

PREP is widely distributed throughout the brain and other tissues and it normally functions to hydrolyze peptides smaller than 30 amino acids (Mantle et al., 1996). PREP inhibition has been proposed as a therapeutic approach to counteract low levels of some neuropeptides in an effort to enhance memory deficits (Yoshimoto et al., 1987). In fact, PREP inhibitors do mildly improve memory in some animal models of cognitive decline (Jalkanen et al., 2007, Kato et al., 1997, Männistö et al., 2007, Marighetto et al., 2000, Miyazaki et al., 1998, Morain et al., 2002, Peltonen et al., 2010, Shinoda et al., 1999, Toide et al., 1997). PREP's role in protein aggregation, a common denominator in neurodegeneration, remains to be fully explored.

As mentioned above, PREP has been shown in vitro and in vivo to play a role in aSyn protein aggregation (Brandt et al., 2008, Lambeir, 2011, Myöhänen et al., 2012). We have also shown that the PREP inhibitor, KYP-2047, effectively prevents the formation of aSyn aggregates in aSyn overexpressing cell lines and increases the clearance of aSyn in two mouse strains carrying the pathogenic human A30P aSyn gene (Myöhänen et al., 2012). Our studies suggest that PREP may enhance aSyn aggregation by influencing the nucleation rate and/or possibly by slowing down the degradation of misfolded proteins.

Several mouse models that overexpress Wt aSyn or mutant aSyn (A30P and A53T) have been generated (Fleming and Chesselet, 2006, Kahle et al., 2000, Rockenstein et al., 2002). In this study, we further characterized the role of PREP on the aSyn accumulation process and the effect of PREP inhibition on dopaminergic system in vivo using an A30P point-mutated transgenic mouse strain (Plaas et al., 2008). Moreover, since we have previously shown that a PREP inhibitor, KYP-2047, decreases the amount of aSyn in the brain of aged transgenic mice, here we examined the mechanism of this beneficial effect of aSyn clearance by characterizing relevant markers of proteasomal and autophagy–lysosomal protein degradation pathways in vitro and in vivo.