Prolyl oligopeptidase acts as a link between chaperone-mediated autophagy and macroautophagy

The accumulation of aggregated alpha-synuclein (alpha-syn) has been identified as the primary component of Lewy bodies that are the pathological hallmarks of Parkinson's disease (PD). Several preclinical studies have shown alpha-syn aggregation, and particularly the intermediates formed during the aggregation process to be toxic to cells. Current PD treatments only provide symptomatic relief, and alpha-syn serves as a promising target to develop a disease-modifying therapy for PD. Our previous studies have revealed that a small-molecular inhibitor for prolyl oligopeptidase (PREP), KYP-2047, increases alpha-syn degradation by accelerating macroautophagy (MA) leading to disease-modifying effects in preclinical PD models. However, alpha-syn is also degraded by chaperone-mediated autophagy (CMA). In the present study, we tested the effects of PREP inhibition or deletion on CMA activa-tion and alpha-syn degradation. HEK-293 cells were transfected with alpha-syn and incubated with 1 & 10 mu M KYP-2047 for 24 h. Both 1 & 10 mu M KYP-2047 increased LAMP-2A levels, induced alpha-syn degradation and reduced the expression of Hsc70, suggesting that the PREP inhibitor prevented alpha-syn aggregation by activating the CMA pathway. Similarly, KYP-2047 increased the LAMP-2A immunoreactivity and reduced the Hsc70 levels in mouse primary cortical neurons. When LAMP-2A was silenced by a siRNA, KYP-2047 increased the LC3BII/LC3BI ratio and accelerated the clearance of alpha-syn. Additionally, KYP-2047 induced CMA effectively also when MA was blocked by bafilomycin A1. Based on our results, we suggest that PREP might function as a core network node in MA-CMA crosstalk, and PREP inhibition can reduce alpha-syn levels via both main autophagy systems.Peer reviewe

Deletion or inhibition of prolyl oligopeptidase blocks lithium-induced phosphorylation of GSK3b and Akt by activation of protein phosphatase 2A

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Removal of prolyl oligopeptidase reduces alpha-synuclein toxicity in cells and in vivo

Prolyl oligopeptidase (PREP) inhibition by small-molecule inhibitors can reduce alpha-synuclein (aSyn) aggregation, a key player in Parkinson's disease pathology. However, the significance of PREP protein for aSyn aggregation and toxicity is not known. We studied this in vivo by using PREP knock-out mice with viral vector injections of aSyn and PREP. Animal behavior was studied by locomotor activity and cylinder tests, microdialysis and HPLC were used to analyze dopamine levels, and different aSyn forms and loss of dopaminergic neurons were studied by immunostainings. Additionally, PREP knock-out cells were used to characterize the impact of PREP and aSyn on autophagy, proteasomal system and aSyn secretion. PREP knock-out animals were nonresponsive to aSyn-induced unilateral toxicity but combination of PREP and aSyn injections increased aSyn toxicity. Phosphorylated p129, proteinase K resistant aSyn levels and tyrosine hydroxylase positive cells were decreased in aSyn and PREP injected knock-out animals. These changes were accompanied by altered dopamine metabolite levels. PREP knock-out cells showed reduced response to aSyn, while cells were restored to wild-type cell levels after PREP overexpression. Taken together, our data suggests that PREP can enhance aSyn toxicity in vivo.Peer reviewe

Prolyl oligopeptidase inhibition attenuates the toxicity of a proteasomal inhibitor, lactacystin, in the alpha-synuclein overexpressing cell culture

Lewy bodies, the histopathological hallmarks of Parkinson's disease (PD), contain insoluble and aggregated alpha-synuclein (aSyn) and many other proteins, proposing a role for failure in protein degradation system in the PD pathogenesis. Proteasomal dysfunction has indeed been linked to PD and aSyn oligomers have been shown to inhibit proteasomes and autophagy. Our recent studies have shown that inhibitors of prolyl oligopeptidase (PREP) can prevent the aggregation and enhance the clearance of accumulated aSyn, and therefore, we wanted to study if PREP inhibition can overcome the aSyn aggregation and toxicity induced by lactacystin, a proteasomal inhibitor. The cells overexpressing human A30P or A53T mutated aSyn were incubated with lactacystin and a PREP inhibitor, KYP-2047, for 48 h. Theafter, the cells were fractioned, and the effects of lactacystin with/without 1 mu M KYP-2047 on aSyn aggregation and ubiquitin accumulation, cell viability and on autophagic markers (p62, Beclin1 and LC3BII) were studied. We found that KYP-2047 attenuated lactacystin-induced cell death in mutant aSyn overexpressing cells but not in non-overexpressing control cells. KYP-2047 reduced significantly SDS-insoluble high-molecular-weight aSyn oligomers that were in line with the cell viability results. In addition, significant reduction in protein accumulation marker, p62, was seen in SDS fraction while LC3BII, a marker for autophagosome formation, was increased, indicating to enhanced autophagy. Our results further streghten the possibilities for PREP inhibitors as a potential drug therapy against synucleinopathies and other protein aggregating diseases. (C) 2016 Elsevier Ireland Ltd. All rights reserved.Peer reviewe

Prolyl oligopeptidase inhibition activates autophagy via protein phosphatase 2A

Prolyl oligopeptidase (PREP) is a serine protease that has been studied particularly in the context of neurode-generative diseases for decades but its physiological function has remained unclear. We have previously found that PREP negatively regulates beclinl-mediated macroautophagy (autophagy), and that PREP inhibition by a small-molecule inhibitor induces clearance of protein aggregates in Parkinson's disease models. Since autophagy induction has been suggested as a potential therapy for several diseases, we wanted to further characterize how PREP regulates autophagy. We measured the levels of various kinases and proteins regulating beclin1-autophagy in HEK-293 and SH-SY5Y cell cultures after PREP inhibition, PREP deletion, and PREP overexpression and restoration, and verified the results in vivo by using PREP knock-out and wild-type mouse tissue where PREP was restored or overexpressed, respectively. We found that PREP regulates autophagy by interacting with protein phosphatase 2A (PP2A) and its endogenous inhibitor, protein phosphatase methylesterase 1 (PME1), and activator (protein phosphatase 2 phosphatase activator, PTPA), thus adjusting its activity and the levels of PP2A in the intracellular pool. PREP inhibition and deletion increased PP2A activity, leading to activation of deathassociated protein kinase 1 (DAPK1), beclin1 phosphorylation and induced autophagy while PREP overexpression reduced this. Lowered activity of PP2A is connected to several neurodegenerative disorders and cancers, and PP2A activators would have enormous potential as drug therapy but development of such compounds has been a challenge. The concept of PREP inhibition has been proved safe, and therefore, our study supports the further development of PREP inhibitors as PP2A activators.Peer reviewe

Deletion or inhibition of prolyl oligopeptidase blocks lithium-induced phosphorylation of GSK3b and Akt by activation of protein phosphatase 2A

Alterations in prolyl oligopeptidase (PREP) activity have been connected, for example, with bipolar and major depressive disorder, and several studies have reported that lack or inhibition of PREP blocks the effects of lithium on inositol 1,4,5-triphosphate (IP3) levels. However, the impact of PREP modulation on other intracellular targets of lithium, such as glycogen synthase kinase 3 beta (GSK3b) or protein kinase B (Akt), has not been studied. We recently found that PREP regulates protein phosphatase 2A (PP2A), and because GSK3b and Akt are PP2A substrates, we studied if PREP-related lithium insensitivity is dependent on PP2A. To assess this, HEK-293 and SH-SY5Y cells with PREP deletion or PREP inhibition (KYP-2047) were exposed to lithium, and thereafter, the phosphorylation levels of GSK3b and Akt were measured by Western blot. As expected, PREP deletion and inhibition blocked the lithium-induced phosphorylation on GSK3b and Akt in both cell lines. When lithium exposure was combined with okadaic acid, a PP2A inhibitor, KYP-2047 did not have effect on lithium-induced GSK3b and Akt phosphorylation. Therefore, we conclude that PREP deletion or inhibition blocks the intracellular effects of lithium on GSK3b and Akt via PP2A activation

Prolyl oligopeptidase inhibition reduces alpha-synuclein aggregation in a cellular model of multiple system atrophy

Multiple system atrophy (MSA) is a fatal neurodegenerative disease where the histopathological hallmark is glial cytoplasmic inclusions in oligodendrocytes, rich of aggregated alpha-synuclein (aSyn). Therefore, therapies targeting aSyn aggregation and toxicity have been studied as a possible disease-modifying therapy for MSA. Our earlier studies show that inhibition of prolyl oligopeptidase (PREP) with KYP-2047 reduces aSyn aggregates in several models. Here, we tested the effects of KYP-2047 on a MSA cellular models, using rat OLN-AS7 and human MO3.13 oligodendrocyte cells. As translocation of p25α to cell cytosol has been identified as an inducer of aSyn aggregation in MSA models, the cells were transiently transfected with p25α. Similar to earlier studies, p25α increased aSyn phosphorylation and aggregation, and caused tubulin retraction and impaired autophagy in OLN-AS7 cells. In both cellular models, p25α transfection increased significantly aSyn mRNA levels and also increased the levels of inactive protein phosphatase 2A (PP2A). However, aSyn or p25α did not cause any cellular death in MO3.13 cells, questioning their use as a MSA model. Simultaneous administration of 10 µM KYP-2047 improved cell viability, decreased insoluble phosphorylated aSyn and normalized autophagy in OLN-AS7 cells but similar impact was not seen in MO3.13 cells.Peer reviewe

Prolyl oligopeptidase inhibition reduces alpha-synuclein aggregation in a cellular model of multiple system atrophy

Multiple system atrophy (MSA) is a fatal neurodegenerative disease where the histopathological hallmark is glial cytoplasmic inclusions in oligodendrocytes, rich of aggregated alpha-synuclein (aSyn). Therefore, therapies targeting aSyn aggregation and toxicity have been studied as a possible disease-modifying therapy for MSA. Our earlier studies show that inhibition of prolyl oligopeptidase (PREP) with KYP-2047 reduces aSyn aggregates in several models. Here, we tested the effects of KYP-2047 on a MSA cellular models, using rat OLN-AS7 and human MO3.13 oligodendrocyte cells. As translocation of p25 alpha to cell cytosol has been identified as an inducer of aSyn aggregation in MSA models, the cells were transiently transfected with p25 alpha. Similar to earlier studies, p25 alpha increased aSyn phosphorylation and aggregation, and caused tubulin retraction and impaired autophagy in OLN-AS7 cells. In both cellular models, p25 alpha transfection increased significantly aSyn mRNA levels and also increased the levels of inactive protein phosphatase 2A (PP2A). However, aSyn or p25 alpha did not cause any cellular death in MO3.13 cells, questioning their use as a MSA model. Simultaneous administration of 10 mu M KYP-2047 improved cell viability, decreased insoluble phosphorylated aSyn and normalized autophagy in OLN-AS7 cells but similar impact was not seen in MO3.13 cells

Developing Automation for Microbial Engineering:Automated Yeast Transformation Protocol

Tutkimuslaboratoriomenetelmien automatisointi yleistyy, koska nesteiden käsittely vie aikaa ja on työlästä. Manuaalisessa pipetoinnissa on variaatiota, ja vaihtelu on mahdollista poistaa automaatiolla. Manuaaliset menetelmät voivat kuitenkin olla helpommin toteutettavissa silloin kun näytemäärät ovat pieniä ja pipetoijat tottumattomia automaatioon. Mitä suuremmat näytemäärät ja pienemmät pipetointitilavuudet ovat kyseessä, sitä hyödyllisempää automaatiosta tulee. Nesteannostelijarobottien ohjelmointi voi olla alussa vaikeaa kokemattomille käyttäjille mutta toteutettavissa, kun käyttäjä tuntee ohjelmiston ja robotin. Yksinkertaisimmissa roboteissa on valmiita ohjelmia ladattaviksi ja käyttö opitaan muutamien tuntien tai päivien aikana. Monimutkaisemmat robotit ohjelmoidaan yleensä itse, joten robotin kokonaisuuden ymmärtäminen ja ohjelmoinnin oppiminen vie aikaa. Tutkimuksen tavoitteena oli selvittää, onko hiivatransformaatioprotokollan automatisoinnista hyötyä ja mitä etuja ja haittoja sillä voi olla manuaaliseen menetelmään verrattuna. Hiivakannaksi valittiin Saccharomyces cerevisia, joka transformoitiin tyhjällä pYX212-plasmidilla käyttäen litiumasetaatti/ PEG- menetelmää. Hiivan kasvatus standardoitiin optiseen tiheyteen 1, jotta voitiin varmistaa, että solut olivat optimaalisessa kasvuvaiheessa transformaatioita varten. Transformaatioliuos tehtiin pipetoimalla erilaisia nesteitä joko käsin pipetoimalla tai nesteenkäsittelyrobotteja käyttäen. Tämän jälkeen transformoidut solut laitettiin käsin kasvamaan SCD-URA-maljoille kahdeksi päiväksi 30°C:n lämpötilaan. Pesäkkeet laskettiin käyttämällä solulaskuria, minkä jälkeen transformaatiotehokkuutta, aktiivista aikaa ja kokonaisaikaa verrattiin manuaalisten ja automaattisten menetelmien välillä. Robottien ohjelmointi vei muutaman kuukauden, koska nesteet käyttäytyvät eri tavalla keskenään. Piti huomioida myös käyttöturvallisuustiedotteet ja mahdolliset robottialustan sisäiset törmäykset sekä nesteiden fysikaaliset ja kemialliset ominaisuudet, jotka vaikuttivat pipettikärkien valintaan. Manuaalisella menetelmällä transformaatiotehokkuus oli parempi, mutta automaatiomenetelmää voidaan kehittää vielä eteenpäin. Hiivatransformaatioprotokollan automatisointi hyödyttää etenkin suurissa näytesarjoissa, mikä säästää aktiivista aikaa ja vähentää rasitusvammoja. Käyttäjän pitää kuitenkin olla perehtynyt robottien käsittelyyn.The aim of this thesis was to examine whether it is beneficial to automate yeast transformation protocol and what benefits and limitations it might have. There is variation in pipetting techniques between people, and with automation it is possible to remove the variation. In addition, larger the sample sizes and smaller the pipetting volumes, the more useful automation becomes especially with high-throughput analysis. A quantitative research method was used in this study as the manual and automated transformation methods were compared. It was possible to get clear results and data from the methods. Saccharomyces cerevisiae yeast was selected to be transformed with an empty plasmid pYX212 with lithium acetate/ PEG method. Transformation mix was done with pipetting of different liquids either manually or with using liquid-handling robots. Colonies were calculated using a cell clicker, and transformation efficiency, active time and total time were compared between manual and automated methods. It is concluded that automation of yeast transformation is useful especially with larger sample sets saving active time and preventing from work-related repetitive strain injuries. Transformation efficiency was better with manual method, but the time for developing automated method was limited and the protocol can be further developed to work better

Prolyl oligopeptidase inhibition reduces PolyQ aggregation and improves cell viability in cellular model of Huntington’s disease

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