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

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

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 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 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 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

Removal of proteinase K resistant alpha Syn species does not correlate with cell survival in a virus vector-based Parkinson's disease mouse model

Parkinson's disease (PD) is characterized by degeneration of nigrostriatal dopaminergic neurons and accumu-lation of alpha-synuclein (alpha Syn) as Lewy bodies. Currently, there is no disease-modifying therapy available for PD. We have shown that a small molecular inhibitor for prolyl oligopeptidase (PREP), KYP-2047, relieves alpha Syn-induced toxicity in various PD models by inducing autophagy and preventing alpha Syn aggregation. In this study, we wanted to study the effects of PREP inhibition on different alpha Syn species by using cell culture and in vivo models.We used Neuro2A cells with transient alpha Syn overexpression and oxidative stress or proteasomal inhibition -induced alpha Syn aggregation to assess the effect of KYP-2047 on soluble alpha Syn oligomers and on cell viability. Here, the levels of soluble alpha Syn were measured by using ELISA, and the impact of KYP-2047 was compared to anle138b, nilotinib and deferiprone. To evaluate the effect of KYP-2047 on alpha Syn fibrillization in vivo, we used unilateral nigral AAV1/2-A53T-alpha Syn mouse model, where the KYP-2047 treatment was initiated two-or four -weeks post injection.KYP-2047 and anle138b protected cells from alpha Syn toxicity but interestingly, KYP-2047 did not reduce soluble alpha Syn oligomers. In AAV-A53T-alpha Syn mouse model, KYP-2047 reduced significantly proteinase K-resistant alpha Syn oligomers and oxidative damage related to alpha Syn aggregation. However, the KYP-2047 treatment that was initiated at the time of symptom onset, failed to protect the nigrostriatal dopaminergic neurons. Our results emphasize the importance of whole alpha Syn aggregation process in the pathology of PD and raise an important question about the forms of alpha Syn that are reasonable targets for PD drug therapy.Peer reviewe

Combination of CDNF and Deep Brain Stimulation Decreases Neurological Deficits in Late-stage Model Parkinson's Disease

Several neurotrophic factors ( NTF) are shown to be neuroprotective and neurorestorative in pre-clinical animal models for Parkinson's disease ( PD), particularly in models where striatal dopamine neuron innervation partially exists. The results of clinical trials on late-stage patients have been modest. Subthalamic deep brain stimulation ( STN DBS) is a proven treatment for a selected group of advanced PD patients. The cerebral dopamine neurotrophic factor ( CDNF) is a promising therapeutic protein, but its effects in animal models of late-stage PD have remained under-researched. The interactions of NTF and STN DBS treatments have not been studied before. We found that a nigral CDNF protein alone had only a marginal effect on the behavioral deficits in a late-stage hemiparkinsonian rat model ( 6-OHDA MFB). However, CDNF improved the effect of acute STN DBS on front limb use asymmetry at 2 and 3 weeks after CDNF injection. STN lesion-modeling chronic stimulation-had an additive effect in reducing front limb use in the cylinder test and apomorphine-induced rotation. The combination of CDNF and acute STN DBS had a favorable effect on striatal tyrosine hydroxylase. This study presents a novel additive beneficial effect of NTF and STN DBS, which might be explained by the interaction of DBS-induced endogenous NTFs and exogenously injected CDNF. SNpc can be reached via similar trajectories used in clinical STN DBS, and this interaction is an important area for future studies. (C) 2018 The Authors. Published by Elsevier Ltd on behalf of IBRO. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/).Peer reviewe

Säädelty geeniterapia Parkinsonin taudin hoidossa

Parkinson's disease is a progressive neurodegenerative disease. The incidence of the disease is 1.5-2 per cent after age 60. Typical symptoms are tremor, rigidity and bradykinesia. At the late stage of the disease patients have psychic disorders, for example dementia, anxiety and depression. Motor impairment is caused by degenerative loss of dopamine cells in nigrostriatal tract. Current treatment of the disease relieves the symptoms but it cannot stop or slow the progress of the disease. Neurotrophic factors and gene therapy have been trialled to improve the treatment of Parkinson's disease and the results have been encouraging. Neurotrophic factors are proteins that regulate actions of neurons. It has been discovered that they are neuroprotective and neurorestorative. The results with glial cell line-derived neurotrophic factor (GDNF) have been encouraging in in vivo studies of Parkinson's disease. There has been variability in success of clinical trials though. GDNF degrades quickly in vivo but overexpression of GDNF in cells can be produced with viral vector adeno-associated virus. Two different forms of GDNF, pre-α-pro-GDNF (α-GDNF) and pre-β-pro-GDNF (β-GDNF), are produced as precursors and they are activated proteolytically. Based on in vitro studies, some differences in secretion of precursors have been discovered. α-GDNF is secreted constitutively and secretion of β-GDNF is dependent on physiological stimulation. Previous in vitro studies have focused on α-GDNF, but β-GDNF might be a better solution for treating Parkinson's disease based on physiological regulation system. Cerebral dopamine neurotrophic factor (CDNF) is recently discovered and less studied than GDNF. It has been discovered that CDNF also has neuroprotective and neurorestorative effects in animal models of Parkinson's disease. The aim of the first part of this study was to discover the neurorestorative effect of single injection of CDNF injected above substantia nigra for rats that received injection of 6-hydroxydopamine (6-OHDA) into medial forebrain bundle. One week later rats received PBS, GDNF or CDNF injection. The degree of the lesion was estimated with apomorphine (0.1 mg/kg s.c.) or d-amphetamine sulphate (2.5 mg/kg) induced rotation test. The rats were perfused nine weeks post-lesion and their brains were sliced. Tyrosine hydroxylase (TH) positive dopamine cells were stained by immunohistochemistry. The amount of TH positive cells in substantia nigra was counted and optical density of TH positive fibres in striatum was measured. The aim of the second part of the study was to research the neuroprotective effect of two different precursors of GDNF, dsAAV1-pre-α-pro-GDNF and dsAAV1-pre-β-pro-GDNF, given with viral vectors. The dopamine cells in nigrostriatal tract were destroyed with a 6-OHDA injection into striatum and viral vectors were injected two weeks later. Rats in control group received injection of dsAAV1-GFP. The degree of the lesion was evaluated with d-amphetamine sulphate (2.5 mg/kg) induced rotation tests and cylinder test. The rats were perfused eight weeks post-lesion and their brains were processed for immunohistochemistry. The results of the study were interesting and supporting previous studies. The success of the neurotrophic factor treatment is dependent on a successful injection of protein or viral vector, and the dose is dependent on the size of the lesion. Neurotrophic factors and gene therapy needs to be studied more before wide clinical usage.Parkinsonin tauti on etenevä aivoja rappeuttava sairaus, johon sairastuu noin 1,5-2 % yli 60-vuotiaista. Parkinsonin taudin oireita ovat heikentynyt lihasvoima, lihasjäykkyys, lepovapina ja liikkeiden aloittamisen vaikeus. Edetessään tauti voi aiheuttaa motoristen oireiden lisäksi myös psyykkisiä oireita, kuten muistihäiriöitä, ahdistusta ja masennusta. Taudin motoriset oireet johtuvat dopamiinisolujen tuhoutumisesta aivojen nigrostriataalisessa radassa. Nykyisillä lääkkeillä voidaan lievittää Parkinsonin taudin oireita, mutta taudin etenemistä ei voida hidastaa tai pysäyttää. Parkinsonin taudin hoidon kehittämiseksi on tutkittu hermokasvutekijöitä ja geeniterapiaa, joilla on saatu lupaavia tuloksia. Hermokasvutekijät ovat hermosolujen toimintaa sääteleviä proteiineja, joilla on havaittu solujen toimintaa suojaavia ja palauttavia vaikutuksia. Gliasolulinjaperäisellä hermokasvutekijällä (engl. glial cell line -derived neurotrophic factor, GDNF) on saatu lupaavia tuloksia Parkinsonin taudin eläinmalleissa. Kliinisissä kokeissa GDNF-hoidon tulokset ovat kuitenkin olleet vaihtelevia. GDNF hajoaa elimistössä nopeasti, mutta solut voidaan saada pitkäaikaisesti tuottamaan ylimäärin GDNF:ää käyttämällä virusvektoreita, kuten AAV-virusta (engl. adeno-associated virus). GDNF:n kaksi erilaista muotoa, pre-α-pro-GDNF (α-GDNF) ja pre-β-pro-GDNF (β-GDNF), tuotetaan soluissa esiasteina, jotka aktivoidaan proteolyyttisesti. Soluilla tehtyjen kokeiden perusteella nämä kaksi muotoa eroavat erityksensä suhteen. α-muotoa erittyy hermosoluista jatkuvasti ja β-muoto vapautuu hermosoluista fysiologisen stimulaation seurauksena. Aiemmat in vivo -tutkimukset ovat keskittyneet pääosin α-muotoon, mutta β-muoto saattaisi soveltua Parkinsonin taudin hoitoon paremmin kuin α- muoto, koska sen erityksen säätely on fysiologista. Dopamiinisolujen hermokasvutekijä (engl. cerebral dopamine neurotrophic factor, CDNF) on uudempi ja siten vähemmän tutkittu hermokasvutekijä kuin GDNF. Myös CDNF:llä on havaittu dopaminergisiä hermosoluja suojaavia ja palauttavia vaikutuksia Parkinsonin taudin in vivo -malleissa. Tutkimuksen ensimmäisen osan tarkoituksena oli tutkia keskiaivojen mustatumakkeen yläpuolelle annetun CDNF-proteiini-injektion dopamiinihermosoluja palauttavaa vaikutusta rotilla, joiden nigrostriataaliset dopamiinisolut tuhottiin 6-hydroksidopamiini-injektiolla (6-OHDA) mediaaliseen etuaivojuosteeseen (engl. medial forebrain bundle, MFB). Viikko tämän jälkeen rotille annettiin CDNF-, GDNF- tai PBS-injektio. Solutuhon laajuutta ja kehitystä arvioitiin apomorfiinilla (0,1 mg/kg s.c.) ja d-amfetamiinisulfaatilla (2,5 mg/kg s.c.) indusoitujen pyörityskokeiden avulla. Rotat perfusoitiin yhdeksän viikon kuluttua 6-OHDA-injektiosta ja niiden aivoista tehtiin leikkeitä. Tyrosiinihydroksylaasia (TH) sisältävät dopamiinihermosolut värjättiin immunohistokemiallisella menetelmällä. Aivoleikkeistä laskettiin mustatumakkeiden TH-positiiviset solut ja määritettiin TH-positiivisten solujen optinen tiheys aivojuovioissa. Tutkimuksen toisen osan tarkoituksena oli tutkia virusvektorin avulla aivojuovioon annetun GDNF:n esiasteen, dsAAV1-pre-α-pro-GDNF:n ja dsAAV1-pre-β-pro-GDNF:n, suojaavaa vaikutusta. Tässä kokeessa nigrostriataaliset dopamiinisolut tuhottiin injektoimalla 6-OHDA aivojuovioon ja virusvektorit injektoitiin kaksi viikkoa 6-OHDA:n jälkeen. Kontrolliryhmän rotilleannettiin dsAAV1-GFP-injektio. Solutuhon laajuutta ja kehitystä arvioitiin d-amfetamiinisulfaatilla (2,5 mg/kg s.c.) indusoitujen pyörityskokeiden sekä sylinterikokeen avulla. Rotat perfusoitiin kahdeksan viikon kuluttua 6-OHDA-injektiosta ja niiden aivot otettiin talteen immunohistokemian tutkimuksia varten. Tutkimuksen tulokset olivat mielenkiintoa herättäviä ja tukevat aiempia tutkimustuloksia. Hermokasvutekijähoidon onnistuminen riippuu suurelta osin proteiini- ja virusinjektion onnistumisesta ja hoidon annos ja teho on riippuvainen dopamiinivajeen suuruudesta. Hermokasvutekijöitä ja geeniterapiaa täytyy vielä tutkia lisää ennen kuin niitä voidaan ottaa laajaan kliiniseen käyttöön Parkinsonin taudin hoidossa