Prolyl oligopeptidase and alpha-synuclein in the regulation of nigrostriatal dopaminergic neurotransmission

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http://urn.fi/URN:ISBN:978-951-51-5243-5
Title: Prolyl oligopeptidase and alpha-synuclein in the regulation of nigrostriatal dopaminergic neurotransmission
Author: Julku, Ulrika
Contributor: University of Helsinki, Faculty of Pharmacy, Division of Pharmacology and Pharmacotherapy
Doctoral Programme in Drug Research
Publisher: Helsingin yliopisto
Date: 2019-05-31
Belongs to series: URN:ISSN:2342-317X
URI: http://urn.fi/URN:ISBN:978-951-51-5243-5
http://hdl.handle.net/10138/301607
Thesis level: Doctoral dissertation (article-based)
Abstract: Dopamine is one of the main neurotransmitters in the brain. Dopaminergic signalling regulates reward, memory, attention and motor functions. In the synapses of dopaminergic neurons, dopamine transporter (DAT) re-uptakes dopamine into the presynaptic nerve terminals after dopamine release terminating the dopaminergic signal and acting as one of the main regulators for kinetics of dopaminergic neurotransmission. Loss of dopaminergic neurons in the nigrostriatal pathway and protein aggregates called Lewy bodies are the main pathological findings in Parkinson´s disease. Lewy bodies are mainly composed of a protein called α-synuclein. The physiological role of α-synuclein has remained unclear but it has been suggested that the main function is regulation of dopaminergic neurotransmission since α-synuclein has been shown to participate in the regulation of dopamine synthesis, storage, release, and metabolism. α-synuclein-regulated functions in dopaminergic signaling are described in the literature review of this thesis. Prolyl oligopeptidase (PREP) is a serine protease that binds to α-synuclein and induces its aggregation. PREP inhibitors have beneficial effects in cellular and in vivo models of Parkinson´s disease by reducing α-synuclein aggregates and oligomers, and improving motor functions. Additionally, PREP inhibitors alter striatal dopamine level in mice and rats, and decrease immunoreactive DAT in the mouse striatum suggesting that PREP could have an effect on dopaminergic function. The aim of this study was to characterize the role of PREP in dopaminergic signaling and the effect of α-synuclein in PREP-mediated changes of the dopaminergic system. In the first study, the effect of PREP and α-synuclein on DAT phosphorylation and function was studied in DAT transfected HEK-293 cells. PREP altered DAT function and dopamine uptake, but the changes were not dependent on ERK phosphorylation or PKC activity. α-synclein had an effect on DAT phosphorylation in the absence of PREP but this was also independent of phosphorylation of ERK indicating that both α-synuclein and PREP are able to modulate DAT function via an ERK¬-independent mechanism. In the second study, the role of PREP in dopaminergic signaling was characterized in the nigrostriatal pathway of mouse. The influence of PREP was investigated by comparing the dopaminergic function of PREP knock-out mice and wild-type littermates. Lack of PREP elevated extracellular dopamine concentration, delayed re-uptake of dopamine, and increased phosphorylation of DAT in the mouse striatum indicating that PREP is able to regulate DAT function by modulating phosphorylation and localization of DAT. The effect of PREP inhibition on dopaminergic function, behavior, and α-synuclein in a Parkinson´s disease mouse model was investigated in the third study. Overexpression of α-synuclein was induced by supranigral microinjection of AAV-α-synuclein and mice were treated with the PREP inhibitor KYP-2047 after the onset of the behavioral symptoms. KYP-2047 treatment did not restore α-synuclein-induced reduction in striatal dopamine but behavioral improvement and reduction in α-synuclein oligomers indicated restoration of dopamine release and recycling. The aim of the fourth study was to investigate if α-synuclein-induced toxicity in the nigrostriatal pathway is dependent on PREP expression. The main finding was that α-synuclein toxicity was reduced in the absence of PREP and restoration of PREP expression increased toxicity in the behavioral tests. However, nigrostriatal dopamine level was not affected suggesting that lack of PREP protects dopamine release and recycling from α-synuclein-induced toxicity. In conclusion, PREP regulates DAT function in cells and in the mouse nigrostriatal pathway, but the mechanism is not dependent on ERK and PKC activation. Deletion of PREP or PREP inhibition do not have effects on α-synuclein-induced dopaminergic cell loss, but they are able to restore behavior and dopaminergic function in the mouse brain suggesting that PREP inhibitors could provide a novel treatment for Parkinson´s disease.Dopamiini kuuluu aivojen tärkeimpiin hermovälittäjäaineisiin, ja se säätelee mm. tahdonalaisia liikkeitä ja mielihyvää. Parkinsonin taudissa dopamiinihermosolut tuhoutuvat nigrostriataaliradaksi kutsutulla aivoalueella, mikä aiheuttaa vapinaa ja liikehäiriöitä. Nykyisillä hoitomenetelmillä, jotka perustuvat aivojen dopamiinin korvaamiseen, voidaan lievittää taudin oireita, mutta ei hidastaa tai pysäyttää sairauden etenemistä. Terveissä aivoissa α-synukleiiniksi kutsuttu proteiini osallistuu dopamiinin muodostumisen, varastoitumisen, vapautumisen ja hajoamisen säätelyyn, mutta Parkinsonin tautia sairastavien aivoissa α-synukleiinista muodostuu proteiinikertymiä, jotka ovat haitallisia solujen normaalille toiminnalle. Prolyylioligopeptidaasi (PREP) on entsyymi, joka lisää α-synukleiinikertymien muodostumista, ja aiemmissa tutkimuksissa on selvinnyt, että PREP-entsyymin toimintaa estämällä voidaan vähentää α-synukleiinikertymiä Parkinsonin taudin solu- ja eläinmalleissa. PREP-estäjien on myös havaittu aiheuttavan muutoksia dopamiinijärjestelmän toimintaan aivojen nigrostriataaliradassa hiirillä ja rotilla. Tämän väitöskirjatyön tarkoituksena oli tutkia tarkemmin PREP:n roolia aivojen nigrostriataaliradan dopamiinijärjestelmässä. Lisäksi tutkittiin, että voidaanko PREP-entsyymin toimintaa estämällä korjata α-synukleiinin aiheuttamia hermosoluvaurioita Parkinsonin taudin hiirimallissa ja suojaako PREP:n poistaminen hiiriä α-synukleiinin vahingollisilta vaikutuksilta. Tärkeimpänä löydöksenä oli PREP:n osallistuminen dopamiininkuljettajaproteiinin toiminnan säätelyyn sekä soluissa että hiiren aivoissa. Solukokeet kuitenkin osoittivat, että PREP ei säätele dopamiininkuljettajaproteiinin toimintaa sen keskeisimpänä pidettyjen säätelijöiden kautta. Parkinsonin taudin hiirimallissa PREP-estäjällä saatiin korjattua käytösmuutoksia ja dopamiinijärjestelmän toimintaa, vaikka hoidolla ei ollutkaan vaikutusta aivojen dopamiinisolutuhoon. Lisäksi havaittiin, että PREP:n puuttuminen vähensi α-synukleiinin haitallisia vaikutuksia hiirillä. Yhteenvetona näiden löydösten perusteella voidaan todeta, että PREP osallistuu aivojen dopamiinijärjestelmän säätelyyn, ja että PREP-estäjät ovat lupaava uusi keino Parkinsonin taudin hoitoon.
Subject: Farmasia
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