Repeated Ethanol Exposure Alters DNA Methylation Status and Dynorphin/Kappa-Opioid Receptor Expression in Nucleus Accumbens of Alcohol-Preferring AA Rats

Growing evidence suggests that epigenetic mechanisms, such as DNA methylation and demethylation, and histone modifications, are involved in the development of alcohol and drug addiction. However, studies of alcohol use disorder (AUD) that are focused on epigenetic DNA modifications and gene expression changes remain conflicting. Our aim was to study the effect of repeated ethanol consumption on epigenetic regulatory enzymes such as DNA methyltransferase and demethylase enzymes and whether those changes affected dynorphin/kappa-opioid receptor system in the Nucleus Accumbens (NAc). Two groups of male alcohol-preferring Alko Alcohol (AA) rats, rats which are selectively bred for high voluntary alcohol consumption and one group of male Wistar rats were used. The first group of AA rats had access to alcohol (10% ethanol solution) for 90 min on Mondays, Wednesdays and Fridays over a period of 3 weeks to establish a stable baseline of ethanol intake (AA-ethanol). The second group of AA rats (AA-water) and the Wistar rats (Wistar-water) were provided with water. Using qPCR, we found that voluntary alcohol drinking increased Dnmt1, -3a, and -3b mRNA levels and did not affect Tet family transcripts in the AA-ethanol group when compared with AA- and Wistar-water rats. DNMT and TET enzymatic activity measurements showed similar results to qPCR, where DNMT activity was increased in AA-ethanol group compared with AA-water and Wistar-water groups, with no statistically significant difference between groups in TET enzyme activity. In line with previous data, we found an increased percentage of global DNA methylation and hydroxymethylation in the AA-ethanol group compared with control rats. Finally, we investigated changes of selected candidate genes from dynorphin/kappa-opioid receptor system (Pdyn, Kor) and Dnmt3a genes that might be important in AUD-related behaviour. Our gene expression and promoter methylation analysis revealed a significant increase in the mRNA levels of Pdyn, Kor, and Dnmt3a in the AA-ethanol group, however, these changes can only be partially associate with the aberrant DNA methylation in promoter areas of the selected candidate genes. Thus, our findings suggest that the aberrant DNA methylation is rather one of the several mechanisms involved in gene expression regulation in AA rat model.Peer reviewe

Tetrazole as a Replacement of the Electrophilic Group in Characteristic Prolyl Oligopeptidase Inhibitors

4-Phenylbutanoyl-aminoacyl-2(S)-tetrazolylpyrrolidines were studied as prolyl oligopeptidase inhibitors. The compounds were more potent than expected from the assumption that the tetrazole would also here be a bioisostere of the carboxylic acid group and the corresponding carboxylic acids are at their best only weak inhibitors. The aminoacyl groups L-prolyl and L-alanyl gave potent inhibitors with IC50 values of 12 and 129 nM, respectively. This was in line with typical prolyl oligopeptidase inhibitors; however, we did observe a difference with N-methyl-L-alanyl, which gave potent inhibitors in typical prolyl oligopeptidase inhibitors but not in our novel compound series. Furthermore, all studied 4-phenylbutanoyl-aminoacyl-2(S)-tetrazolylpyrrolidines decreased alpha-synuclein dimerization at the concentration of 10 mu M, also when they were only weak inhibitors of the proteolytic activity of the enzyme with an IC50 value of 205 mu M. Molecular docking studies revealed that the compounds are likely to bind differently to the enzyme compared to typical prolyl oligopeptidase inhibitors represented in this study by 4-phenylbutanoyl-aminoacyl-2(S)-cyanopyrrolidines.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

Prolyylioligopeptidaasi-inhibition vaikutus proteiiniaggregaatioon sekä hapetusstressiin hermorappeumasairauksien malleissa

Neurodegenerative diseases are a major public health problem for aging population around the world. Currently there are no effective treatment available that could prevent, slow, or halt the progression of these diseases. Although there are various neurodegenerative diseases with differing clinical and histopathological presentations, most of them share several common toxic features, such as protein aggregation and accumulation, dysfunctional protein clearance mechanisms, and oxidative stress (OS). Prolyl oligopeptidase (PREP) is a serine protease that participates in neurodegeneration by regulating various proteins via direct protein-protein interactions. PREP induces alpha-synuclein (αSyn) aggregation and reduces autophagy-mediated protein clearance in various Parkinson’s disease models, whereas its inhibition by small molecules has been able to revert these harmful effects. It has also been reported that PREP inhibition has beneficial effect on OS. Furthermore, PREP inhibition induces autophagy by activating protein phosphatase 2A (PP2A) that is also essential in regulation of the microtubule-stabilizing protein Tau, suggesting potential beneficial impacts also on Tau-related neurodegeneration. In this project, we established the role of PREP in cellular redox-regulation and the ability of PREP inhibition to reduce production of reactive oxygen species (ROS) in cellular models. We also demonstrated that PREP inhibition reduced ROS production by decreasing activity of NADPH oxidase via PP2A activation. Moreover, we could reduce OS in an AAV-A53T-αSyn-based mouse model for Parkinson’s disease as well as in human mutant P301S-Tau transgenic (PS19) mice. In addition, chronic PREP inhibitor treatment reduced αSyn levels, particularly large, proteinase K-resistant aggregates in the brains of AAV-A53T-αSyn injected mice, while enhancing autophagy, and reducing OS. However, this did not protect the nigrostriatal dopaminergic neurons, indicating that the rapidly oligomerizing A53T-αSyn produced toxic oligomers in such a fast rate that beneficial effect by PREP inhibition were not able to keep up. According to these results, soluble αSyn oligomers are responsible of the toxicity in Parkinson’s disease. Finally, we demonstrated by a multi-model approach, from cells to animals, that PREP inhibition reduces Tau phosphorylation, aggregation, and accumulation by activating PP2A and enhancing autophagy. In addition, chronic PREP inhibition prevented deterioration of cognitive skills in the PS19 mice. The results of this project together with previous findings suggest that PREP inhibition could provide a potential and safe multi-target approach to tackle several common toxic features of neurodegeneration, even beyond αSyn-related indications.Hermorappeumasairaudet ovat merkittävä kansanterveysongelma maailman ikääntyvälle väestölle, eikä näiden sairauksien kulkuun vaikuttavia tehokkaita hoitoja ole vielä saatavilla. Vaikka hermorappeumasairaudet ovat moninainen ryhmä erilaisia sairauksia, joilla on erilaisia kliinisiä sekä histopatologisia löydöksiä, jakavat nämä myös lukuisia yhteisiä haitallisia mekanismeja. Näitä mekanismeja ovat mm. proteiinien aggregaatio ja kertyminen, häiriintyneet proteiinien puhdistusmekanismit sekä hapetusstressi. Prolyylioligopeptidaasi (PREP) on seriiniproteaasi, jonka on osoitettu osallistuvan hermorappeumasairauksiin säätelemällä lukuisia proteiineja suorien proteiini-proteiini interaktioiden välityksellä. PREP lisää alfa-synukleiinin (αSyn) aggregaatiota ja vähentää autofagia-välitteistä proteiinien hajotusta Parkinsonin taudin malleissa, kun taas pienmolekyylisen PREP estäjän on osoitettu kumoavan nämä haitalliset vaikutukset. PREP:n estolla on myös raportoitu olevan hyödyllisiä vaikutuksia hapetusstressiin. Lisäksi PREP esto tehostaa autofagiaa aktivoimalla proteiinifosfataasi 2A:ta (PP2A), jonka tiedetään olevan tärkeä mikrotubuluksia stabiloivan Tau-proteiinin säätelijä. Tämän perusteella PREP esto voisi osoittautua hyödylliseksi myös Tau-proteiiniin liittyvissä hermorappeumasairauksissa. Tässä projektissa osoitimme PREP:n osuuden solujen hapetus-pelkistystasapainon säätelijänä, ja sen, että PREP:n esto vähensi happiradikaalien tuotantoa vähentämällä NADPH-oksidaasin aktiivisuutta PP2A-välitteisesti. PREP:n esto vähensi hapetusstressiä myös Parkinsonin taudin AAV-A53T-αSyn hiirimallissa sekä P301S-Tau siirtogeenisissä hiirissä. AAV-A53T-αSyn injektoitujen hiirien aivoissa PREP eston vaikutus näkyi eritysesti suurissa proteinaasi K -käsittelyä kestävissä aggregaateissa. Tämä ei kuitenkaan suojannut nigrostriataalisia dopaminergisiä hermosoluja, mikä todennäköisimmin johtui A53T-αSyn:n nopeasta kyvystä tuottaa toksisia oligomeerejä, jolloin PREP eston hyödylliset vaikutukset tulivat liian myöhään. Tämä havainto vahvisti liukoisten αSyn oligomeerien merkitystä Parkinsonin taudin patofysiologiassa. Tämän lisäksi osoitimme soluista aina eläinmalleihin asti, että PREP estolla kyetään vähentämään Tau-proteiinin fosforylaatiota, aggregaatiota ja kertymistä PP2A aktivaation ja autofagian tehostamisen välityksellä. Lisäksi pitkäkestoinen PREP:n esto ehkäisi kognitiivisten taitojen rappeutumista Tau-siirtogeenisessä hiirimallissa. Yhdessä tämän projektin tulokset sekä aikaisemmat löydökset viittaavat siihen, että PREP:n esto voisi tarjota potentiaalisen ja turvallisen monivaikutteisen lähestymistavan, jolla hoidon vaikutuksen voisi kohdentaa useaan hermorappeumasairauksissa esiintyvään yleiseen toksiseen mekanismiin, myös αSyn-indikaation ulkopuolella

κ-Opioidireseptoriantagonistien vaikutus rottien alkoholin juomisen retkahtamisalttiuteen

lcohol addiction is a significant public health problem worldwide, and its treatment is extremely challenging. One major problem in the treatment of alcohol addiction are the later relapses to uncontrollable drinking. Approximately 60-70 % of addicts relapse to drinking within a year from the beginning of the treatment. The current treatment of alcohol addiction is based on a combined psychotheraphy and pharmacological treatments, but even at the best the efficacy remains quite modest. This is why further studies on the underlying mechanisms behind alcohol addiction and development of more effective pharmaceuticals to treat it are an important field of research. Chronic exposure to the rewarding effects of alcohol causes neurochemical adaptations in the brain reward system. These adaptations strive to restrain the recurring rewarding signals caused by alcohol and lead eventually to increased reward thresholds in the reward system. As the reward thresholds increase, the individual develops tolerance to the rewarding effects of alcohol, but also craving for the substance and a dysphoric mental state which are highlighted especially during periods of abstinence. It is known that the increase in reward thresholds is an important factor leading to relapses, but the exact nature of the neurochemical adaptations behind it are not known. According to recent studies dynorphin -peptides (DYN) and κ-opioid receptors (KOR) of the endogenous opioid system seem to have an important role in these neurochemical adaptations. It has been shown that chronic alcohol exposure increases the activity of DYN/ KOR -system especially within the nucleus accumbens (NAc), which is an essential structure of the brain reward system. The increased activity of the DYN/ KOR -system in the NAc has been shown to inhibit the development of rewarding signals. Previous studies have shown that inhibiting the increased activity of the DYN/ KOR -system with a selective KOR-antagonist, reduces voluntary alcohol intake and relapse-like alcohol seeking behavior during periods of abstinence, especially in physically addicted animals. In this study we studied the relapse-like alcohol drinking of Long-Evans rats in the alcohol deprivation effect (ADE) model. The effects of selective and long-acting KOR-antagonists, JDTic and nor-BNI, were tested on the ADE-effect which occurs after a period of deprivation. The ADE is defined as a transient increase in alcohol intake after a forced period of abstinence and it has been shown both in rodents with a history of alcohol consumption, and human alcohol addicts. In this study the rats were allowed to consume alcohol (10% ethanol-water solution) voluntarily during 90 minutes for 10 consecutive days after which followed a six days long deprivation period. According to results, both intra-accumbally (15 µg/ 0,3 µl/min/ site) or subcutaneously (10 mg/kg) administered JDTic decreased the ADEeffect significantly compared to vehicle, when administered 24 hours prior the end of the deprivation period. Also intra-accumbally administered nor-BNI (3 µg/0,3 µl/min/site) decreased the ADE-effect significantly compared to vehicle when administered 24 hour prior the end of deprivation. The results are in line with the theory that alcohol induces sensitization of the DYN/ KOR -system within the brain structures involved in reward. In theory it can be speculated that by suppressing the activity of the DYN/ KOR -system, KOR-antagonists can relieve craving for alcohol. This can be seen as a decrease in relapse-like consumption of alcohol. In conclusion, it can be suggested that by suppressing the increased activity of the DYN/ KOR -system induced by chronic alcohol exposure with a selective KOR-antagonist, like JDTic or nor-BNI, it could be possible to reduce the risk of relapse during abstinence and thus improve the efficacy of treatments for alcohol addiction.Alkoholiriippuvuus on vaikeahoitoinen sairaus, joka on merkittävä maailmanlaajuinen kansanterveysongelma. Alkoholiriippuvuuden hoidon suurin ongelma ovat myöhemmät hallitsemattomaan juomiseen retkahtamiset. Arvioiden mukaan jopa noin 60-70 % hoitoon hakeutuneista potilaista retkahtaa vuoden sisällä hoidon aloituksesta. Nykyinen alkoholiriippuvuuden hoito perustuu yhdistettyyn terapiamuotoiseen ja lääkkeillä toteutettuun hoitoon, mutta parhaimmillaankin sen vaikutukset ovat vain kohtalaisia. Tästä syystä alkoholiriippuvuuden taustalla olevien mekanismien tarkempi selvittäminen ja tehokkaampien alkoholiriippuvuuden lääkehoitojen kehittäminen on tärkeää. Pitkäaikainen altistuminen alkoholin palkitseville vaikutuksille saa aivojen palkitsemisjärjestelmässä aikaan neurokemiallisia muutoksia, jotka pyrkivät vastustamaan alkoholin aiheuttamia toistuvia palkitsemisvasteita. Nämä neurokemialliset muutokset saavat aikaan palkitsemisvasteen kynnysarvon kohoamisen, jonka myötä yksilölle kehittyy toleranssi alkoholin palkitsevia vaikutuksia kohtaan sekä alkoholin himo ja aversiivinen henkinen olotila, jotka ovat erityisen korostuneita silloin, kun alkoholia ei ole saatavilla. Palkitsemisvasteen kynnysarvon kohoaminen on merkittävä vieroitushoidon aikaisten retkahdusten taustatekijä, mutta siihen johtavien neurokemiallisten muutosten täydellistä luonnetta ei vielä tunneta. On kuitenkin havaittu, että endogeeniseen opioidijärjestelmään kuuluvat dynorfiinipeptidit (DYN) ja κ-opioidireseptorit (KOR) ovat merkittävässä osassa näissä muutoksissa. DYN/ KOR - järjestelmän aktiivisuuden on osoitettu lisääntyvän pitkäaikaisen alkoholialtistuksen seurauksena etenkin palkitsemisjärjestelmän keskeisessä rakenteessa, akkumbens-tumakkeessa (NAc), jossa se hillitsee palkitsemisvasteiden syntyä. Aiemmat tutkimukset ovat osoittaneet, että DYN/ KOR -järjestelmän lisääntyneen aktiivisuuden hillitseminen selektiivisellä KOR-antagonistilla vähentää alkoholin vapaaehtoista kulutusta sekä vieroitusjakson aikaista alkoholihakuisuutta etenkin fyysisesti alkoholiriippuvaisilla eläimillä. Tässä tutkimuksessa tutkittiin Long-Evans -rottien alkoholin juomiseen retkahtamista alkoholideprivaatioeläinmallissa (ADE-malli). Kokeessa selvitettiin selektiivisten ja pitkävaikutteisten KOR-antagonistien, JDTic:n ja nor-BNI:n, vaikutuksia retkahtamista kuvaavaan ADE-ilmiöön, joka mallintaa vieroitusjakson jälkeistä hetkellistä alkoholin kulutuksen nousua vieroitusta edeltäneeseen juomistasoon nähden. ADE-ilmiön on havaittu esiintyvän niin alkoholia kuluttaneilla jyrsijöillä kuin alkoholiriippuvaisilla ihmisillä. Kokeessa rotat saivat juoda vapaaehtoisesti alkoholia (10 % etanoli-vesi -liuos) 90 min ajan 10 peräkkäisenä päivänä, jonka jälkeen ne siirtyivät kuuden vuorokauden mittaiselle deprivaatiojaksolle. Tulosten perusteella JDTic vähensi vehikkeliin verrattuna merkitsevästi rottien ADE-ilmiötä annosteltuna niin akkumbaalisesti (15 µg/ 0,3 µl/min/aivopuoli) kuin systeemisesti (10 mg/kg) 24 h ennen deprivaatiojakson loppumista. Myös 24 h ennen deprivaatiojakson päättymistä akkumbaalisesti annosteltu nor-BNI (3 µg/0,3 µl/min/aivopuoli) vähensi vehikkeliin verrattuna merkitsevästi ADE-ilmiötä. Tulokset viittaavat alkoholin aikaansaamaan palkitsemisjärjestelmän sisäisen DYN/ KOR -järjestelmän herkistymiseen, sillä teoriassa kokeessa käytetyt KOR-antagonistit ovat vähentäneet kyseisen järjestelmän aktiivisuutta ja näin helpottaneet alkoholiin kohdistuvaa himoa, joka näkyi pienentyneenä retkahdusta kuvaavana ADE-ilmiönä. Johtopäätöksenä voidaan todeta, että pitkäaikaisen alkoholialtistuksen aikaansaaman DYN/ KOR -järjestelmän lisääntyneen aktiivisuuden hillitseminen selektiivisellä KOR-antagonistilla, kuten JDTic:llä tai nor-BNI:llä, voitaisiin todennäköisesti hillitä vieroitushoidon aikaista retkahtamisriskiä ja näin parantaa alkoholiriippuvuuden hoidon tehoa

The selective κ-opioid receptor antagonist JDTic attenuates the alcohol deprivation effect in rats

The mechanisms behind relapse to ethanol intake in recovering alcoholics are still unclear. The negative reinforcing effects contributing to ethanol addiction, including relapse, are considered to be partly driven by the kappa-opioidergic system. As the kappa-opioidergic system interacts with the mesolimbic reward pathway, the aim of the study was to clarify the role of nucleus accumbens shell kappa-opioidergic mechanisms in relapse to ethanol intake by using the alcohol deprivation effect (ADE) paradigm. The ADE is defined as a transient increase in voluntary ethanol intake after a forced period of abstinence. Male Long-Evans rats were trained to voluntarily consume 10% (v/v) ethanol solution. Ethanol access and deprivation cycles were initiated after stable ethanol intake baselines had been reached and bilateral guide cannulas had been implanted above the nucleus accumbens shell. One cycle consisted of 10 days of 90 min access to ethanol followed by 6 days of ethanol deprivation. The ADE was measured in the beginning of a new cycle. Rats received JDTic, a selective kappa-antagonist, either subcutaneously (10 mg/kg) or intra-accumbally (15 mu g/site) or, as a reference substance, systemic naltrexone (0.3 mg/kg) before ethanol re-access, and the effects on the ADE were evaluated. Systemic and intra-accumbal JDTic significantly attenuated the ADE on the first day of ethanol re-access, as did systemic naltrexone. Additionally, naltrexone decreased ethanol intake levels. These results suggest that nucleus accumbens shell kappa-opioidergic mechanisms may have a role in mediating relapse to ethanol intake. Additionally, kappa-antagonism could be a valuable adjunct in ethanol relapse prevention. (C) 2019 Elsevier B.V. and ECNP. All rights reserved.Peer reviewe

Nonpeptidic Oxazole-Based Prolyl Oligopeptidase Ligands with Disease-Modifying Effects on alpha-Synuclein Mouse Models of Parkinson's Disease

Prolyl oligopeptidase (PREP) is a widely distributedserine proteasein the human body cleaving proline-containing peptides; however, recentstudies suggest that its effects on pathogenic processes underlyingneurodegeneration are derived from direct protein-protein interactions(PPIs) and not from its regulation of certain neuropeptide levels.We discovered novel nonpeptidic oxazole-based PREP inhibitors, whichdeviate from the known structure-activity relationship forPREP inhibitors. These new compounds are effective modulators of thePPIs of PREP, reducing alpha-synuclein (alpha Syn) dimerizationand enhancing protein phosphatase 2A activity in a concentration-responsemanner, as well as reducing reactive oxygen species production. Fromthe best performing oxazoles, HUP-55 was selected for in vivo studies. Its brain penetration was evaluated, andit was tested in alpha Syn virus vector-based and alpha Syn transgenicmouse models of Parkinson's disease, where it restored motorimpairment and reduced levels of oligomerized alpha Syn in the striatumand substantia nigra.Peer reviewe

A prolyl oligopeptidase inhibitor reduces tau pathology in cellular models and in mice with tauopathy

Peer reviewe