A prolyl oligopeptidase inhibitor, KYP‐2047, reduces α‐synuclein protein levels and aggregates in cellular and animal models of Parkinson's disease

Abstract

BACKGROUND AND PURPOSE The aggregation of α‐synuclein is connected to the pathology of Parkinson's disease and prolyl oligopeptidase (PREP) accelerates the aggregation of α‐synuclein in vitro. The aim of this study was to investigate the effects of a PREP inhibitor, KYP‐2047, on α‐synuclein aggregation in cell lines overexpressing wild‐type or A30P/A53T mutant human α‐syn and in the brains of two A30P α‐synuclein transgenic mouse strains. EXPERIMENTAL APPROACH Cells were exposed to oxidative stress and then incubated with the PREP inhibitor during or after the stress. Wild‐type or transgenic mice were treated for 5 days with KYP‐2047 (2 × 3 mg·kg−1 a day). Besides immunohistochemistry and thioflavin S staining, soluble and insoluble α‐synuclein protein levels were measured by Western blot. α‐synuclein mRNA levels were quantified by PCR. The colocalization of PREP and α‐synuclein,and the effect of KYP‐2047 on cell viability were also investigated. KEY RESULTS In cell lines, oxidative stress induced a robust aggregation of α‐synuclein,and low concentrations of KYP‐2047 significantly reduced the number of cells with α‐synuclein inclusions while abolishing the colocalization of α‐synuclein and PREP. KYP‐2047 significantly reduced the amount of aggregated α‐synuclein,and it had beneficial effects on cell viability. In the transgenic mice, a 5‐day treatment with the PREP inhibitor reduced the amount of α‐synuclein immunoreactivity and soluble α‐synuclein protein in the brain. CONCLUSIONS AND IMPLICATIONS The results suggest that the PREP may play a role in brain accumulation and aggregation of α‐synuclein, while KYP‐2047 seems to effectively prevent these processes.

Prolyl Oligopeptidase Enhances α-Synuclein Dimerization via Direct Protein-Protein Interaction

“…It was previously shown that PREP accelerates aSyn aggregation and that a PREP inhibitor blocks this in vitro and promotes clearance of aSyn aggregates in aSyn-overexpressing cells and in vivo models (14,22). However, although PREP increased the aggregation of aSyn in a cell-free model, the interaction between PREP and aSyn has not been documented earlier.…”

“…The PREP inhibitor, KYP-2047 (4-phenylbutanoyl-l-prolyl-2(S)-cyanopyrrolidine), was synthesized in the School of Pharmacy, University of Eastern Finland, as previously described (25). KYP-2047 was chosen as a reference compound because the biochemical and pharmacological data indicate that it is potent and selective, enters cells in culture, and crosses the blood-brain barrier effectively in rodents (22,24,26,27).…”

“…PREP Activity Assay-PREP activity assay was used to confirm the enzymatic activity of various PREP constructs, and it was performed as described in Myöhänen et al (22,30). Briefly, the cells transfected with various PREP constructs were homogenized with lysis buffer (50 mM KH 2 PO 4 , 1.5 mM MgCl 2 , 10 mM NaCl, 1 mM EDTA, pH 7.4), and the homogenates were centrifuged at 16,000 ϫ g for 10 min at ϩ4°C.…”

“…in N2A Cells-PREP inhibition was previously shown to reduce chronic oxidative stress-induced accumulation of aSyn aggregates in SH-SY5Y neuroblastoma cells (22). To study the effects of PREP in the earliest stages of the aSyn aggregation process, we first developed an assay for studying aSyn oligomer formation in live cells.…”

“…In addition, PREP colocalizes with aSyn in the substantia nigra in human PD brains (21). PREP inhibition effectively prevents aSyn aggregation in a cell-free system, in aSyn-overexpressing cells, and in transgenic mouse models (14,22). Recently, we have identified PREP as a negative modulator of autophagosome formation, and treatment with PREP inhibitor, KYP-2047, induced macroautophagy and autophagosome formation via a beclin 1-dependent pathway (23).…”