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

Behavioural and dopaminergic changes in double mutated human A30P*A53T alpha-synuclein transgenic mouse model of Parkinson´s disease.

Alpha-synuclein (aSyn) is the main component of Lewy bodies, the histopathological marker in Parkinson's disease (PD), and point mutations and multiplications of the aSyn coding SNCA gene correlate with early onset PD. Therefore, various transgenic mouse models overexpressing native or point-mutated aSyn have been developed. Although these models show highly increased aSyn expression they rarely capture dopaminergic cell loss and show a behavioural phenotype only at old age, whereas SNCA mutations are risk factors for PD with earlier onset. The aim of our study was to re-characterize a transgenic mouse strain carrying both A30P and A53T mutated human aSyn. Our study revealed decreased locomotor activity for homozygous transgenic mice starting from 3 months of age which was different from previous studies with this mouse strain that had behavioural deficits starting only after 7-9 months. Additionally, we found a decreased amphetamine response in locomotor activity and decreased extracellular dopaminergic markers in the striatum and substantia nigra with significantly elevated levels of aSyn oligomers. In conclusion, homozygous transgenic A30P*A53T aSyn mice capture several phenotypes of PD with early onset and could be a useful tool for aSyn studies.Peer reviewe

New tricks of prolyl oligopeptidase inhibitors - A common drug therapy for several neurodegenerative diseases

Changes in prolyl oligopeptidase (PREP) expression levels, protein distribution, and activity correlate with aging and are reported in many neurodegenerative conditions. Together with decreased neuropeptide levels observed in aging and neurodegeneration, and PREP's ability to cleave only small peptides, PREP was identified as a druggable target. Known PREP non-enzymatic functions were disregarded or attributed to PREP enzymatic activity, and several potent small molecule PREP inhibitors were developed during early stages of PREP research. These showed a lot of potential but with variable results in experimental memory models, however, the initial excitement was short-lived and all of the clinical trials were discontinued in either Phase I or II clinical trials for unknown reasons. Recently, PREP's ability to form protein-protein interactions, alter cell proliferation and autophagy has gained more attention than earlier recognized catalytical activity. Of new findings, particularly the aggregation of alpha-synuclein (aSyn) that is seen in the presence of PREP is especially interesting because PREP inhibitors are capable of altering aSyn-PREP interaction in a manner that reduces the aSyn dimerization process. Therefore, it is possible that PREP inhibitors that are altering interactions could have different characteristics than those aimed for strong inhibition of catalytic activity. Moreover, PREP co-localization with aSyn, tau, and amyloid-beta hints to PREP's possible role not only in the synucleinopathies but in other neurodegenerative diseases as well. This commentary will focus on less well-acknowledged non-enzymatic functions of PREP that may provide a better approach for the development of PREP inhibitors for the treatment of neurodegenerative disorders.Peer reviewe

Prolyl Oligopeptidase Regulates Dopamine Transporter Oligomerization and Phosphorylation in a PKC- and ERK-Independent Manner

Prolyl oligopeptidase (PREP) is a serine protease that binds to alpha-synuclein (aSyn) and induces its aggregation. PREP inhibitors have been shown to have beneficial effects in Parkinson's disease models by enhancing the clearance of aSyn aggregates and modulating striatal dopamine. Additionally, we have shown that PREP regulates phosphorylation and internalization of dopamine transporter (DAT) in mice. In this study, we clarified the mechanism behind this by using HEK-293 and PREP knock-out HEK-293 cells with DAT transfection. We tested the effects of PREP, PREP inhibition, and alpha-synuclein on PREP-related DAT regulation by using Western blot analysis and a dopamine uptake assay, and characterized the impact of PREP on protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) by using PKC assay and Western blot, respectively, as these kinases regulate DAT phosphorylation. Our results confirmed our previous findings that a lack of PREP can increase phosphorylation and internalization of DAT and decrease uptake of dopamine. PREP inhibition had a variable impact on phosphorylation of ERK dependent on the metabolic state of cells, but did not have an effect on phosphorylation or function of DAT. PREP modifications did not affect PKC activity either. Additionally, a lack of PREP elevated a DAT oligomerization that is associated with intracellular trafficking of DAT. Our results suggest that PREP-mediated phosphorylation, oligomerization, and internalization of DAT is not dependent on PKC or ERK

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

Prolyl Oligopeptidase Regulates Dopamine Transporter Phosphorylation in the Nigrostriatal Pathway of Mouse

Alpha-synuclein is the main component of Lewy bodies, a histopathological finding of Parkinson's disease. Prolyl oligopeptidase (PREP) is a serine protease that binds to alpha-synuclein and accelerates its aggregation in vitro. PREP enzyme inhibitors have been shown to block the alpha-synuclein aggregation process in vitro and in cellular models, and also to enhance the clearance of alpha-synuclein aggregates in transgenic mouse models. Moreover, PREP inhibitors have induced alterations in dopamine and metabolite levels, and dopamine transporter immunoreactivity in the nigrostriatal tissue. In this study, we characterized the role of PREP in the nigrostriatal dopaminergic and GABAergic systems of wild-type C57Bl/6 and PREP knockout mice, and the effects of PREP overexpression on these systems. Extracellular concentrations of dopamine and protein levels of phosphorylated dopamine transporter were increased and dopamine reuptake was decreased in the striatum of PREP knockout mice, suggesting increased internalization of dopamine transporter from the presynaptic membrane. Furthermore, PREP overexpression increased the level of dopamine transporters in the nigrostriatal tissue but decreased phosphorylated dopamine transporters in the striatum in wild-type mice. Our results suggest that PREP regulates the function of dopamine transporter, possibly by controlling the phosphorylation and transport of dopamine transporter into the striatum or synaptic membrane.Peer reviewe

The effect of prolyl oligopeptidase inhibitors on alpha-synuclein aggregation and autophagy cannot be predicted by their inhibitory efficacy

Previous studies have shown that prolyl oligopeptidase (PREP) negatively regulates autophagy and increases the aggregation of alpha-synuclein (alpha Syn), linking it to the pathophysiology of Parkinson's disease. Our earlier results have revealed that the potent small molecular PREP inhibitor KYP-2047 is able to increase autophagy and decrease dimerization of alpha Syn but other PREP inhibitors have not been systematically studied for these two protein-protein interaction mediated biological functions of PREP. In this study, we characterized these effects for 12 known PREP inhibitors with IC50-values ranging from 0.2 nM to 1010 nM. We used protein-fragment complementation assay (PCA) to assess alpha Syn dimerization and Western Blot of microtubule-associated protein light chain 3B II (LC3B-II) and a GFP-LC3-RFP expressing cell line to study autophagy. In addition, we tested selected compounds in a cell-free alpha Syn aggregation assay, native gel electrophoresis, and determined the compound concentration inside the cell by LC-MS. We found that inhibition of the proteolytic activity of PREP did not predict decreased alpha Syn dimerization or increased autophagy, and we also confirmed that this result did not simply reflect concentration differences of the compounds inside the cell. Thus, PREP ligands regulate the effect of PREP on autophagy and alpha Syn aggregation through a conformational stabilization of the enzyme that is not equivalent to inhibiting its proteolytic activity.Peer reviewe

The Effect of Prolyl Oligopeptidase Inhibitors on Alpha-Synuclein Aggregation and Autophagy Cannot Be Predicted by Their Inhibitory Efficacy

Previous studies have shown that prolyl oligopeptidase (PREP) negatively regulates autophagy and increases the aggregation of alpha-synuclein (αSyn), linking it to the pathophysiology of Parkinson’s disease. Our earlier results have revealed that the potent small molecular PREP inhibitor KYP-2047 is able to increase autophagy and decrease dimerization of αSyn but other PREP inhibitors have not been systematically studied for these two protein-protein interaction mediated biological functions of PREP. In this study, we characterized these effects for 12 known PREP inhibitors with IC50-values ranging from 0.2 nM to 1010 nM. We used protein-fragment complementation assay (PCA) to assess αSyn dimerization and Western Blot of microtubule-associated protein light chain 3B II (LC3B-II) and a GFP-LC3-RFP expressing cell line to study autophagy. In addition, we tested selected compounds in a cell-free αSyn aggregation assay, native gel electrophoresis, and determined the compound concentration inside the cell by LC-MS. We found that inhibition of the proteolytic activity of PREP did not predict decreased αSyn dimerization or increased autophagy, and we also confirmed that this result did not simply reflect concentration differences of the compounds inside the cell. Thus, PREP ligands regulate the effect of PREP on autophagy and αSyn aggregation through a conformational stabilization of the enzyme that is not equivalent to inhibiting its proteolytic activity.</p

Underlying Mechanisms of Prolyl Oligopeptidase Inhibition, Deletion, and Restoration on the α-Synuclein Aggregation Process

Neurodegenerative disorders are characterized by accumulation of toxic protein species that are followed by a gradual loss of neurons in certain brain regions and person’s loss of movement and dementia. The cause of the protein accumulation is not fully understood but is partially influenced by the disturbances in the protein degradation pathways, post-translational protein modifications that facilitate either gain-of-toxicity or loss-of-function of these proteins. In Parkinson’s disease, the best-known aggregation prone protein is alpha-synuclein (aSyn) that is the main component of Lewy bodies, the histopathological hallmarks of Parkinson’s disease and other synucleinopathies. Several studies suggest that aSyn aggregates can damage neurons by various mechanisms, and propagate toxicity by cell-to-cell transfer thus making it a tempting target for drug therapy. Current drug therapies can only relieve symptoms of neurodegenerative diseases but do not address, for example, protein aggregate clearance or pharmacological deceleration of the inclusion formation. In previous studies, prolyl oligopeptidase (PREP) has been shown to enhance the aggregation of aSyn. PREP inhibitors have been shown to reduce the aggregation and increase the clearance of aggregates via enhanced autophagy. However, the mechanisms of how PREP affects aSyn aggregation and regulates autophagy, and if this has a long-term impact on aSyn toxicity, have not been studied. The aim of this study was to investigate the role of PREP deletion, restoration, overexpression, and catalytical inhibition on the cellular signaling pathways and aSyn aggregation. The first part of the work was done in PREP knockout cells and knockout mice where the aSyn protein was overexpressed alone or together with PREP. We showed that absence of PREP decreases aSyn-overexpression mediated behavioral and cellular toxicity in mouse brain. Additionally, we found that PREP knockout cells exhibit reduced stress response and toxicity in the presence of protein overload, have increased autophagic activity, and remove excess aSyn into the cell media. The second part studied effects of chronic PREP inhibition by KYP-2047 on aSyn aggregation and on motor disturbances in the aSyn viral vector overexpression PD mouse model. The main finding showed that after chronic PREP inhibition, animals lost pathological unilateral motor behavior due to reduction in aSyn oligomer species in the nigrostriatal pathway. The third part concentrated on mapping the role of PREP in the pathways responsible for the autophagy initiation. The main finding was discovery of PREP’s role in negatively regulating one of the most important protein phosphatase complexes, protein phosphatase 2A (PP2A), via direct protein-protein interaction. Besides, this interaction could be altered with PREP inhibitor treatment that resulted in upregulation of PP2A activity and explained the functional results of autophagy induction after PREP inhibition. In summary, the findings of this study underline mechanisms through which PREP might be mediating aSyn related pathology and underlines the potential of PREP inhibition as an attractive drug target in reducing aSyn aggregate formation and boosting clearance from the affected brains. PREP involvement in the PP2A network regulation and additional functional data warrants further PREP investigation in the context of other neurodegenerative disorders and PP2A-related ailments.Hermostoa rappeuttavat sairaudet, kuten Parkinsonin ja Alzheimerin tauti, koskettavat yhä suurempaa joukkoa ihmisiä väestön ikääntymisen myötä. Näissä sairauksissa eri aivoalueilla tapahtuu huomattavaa hermosolukatoa, joka johtaa näkyviin oireisiin, kuten Parkinsonin taudissa liikehäiriöihin ja Alzheimerin taudissa muistin häiriöihin. Hermosolukato myös etenee jatkuvasti, ja oireet vaikeutuvat ja lisääntyvät tämän myötä. Nykyiset hoidot eivät kykene pysäyttämään tai edes hidastamaan hermosolutuhoa, joten uusien lääkevaikutuskohteiden ja taudin kulkua muuttavien hoitojen tutkiminen on erittäin tärkeää. Suurin ongelma uusien hoitojen kehittämisessä on ollut se, etteivät hermosolujen tuhoutumisen syyt ole vieläkään kovin hyvin selvillä. Kuitenkin yksi tärkeimmistä tekijöistä näyttää olevan tiettyjen hermosolujen sisäisten proteiinien muuntuminen virheelliseen muotoon ja tästä seuraava kertyminen hermosoluihin. Näiden kertymien olemassaolo esim. Parkinsonin taudin potilaiden aivonäytteissä on tunnettu jo pitkään, mutta vasta viimeisen kahden vuosikymmenen aikana on havaittu, että nämä ovat muodostuessaan myrkyllisiä hermosoluille ja voivat jopa levittää hermosolutuhoa solusta soluun liikkuessaan. Tämän vuoksi proteiinikertymät ovat nousseet keskeisiksi lääkekehityskohteiksi hermostoa rappeuttavissa sairauksissa. Tässä väitöskirjatyössä tutkittiin, miten prolyylioligopeptidaasi (PREP)-entsyymi vaikuttaa erityisesti Parkinsonin taudissa keskeisen alfa-synukleiini-proteiinin (a-synukleiini) kertymiseen ja myrkyllisyyteen. PREP:iä on tutkittu jo 1980-luvulta lähtien hermostoa rappeuttavien sairauksien parissa, mutta vasta 10 vuotta sitten havaittiin, että se voi lisätä a-synukleiinin kertymistä. Tämän jälkeen Helsingin yliopistossa toimiva dosentti Timo Myöhäsen ryhmä on havainnut, että PREP-estäjät voivat vähentää a-synukleiinin kertymistä Parkinsonin taudin solu- ja eläinmalleissa, ja tämän pohjalta väitöskirjaprojektissa keskityttiin seuraaviin kysymyksiin; 1) Miten merkittävä PREP on a-synukleiinin kertymiselle, 2) voidaanko PREP-estäjillä hoitaa a-synukleeinin aiheuttamaa liikehäiriötä eläinallissa ja 3) mihin tämä vaikutus perustuu? Tutkimuksessa havaittiin, että PREP ei ole välttämätön tekijä a-synukleiinin kertymiselle, mutta se lisää erityisesti kaikista myrkyllisimpien a-synukleiini-muotojen määrää, ja vaikuttaa tätä kautta huomattavasti a-synukleiinin myrkyllisyyteen. Jatkotutkimuksessa haluttiin selvittää, voidaanko PREP-estäjää käyttämällä vaikuttaa Parkinsonin taudin eläinmallin liikehäiriöön. Tätä varten pystytettiin a-synukleiinin ylimäärään perustuva hiirimalli, jota alettiin hoitaa PREP-estäjällä liikehäiriöoireiden ilmaannuttua. PREP-estäjä vaikutti jo 2 viikon kuluttua hoidon aloittamisesta niin, että Parkinson-hiirelle syntynyt liikehäiriö palautui normaalin hiiren tasolle, ja kudosanalyysissa havaittiin a-synukleiinikappaleiden selvästi vähentyneen hoidon myötä. Vaikutuksen takana oli PREP-estäjän kyky estää a-synukleiinin kertymistapahtumaa sekä lisätä jo syntyneiden kertymien hajotusta solujen ”kierrätyskeskuksen”, autofagian, kautta. Projektin viimeisessä osassa haluttiin selvittää miten PREP vaikuttaa solujen kierrätyskeskuksen toimintaan, ja tässä selvisi että PREP säätelee erittäin tärkeää solun toiminnan säätelijää, proteiinifosfataasi 2A:ta. Tutkimuksessa selvisi myös, että PREP-estäjillä tämä fosfataasi voidaan aktivoida, mikä lisää huomattavasti PREP-estäjien mahdollisuuksia, sillä proteiinifosfataasi 2A:n toiminnan häiriöt on yhdistetty mm. Parkinsonin ja Alzheimerin tauteihin sekä useisiin syöpiin. Väitöstutkimus osoitti selvästi, että PREP ja PREP-estäjät ovat lupaavia lääkekehityskohteita Parkinsonin tautiin, mutta lisäksi projektissa paljastui, että PREP:illä on potentiaalia myös usean muun sairauden lääkekehityskohteena

Prolyl Oligopeptidase Regulates Dopamine Transporter Oligomerization and Phosphorylation in a PKC- and ERK-Independent Manner

Prolyl oligopeptidase (PREP) is a serine protease that binds to alpha-synuclein (aSyn) and induces its aggregation. PREP inhibitors have been shown to have beneficial effects in Parkinson's disease models by enhancing the clearance of aSyn aggregates and modulating striatal dopamine. Additionally, we have shown that PREP regulates phosphorylation and internalization of dopamine transporter (DAT) in mice. In this study, we clarified the mechanism behind this by using HEK-293 and PREP knock-out HEK-293 cells with DAT transfection. We tested the effects of PREP, PREP inhibition, and alpha-synuclein on PREP-related DAT regulation by using Western blot analysis and a dopamine uptake assay, and characterized the impact of PREP on protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) by using PKC assay and Western blot, respectively, as these kinases regulate DAT phosphorylation. Our results confirmed our previous findings that a lack of PREP can increase phosphorylation and internalization of DAT and decrease uptake of dopamine. PREP inhibition had a variable impact on phosphorylation of ERK dependent on the metabolic state of cells, but did not have an effect on phosphorylation or function of DAT. PREP modifications did not affect PKC activity either. Additionally, a lack of PREP elevated a DAT oligomerization that is associated with intracellular trafficking of DAT. Our results suggest that PREP-mediated phosphorylation, oligomerization, and internalization of DAT is not dependent on PKC or ERK.Peer reviewe