Parkinson hastalığı'nın hayvan modelinde PI3K/Akt yolağı ile mitokondriyal, oksidatif ve apoptotik parametrelerin ilişkisi
The relationship between mitochondrial, oxidative, apoptotic parameters and PI3K/Akt pathway in animal model of parkinson's disease
Yazar: HAZAL HAYTURAL
Danışman: DOÇ. DR. ERDEM TÜZÜN
İstanbul Üniversitesi / Sağlık Bilimleri Enstitüsü / Deneysel Tıp Araştırma Bölümü / Sinir Bilimi Ana Bilim Dalı
Derece: Yüksek Lisans, 2014
Konu: Biyoloji = Biology ; Nöroloji = Neurology
Dizin: Apoptozis = Apoptosis ; Fosfatidilinozitoller = Phosphatidylinositols ; Genler-Bcl-2 = Genes-Bcl-2 ; Mitokondriler = Mitochondria ; Oksidanlar = Oxidants ; Parkinson hastalığı = Parkinson disease ; Rotenon = Rotenone

Özet:
Parkinson hastalığı (PH) dopaminerjik nöronlarda ilerleyici yıkımla seyreden nörodejeneratif bir hastalıktır. PH'nin toksik maddelerle oluşturulan hayvan modelleri, klinik ve patolojik  bulgularıyla PH'yi taklit etmektedir. PH'de nöroprotektif etkiye sahip yeni terapötik stratejiler ve prognozu belirleyecek biyobelirteçler büyük ilgi odağıdır. Bu bağlamda fosfoinositidil-3-kinaz (PI3K)/Akt yolağı PH'de dikkat çeken sağkalım yolaklarından biridir. Bu yolağa ait moleküler değişimlerin araştırılması PH'ya yönelik tedavi ve biyobelirteçlerin bulunmasına yardımcı olabilir. Ancak PH olgularının hastalığın farklı dönemlerindeki beyin dokularında gerçekleşen moleküler değişikliklerin bilinmesi mümkün değildir. Bu çalışmada PH'nin klinik seyrinde PI3K/Akt yolağında ortaya çıkan değişimlerin ortaya konulması ve klinik bulgular ilişkisinin araştırılması planlanmıştır. Bu amaçla, C57BL/6 farelerinde oksidatif stresi arttırdığı bilinen rotenon ile hayvan modeli oluşturulmuş ve PI3K/Akt yolağında yer alan ve Akt tarafından regüle edilen kaspaz-9, kaspaz-3 ve Bcl-2 ile Akt'yi kontrol eden mTOR, DJ-1 ve PTEN genlerinin ekspresyon düzeyleri ardışık haftalarda elde edilen beyin dokularında incelenmiştir. 
Rotenon uygulanan farelerde rijidite, bradikinezi ve postür değişikliği saptandı. Substantia nigra´da bulunan tirozin hidroksilaz pozitif hücrelerin azaldığı ve serebral apoptotik nöronların arttığı gözlendi. Ekspresyon düzeyleri incelenen genlerden sadece Bcl-2'nin kontrol grubuna göre anlamlı derecede azaldığı, diğer genlerden Akt-1'de hafif azalma ve mTOR'da hafif artış olduğu kaspaz-3, kaspaz-9, PTEN ve DJ-1 genlerinde ise düzey değişikliği olmadığı saptandı. Kaspaz-3, mTOR ve Bcl-2 ile klinik parametreler arasında zayıf korelasyon belirlendi. 
Sadece Bcl-2 ekspresyonunda belirgin azalma görülmesi rotenonun PI3K/Akt yolağı dışındaki faktörlerle etki edebileceğini düşündürmüştür. Kaspaz-3 ve kaspaz-9 ekspresyonlarının değişmemesi PH'de kaspazdan bağımsız apoptoz yolaklarının rol oynadığını düşündürmektedir. Özellikle ağır klinik bulguları olan farelerde ekspresyon düzeyinin düşük olması, Bcl-2'nin, PH olgularında prognostik biyobelirteç olarak kullanılabileceğini göstermektedir.

Abstract:
Parkinson's disease (PD) is a neurodegenerative disorder, characterized with progressive loss of dopamine-generating neurons. Neurotoxin-induced animal models of PD mimic clinical and pathological findings of PD. Lately researchers have started focusing on the novel therapeutical strategies that have neuroprotective effects and prognostic biomarkers. Thus, the PI3K/Akt signaling pathway, which regulates cell survival, is attracting increasing amounts of attention. Investigation of molecular alterations related with this pathway might aid in improvisation of novel treatment methods and biomarkers for PD. However, it is not possible to determine molecular changes in brain tissues of PD cases in different stages of the disease. In this study, we aimed to examine the changes in the expression of PI3K/Akt factors during the course of PD and their association with clinical features. Thus, we established a C57BL/6 mouse model of PD by administrating rotenone, which has been shown to increase oxidative stress. Gene expression levels of mTOR, DJ-1 and PTEN that regulate Akt-1, and caspase-3, caspase-9 and Bcl-2 that are regulated by Akt-1 were examined in brain tissues obtained in consecutive weeks.
Rigidity, bradikinesia and postural changes were observed in rotenone-treated mice. Moreover tyrosine hydroxylase positive neurons were decreased in substantia nigra, whereas cerebral apoptotic neurons were increased. Levels of Bcl-2 significantly decreased in rotenone-treated mice as compared to the control group. On the other hand Akt-1 was found to decrease slightly and mTOR was found to increase slightly, whereas there were no changes in caspase-3, caspase-9, DJ-1 and PTEN expression levels. There was a weak correlation between caspase-3, mTOR, Bcl-2 levels and clinical parameters. 
The sole occurrence of significant expression change in Bcl-2 suggests that rotenone induces PH through non-PI3K/Akt mechanisms. The absence of alteration in caspase-3 and caspase-9 expression levels implies that caspase-independent apoptosis is involved in PD pathogenesis. Mice with severe clinical symptoms showed down-regulated Bcl-2 expression suggesting that Bcl-2 might be used as a prognostic biomarker in PD cases. 
Parkinson's disease (PD) is a neurodegenerative disorder, characterized with progressive loss of dopamine-generating neurons. Neurotoxin-induced animal models of PD mimic clinical and pathological findings of PD. Lately researchers have started focusing on the novel therapeutical strategies that have neuroprotective effects and prognostic biomarkers. Thus, the PI3K/Akt signaling pathway, which regulates cell survival, is attracting increasing amounts of attention. Investigation of molecular alterations related with this pathway might aid in improvisation of novel treatment methods and biomarkers for PD. However, it is not possible to determine molecular changes in brain tissues of PD cases in different stages of the disease. In this study, we aimed to examine the changes in the expression of PI3K/Akt factors during the course of PD and their association with clinical features. Thus, we established a C57BL/6 mouse model of PD by administrating rotenone, which has been shown to increase oxidative stress. Gene expression levels of mTOR, DJ-1 and PTEN that regulate Akt-1, and caspase-3, caspase-9 and Bcl-2 that are regulated by Akt-1 were examined in brain tissues obtained in consecutive weeks.
Rigidity, bradikinesia and postural changes were observed in rotenone-treated mice. Moreover tyrosine hydroxylase positive neurons were decreased in substantia nigra, whereas cerebral apoptotic neurons were increased. Levels of Bcl-2 significantly decreased in rotenone-treated mice as compared to the control group. On the other hand Akt-1 was found to decrease slightly and mTOR was found to increase slightly, whereas there were no changes in caspase-3, caspase-9, DJ-1 and PTEN expression levels. There was a weak correlation between caspase-3, mTOR, Bcl-2 levels and clinical parameters. 
The sole occurrence of significant expression change in Bcl-2 suggests that rotenone induces PH through non-PI3K/Akt mechanisms. The absence of alteration in caspase-3 and caspase-9 expression levels implies that caspase-independent apoptosis is involved in PD pathogenesis. Mice with severe clinical symptoms showed down-regulated Bcl-2 expression suggesting that Bcl-2 might be used as a prognostic biomarker in PD cases.