Inhibition of prolyl oligopeptidase increases the survival of alpha-synuclein overexpressing cells after rotenone exposure by reducing alpha-synuclein oligomers

Neuroscience Letters 583 (2014) 37–42

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Inhibition of prolyl oligopeptidase increases the survival of alpha-synuclein overexpressing cells after rotenone exposure by reducing alpha-synuclein oligomers Lana Dokleja 1 , Mirva J. Hannula 1 , Timo T. Myöhänen ∗ Division of Pharmacology and Toxicology, Faculty of Pharmacy, , Viikinkaari 5E, PO Box 56, FIN-00014 University of Helsinki, Finland

h i g h l i g h t s • • • •

Rotenone induced ␣-synuclein (aSyn) aggregation in [A53T]aSyn overexpressing cells. Prolyl oligopeptidase (PREP) inhibition reduced the levels of aSyn oligomers. PREP inhibition decreased ROS production by reducing aSyn oligomers. These effects of PREP inhibition led to increased cell survival.

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Article history: Received 14 May 2014 Received in revised form 3 September 2014 Accepted 9 September 2014 Available online 20 September 2014 Keywords: Protein misfolding Serine protease Parkinson’s disease Autophagy Reactive oxygen species

a b s t r a c t ␣-Synuclein (aSyn) aggregation, mitochondrial dysfunction and oxidative damage has been shown to be related to the death of dopaminergic neurons in Parkinson’s disease (PD). Prolyl oligopeptidase (PREP) is proposed to increase aSyn aggregation, and PREP inhibition has been shown to inhibit the aggregation process in vitro and in vivo. In this study, we investigated the effects of a specific PREP inhibitor, KYP-2047, on rotenone induced aSyn aggregation and increased the production of reactive oxygen species (ROS) in cells overexpressing A53T mutation of aSyn. Rotenone, a mitochondrial toxin that induces oxidative damage and aSyn aggregation, associated with PD pathology, was selected as a model for this study. The results showed that rotenone induced the formation of high-molecular-weight aSyn oligomers, and this was countered by simultaneous incubation with KYP-2047. Inhibition of PREP also decreased the production of ROS in [A53T]aSyn overexpressing cells, leading to improved cell viability. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Parkinson’s disease (PD) is a neurodegenerative disorder where dopaminergic neurons of the nigrostriatal pathway degenerate for unknown reasons. This causes instability in neuronal circuits leading to clinical symptoms including tremor, rigor and bradykinesia [1]. One of the key players in neuronal death may be the aggregation

Abbreviations: aSyn, ␣-synuclein; DMSO, dimethylsulfoxide; DMEM, Dulbecco’s modified Eagle medium; HMW, high-molecular-weight; LC3BI-II, microtubuleassociated protein light chain 3B I-II; OD, optical density; PD, Parkinson’s disease; PBS, phosphate-buffered saline; PREP, prolyl oligopeptidase; ROS, reactive oxygen species; WB, Western blot; WT, wild type. ∗ Corresponding author. Tel.: +358 2941 59459; fax: +358 02941 59138. E-mail addresses: lana.dokleja@helsinki.fi (L. Dokleja), mirva.hannula@helsinki.fi (M.J. Hannula), timo.myohanen@helsinki.fi (T.T. Myöhänen). 1 Equal contribution. http://dx.doi.org/10.1016/j.neulet.2014.09.026 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.

of ␣-synuclein (aSyn), a brain protein with functions in neurotransmitter packing and release [2]. In 1998, Spillantini et al. [3] found that Lewy bodies, histopathological hallmarks of PD, consisted mostly of aggregated aSyn. However, recent studies are proposing that fibrillar aggregates in Lewy bodies may be protective for cells, and aSyn oligomers are actually the toxic species [4]. In addition, mutations in the aSyn gene are shown to be risk factors for PD to further support the role of aSyn in this disease [2]. Post mortem studies in patients with PD have shown systemic defects in the mitochondrial respiratory complex I, especially in the substantia nigra (SN) [5]. Mitochondrial damage causes a reduction in ATP production and increases the levels of reactive oxygen species (ROS), and dopaminergic neurons in the SN seem to be particularly sensitive to oxidative damage [5–7]. In addition, the aggregation of aSyn increases in the presence of ROS, leading to further ROS overproduction [8]. aSyn oligomers can interact with the mitochondrial membrane and cause fragmentation of mitochondria, followed by cell death [9]. Rotenone is a toxin used as a

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pesticide, acting as a high-affinity inhibitor of the mitochondrial complex I and causing an increase in intracellular ROS [7]. Therefore, rotenone has been used as a toxin model for PD, and systemic administration of rotenone causes PD-like symptoms with aSyn aggregation and dopaminergic cell death in vivo [7]. Brandt et al. [10] showed in an in vitro turbidity assay that the aggregation rate of aSyn is increased in the presence of prolyl oligopeptidase (PREP), and this action can be blocked by a specific PREP inhibitor. PREP is a serine protease that hydrolyzes proline containing peptides shorter than 30-mer at least in vitro, and several functions in cell proliferation, intracellular signaling and memory functions has been proposed [11]. In the CNS, PREP activity has been reported to increase during aging and neurodegenerative disorders, supporting the possible role in neuropeptide catabolism, and this has led to PREP inhibitor development. However, the results of PREP inhibition on neuropeptide levels in vivo are inconsistent [12]. Moreover, Tenorio-Laranga et al. [13] showed that 4-day PREP inhibition changed the amount of cytosolic peptides but they were not derived from traditional PREP substrates such as substance P etc., and most of them were not cleaved after proline specifically. Recently, studies with knock-out models have shown that lack of PREP leads to decreased neuronal growth and impaired memory but these functions seem to be independent of its hydrolytic activity [14,15], and the true physiological role of PREP remains largely unknown. In our current studies, we have shown that PREP colocalizes with aSyn in brain of PD patients [16], and that PREP inhibition decreases aSyn aggregation and enhances the clearance of aggregates via autophagy in aSyn overexpressing cells and animal models [17,18]. However, the mechanism has remained unclear. By using a cell line overexpressing A53T mutated aSyn with rotenone-induced mitochondrial damage, we were able to study not only the effect of PREP inhibition on aSyn aggregation but also the effect of PREP inhibition on ROS and autophagy markers. This would further explain the mechanisms how PREP inhibition can reduce aSyn aggregation in cells. 2. Materials and methods 2.1. Chemicals Chemicals used were purchased form Sigma–Aldrich (St. Louis, MO, USA) unless otherwise specified. Ethanol was purchased from Altia (Helsinki, Finland). The PREP inhibitor, KYP-2047 (4-phenylbutanoyl-l-prolyl-2(S)-cyanopyrrolidine), was obtained from Dr. Elina Jarho (University of Eastern Finland), and its synthesis was described in [19]. KYP-2047 has been shown to be potent, selective and enter the cells in culture [18,20,21]. KYP-2047 was dissolved in DMSO at a concentration of 100 mM and then further diluted.

effect of KYP-2047 on rotenone-induced aSyn aggregation during 3 days. Groups were as follows: (1) rotenone 50 nM + 0.01% DMSO (rotenone with vehicle), (2) rotenone 50 nM + KYP-2047 10 ␮M in 0.01% DMSO (rotenone with KYP), (3) KYP-2047 10 ␮M in 0.01% DMSO (KYP) and (4) 0.01% DMSO (vehicle). Cells in well plates had an additional group: (5) medium (negative control). WT cells were treated as above. However, no aSyn aggregates were seen, and therefore, this data is not shown. 2.4. aSyn immunocytochemistry Cells cultured in 12-well plates were immunostained for aSyn after the 3-day treatments, using a protocol described in [18]. Briefly, after fixing and blocking, the cells were incubated overnight at +4 ◦ C with a primary antibody against aSyn (rabbit anti-aSyn, #ab52168, AbCam, Cambridge, UK; 1:500 in 1% normal goat serum). On the next day, the cells were incubated for 2 h with a secondary antibody (anti-rabbit fluorescein, #31635, Thermo Fisher Scientific, Waltham, MA, USA; 1:500 in 1% normal goat serum). Cells were photographed using Nikon Eclipse TE300 microscope with Image Pro Plus software (Media Cybernetics, Bethesda, MD, USA). The cell calculations were done as previously reported [18]. 2.5. aSyn fractionation and WB For WB experiments, cells grown in T25 flasks were lysed and homogenized according to the protocol described earlier [18]. The procedure resulted in fractions containing soluble aSyn, its SDSsoluble monomers, and SDS-insoluble oligomers. To detect the levels of different aSyn forms, and autophagy markers p62/SQSTM1 (p62; accumulation marker) and microtubule-associated protein 1 light chain 3 beta (LC3B; autophagosome marker), WB was used [23]. Protein levels were measured using the BCA Protein Assay Kit (#23227, Thermo Fisher Scientific) and the lysates were loaded onto a 12% SDS gel (20 ␮g of protein/well). Standard transfer and blocking techniques were used and the loading control was mouse anti-beta-actin (#ab8226, AbCam; 1:2500). Following primary antibodies were used: mouse anti-aSyn antibody (#ab1903, AbCam; 1:1000), anti-mouse p62 (#ab56416, AbCam; 1:5000) and anti-rabbit LC3B (#L7543; 1:1000). Anti-mouse HRP (#31430, Thermo Fisher Scientific; 1:2000) and anti-rabbit HRP (#31460, Thermo Fisher Scientific; 1:2000) were used as secondary antibodies. All antibodies were diluted in 5% skim milk in 0.05% Tween20 in Tris-buffered saline. The images were captured using the C-Digit imaging system (Licor, Lincoln, NE, USA). Three independent WB experiments were performed. ImageJ (NIH, Bethesda, MD, USA) was used for analyzing bands, and the optical density (OD) value was calculated by comparing the OD value to the corresponding beta-actin OD value. 2.6. Detection of ROS

2.2. Cell lines WT SH-SY5Y human neuroblastoma cell line was cultured as described earlier [20]. The stable cell line expressing A53T aSyn ([A53T]aSyn) was obtained from Prof. V. Baekelandt and Dr. M. Gérard and the development of this line was described in [22]. Cells were grown at 37 ◦ C and 5% CO2 in a humidified atmosphere. 2.3. Induction of ˛-synuclein aggregation by rotenone and the effect of KYP-2047 Cells were cultured in 12-well plates for immunochemistry (8 × 104 cells/well), 96-well plates for cell viability and ROS experiments (6 × 104 cells/well) and in T25 flasks for Western blot (WB) (106 cells/flask). After 24 h, study groups were formed to test the

To detect the effects of rotenone and KYP-2047 on ROS in [A53T]aSyn and WT cells, DCFDA Cellular ROS Detection Assay Kit (#ab113851, AbCam) was used. The formation of 2,7dichlorofluorescein (excitation and emission spectra of 495 nm and 529 nm, respectively) was measured using Wallac 1420 Victor2 fluorescence plate reader (Perkin Elmer, Waltham, MA, USA). 2.7. Cell viability assay Cells were plated and treated with 50 nM and 100 nM rotenone in the presence of KYP-2047/vehicle (150 ␮L/well). To assess cell viability a standard LDH release assay was performed as previously described [10]. Absorbance was measured at 490 nm using Wallac 1420 Victor2 fluorescence plate reader (Perkin Elmer).

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Fig. 1. (A–D) KYP-2047 significantly decreases aSyn immunofluorescence in rotenone-induced aSyn aggregation. Rotenone-induced (50 nM, 3 days) aSyn aggregation (A) was significantly decreased (***P < 0.001, 1-way ANOVA with Tukey post hoc test) with simultaneous PREP inhibition using 10 ␮M KYP-2047 (B and D). Photomicrographs depict the levels of aSyn aggregates using immunofluorescence after 3-day rotenone exposure (A), the effect of incubation simultaneously with KYP-2047 (B) and the negative control (C). Scale bar is 10 ␮m in all pictures. aSyn, ␣-synuclein; KYP 10 ␮M, 3-day 10 ␮M KYP-2047; NC, negative control; Rot 50 nM + vehicle, 3-day rotenone + 0.01% DMSO; Rot 50 nM + KYP 10 ␮M, 3-day 50 nM rotenone + 10 ␮M KYP-2047; Vehicle, 3-day 0.01% DMSO.

2.8. Statistical analysis In order to detect the differences between the groups in aSyn immunofluorescence and ROS formation, 1-way ANOVA followed by a Tukey post hoc test was used. 2-way ANOVA followed by a Bonferroni post hoc test was used in the Western blot analysis. Statistically significant differences were considered at P < 0.05.

significant increase was seen on the levels of SDS-insoluble highmolecular-weight (HMW) aSyn (Fig. 2C; +314%; 2-way ANOVA, rotenone vs. control: F1,7 = 9.075; P = 0.0168). Simultaneous exposure to KYP-2047 caused significant decrease (−250% compared to vehicle) in the levels of HMW aSyn (Fig. 2C; 2-way ANOVA with Bonferroni post hoc test, KYP-2047 vs. vehicle, P < 0.05).

3. Results

3.3. Autophagy marker levels assessed by WB

3.1. aSyn immunofluorescence

Exposure to rotenone had no significant effect on the levels of p62, a protein accumulation marker (Fig. 2D), but significantly elevated the levels of LC3BII (+140%), a marker for autophagosome synthesis (Fig. 2E; 2-way ANOVA, rotenone vs. control: F1,7 = 10.01; P = 0.013). PREP inhibition did not further alter the autophagy markers.

Roteone induced aSyn immunoreactivity (+23%; Fig. 1A-D; 1-way ANOVA with Tukey post hoc test, F1,5 = 82.37; P < 0.001 rotenone + vehicle vs. negative control) in [A53T]aSyn cells. Simultaneous exposure to KYP-2047 significantly decreased this effect (−12%; Fig. 1A–D; 1-way ANOVA with Tukey post hoc test, P < 0.001 rotenone + vehicle vs. rotenone + KYP-2047). WT cells did not show aSyn aggregation caused by rotenone exposure (data not shown). 3.2. WB analysis of soluble and insoluble forms of aSyn Rotenone increased the levels of soluble aSyn 50% over the control level (Fig. 2A; 2-way ANOVA, rotenone vs. control: F1,7 = 8.352; P = 0.0202) in [A53T]aSyn cells. Simultaneous KYP-2047 incubation had no effect on soluble aSyn levels, and SDS-soluble monomers of aSyn were not increased by rotenone (Fig. 2A and B). The most

3.4. ROS levels Exposure to rotenone resulted in a significant increase in ROS, both in [A53T]aSyn cells (+16%; Fig. 3A) and WT cells (+14%; Fig. 3B; 1-way ANOVA with Tukey post hoc test, F1,5 = 29.70; P < 0.001 in both cell lines). PREP inhibition with rotenone reduced the level of ROS in [A53T]aSyn overexpressing cells (−6% compared to vehicle) (P < 0.05, 1-way ANOVA with Tukey post hoc test), but not in WT cells.

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Fig. 2. (A–E) The effect of rotenone and KYP-2047 on aSyn aggregation and autophagy markers. 3-day rotenone exposure (50 nM) increased the levels of soluble aSyn (A; P = 0.0202, 2-way ANOVA, rotenone vs. control) but had no effect on SDS-soluble monomeric aSyn in [A53T]aSyn cells (B). The most robust effect was seen on HMW oligomers (C) where rotenone significantly increased the levels (P = 0.0168, 2-way ANOVA, rotenone vs. control) and simultaneous incubation with 10 ␮M KYP-2047 attenuated the rotenone effect (*P < 0.05, KYP-2047 vs. vehicle, 2-way ANOVA with Bonferroni post hoc test). In autophagy markers, p62 levels showed no changes (D), whereas the levels of LC3BII were significantly increased (E; P = 0.013, 2-way ANOVA, rotenone vs. control). KYP-2047 did not significantly alter the levels of autophagy markers. Study groups as in Fig. 1.

3.5. Cell viability

4. Discussion

In the LDH viability test, rotenone was more toxic to [A53T]aSyn cells ([A53T]aSyn cells: +21% (50 nM) and +42% (100 nM) cell death; Wt cells +13% (50 nM) and +13% (100 nM) cell death Fig. 3C and D), although it caused significant toxicity in both cell lines (2way ANOVA, rotenone + veh/KYP vs. negative control: [A53T]aSyn cells F2,45 = 21.38, P < 0.0001; WT cells F2,47 = 18.17, P < 0.0001). Inhibition of PREP by KYP-2047 during rotenone incubation significantly improved the cell survival in [A53T]aSyn cells (−13% (50 nM) and −29% (100 nM) compared to vehicle), but not in WT cells (Fig. 3C; 2-way ANOVA with Bonferroni post hoc test, Rotenone + KYP vs. Rotenone + vehicle: F1,45 = 16.80, P = 0.0002; interaction F1,45 = 6.222, P = 0.0041).

In this study, rotenone was used as a toxin to mimic the development of PD in cells overexpressing pathological A53T mutated aSyn. We saw a significant increase in aSyn immunostaining after 3 days of rotenone exposure, and the number of cells with high aSyn immunoreactivity was decreased by simultaneous KYP-2047 incubation. In addition, WB analysis revealed an increase in soluble aSyn, suggesting that rotenone upregulates aSyn [24]. As expected, protein levels of aSyn HMW oligomers were significantly increased following exposure to rotenone, while treatment with KYP-2047 significantly reduced this effect similar to our previous cell culture experiments [18]. Moreover, KYP-2047 had no effect on rotenoneinduced increase of soluble aSyn, and we have earlier shown that

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Fig. 3. (A–D) The effect of rotenone and KYP-2047 on reactive oxygen species (ROS) formation and cell viability assayed by lactate-dehydrogenase (LDH) test. Rotenone significantly increased the levels of ROS both in [A53T]aSyn (A) and WT SH-SY5Y cells (B) compared to negative control (***P < 0.001; 1-way ANOVA, rot 50 nM + veh/KYP vs. NC). KYP-2047 had a significant effect on rotenone-induced ROS increase in [A53T]aSyn cells (A; P < 0.05; 1-way ANOVA with Tukey post hoc test, rot 50 nM + veh vs. rot 50 nM + KYP) that was not seen in WT cells (B). KYP-2047 or vehicle itself did not have any effect on ROS and 50 ␮M antimycin served as a positive control (A and B; ***P < 0.001; 1-way ANOVA with Tukey post hoc test, antimycin vs. NC). Incubation of rotenone decreased the cell viability in both cell lines (P < 0.0001 rotenone vs. control, 2-way ANOVA) while simultaneous incubation with KYP-2047 significantly increased the viability of [A53T]aSyn cells (C) (P = 0.0002 KYP-2047 vs. vehicle, 2-way ANOVA). This was more evident for the 100 nM rotenone dose (***P < 0.001 KYP-2047 vs. vehicle, 2-way ANOVA with Bonferroni post-test). In WT cells, no effect of KYP-2047 was seen on cell viability (D). Study groups as in Fig. 1.

KYP-2047 cannot modulate the mRNA levels of aSyn in cell culture [18]. These observations indicate that the strong increase in aSyn immunostaining can be attributed to aSyn aggregation induced by rotenone. Based on our previous results in vitro and in vivo [18,10], it is likely that PREP can serve as a seeding point for aSyn aggregation via direct protein–protein interaction, and that PREP inhibitor can then modulate the interaction leading to decreased aSyn aggregation. Although the interaction mechanism and interaction site are not known, an active form of PREP is needed for this since the inactive S554A PREP mutant did not have effect on aSyn aggregation in vitro [10], and the potency of PREP inhibitor correlated with the reduced aSyn aggregation and apoptosis [25]. To clear larger aSyn oligomers from the cell, autophagy and chaperone-mediated autophagy are needed [26,27]. In a previous study, rotenone toxicity on SH-SY5Y cells was attenuated by rapamycin, an autophagy inducer [28], and since we showed in our recent paper [17] that PREP inhibition has beneficial effects on autophagy, we hypothesized that this could be one of the mechanisms behind the decrease of HMW aSyn oligomers by KYP2047. Rotenone incubation increased autophagosome formation as described earlier [29] but only some, not significant, increase was seen on an accumulation marker p62 [30]. Moreover, although there were some positive changes on LC3B and p62 levels by KYP2047, they did not reach significance in this model.

Inhibition of the mitochondrial complex I by rotenone increased ROS formation [5]. Furthermore, overexpression of aSyn was shown to cause mitochondrial damage in dopaminergic neurons [31], causing increased ROS production in cells [32]. Our results confirmed this effect showing a significant increase in cellular ROS after rotenone incubation, but in our study the effect of rotenone was similar both in [A53T]aSyn overexpressing and WT control cells. Since rotenone incubation with KYP-2047 reduced ROS in [A53T]aSyn overexpressing cells, but not in WT cells, it is likely that the reduction of aSyn oligomers caused the decrease in ROS formation in [A53T]aSyn cells. In a previous study, the presence of mutated aSyn resulted in increased rotenone-induced toxicity [33]. In this study, cells overexpressing [A53T]aSyn were more sensitive to damage caused by rotenone, implicating that the increase in HMW aSyn oligomers is mostly responsible for cell death. Furthermore, PREP inhibition by KYP-2047 caused a significant positive effect on cell viability of [A53T]aSyn cells, but not on WT cells. This emphasizes the role of HMW aSyn oligomers in cell death and the importance of KYP-2047 mediated aSyn reduction on cell viability. 5. Conclusions Collectively, our results point that rotenone incubation (1) induced aSyn aggregation in [A53T]aSyn cells, (2) caused changes

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in autophagosomes, and (3) increased ROS production in cells leading to increased cell death. Simultaneous incubation of cells with a PREP inhibitor, KYP-2047, countered these effects by reducing the HMW aSyn oligomers that led to decreased ROS production and increased cell survival. This proposes a role for PREP inhibition as a potential drug therapy in PD and other synucleinopathies. Acknowledgements This work was supported by the grants of Academy of Finland (no. 138127 and no. 267788), Jane and Aatos Erkko Foundation and Sigrid Juselius Foundation for Timo T. Myöhänen. The authors thank Prof. Veerle Baekelandt, Catholic University of Leuven, Belgium, for cell lines used in this study. References [1] D.T. Dexter, P. Jenner, Parkinson disease: from pathology to molecular disease mechanisms, Free Radic. Biol. Med. 62 (2013) 132–144. [2] A. Surguchov, Molecular and cellular biology of synucleins, Int. Rev. Cell. Mol. Biol. 270 (2008) 225–317. [3] M.G. Spillantini, M.L. Schmidt, V.M. Lee, J.Q. Trojanowski, R. Jakes, M. Goedert, Alpha-synuclein in Lewy bodies, Nature 388 (1997) 839–840. [4] K. Wakabayashi, K. Tanji, S. Odagiri, Y. Miki, F. Mori, H. Takahashi, The Lewy body in Parkinson’s disease and related neurodegenerative disorders, Mol. Neurobiol. 47 (2013) 495–508. [5] C. Perier, M. Vila, Mitochondrial biology and Parkinson’s disease, Cold Spring Harb Perspect. Med. 2 (2012) a009332. [6] D.J. Surmeier, Calcium, ageing, and neuronal vulnerability in Parkinson’s disease, Lancet Neurol. 6 (2007) 933–938. [7] T.N. Martinez, J.T. Greenamyre, Toxin models of mitochondrial dysfunction in Parkinson’s disease, Antioxid. Redox Signal. 16 (2012) 920–934. [8] V. Dias, E. Junn, M.M. Mouradian, The role of oxidative stress in Parkinson’s disease, J. Parkinson’s Dis. 3 (2013) 461–491. [9] K. Nakamura, V.M. Nemani, F. Azarbal, G. Skibinski, J.M. Levy, K. Egami, L. Munishkina, J. Zhang, B. Gardner, J. Wakabayashi, H. Sesaki, Y. Cheng, S. Finkbeiner, R.L. Nussbaum, E. Masliah, R.H. Edwards, Direct membrane association drives mitochondrial fission by the Parkinson disease-associated protein alpha-synuclein, J. Biol. Chem. 286 (2011) 20710–20726. [10] I. Brandt, M. Gerard, K. Sergeant, B. Devreese, V. Baekelandt, K. Augustyns, S. Scharpe, Y. Engelborghs, A.M. Lambeir, Prolyl oligopeptidase stimulates the aggregation of alpha-synuclein, Peptides 29 (2008) 1472–1478. [11] J.A. Garcia-Horsman, P.T. Männistö, J.I. Venäläinen, On the role of prolyl oligopeptidase in health and disease, Neuropeptides 41 (2007) 1–24. [12] P.T. Männistö, J.I. Venäläinen, A.J. Jalkanen, J.A. Garcia-Horsman, Prolyl oligopeptidase: a potential target for the treatment of cognitive disorders, Drugs News Perspect. 20 (2007) 293–305. [13] J. Tenorio-Laranga, P.T. Männistö, M. Storvik, P. Van der Veken, J.A. GarciaHorsman, Four day inhibition of prolyl oligopeptidase causes significant changes in the peptidome of rat brain, liver and kidney, Biochimie 94 (2012) 1849–1859. [14] E. Di Daniel, C.P. Glover, E. Grot, M.K. Chan, T.H. Sanderson, J.H. White, C.L. Ellis, K.T. Gallagher, J. Uney, J. Thomas, P.R. Maycox, A.W. Mudge, Prolyl oligopeptidase binds to GAP-43 and functions without its peptidase activity, Mol. Cell. Neurosci. 41 (2009) 373–382. [15] G. D’Agostino, J.D. Kim, Z.W. Liu, J.K. Jeong, S. Suyama, A. Calignano, X.B. Gao, M. Schwartz, S. Diano, Prolyl endopeptidase-deficient mice have reduced synaptic spine density in the CA1 region of the hippocampus, impaired LTP, and spatial learning and memory, Cereb. Cortex (2012).

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