Astrogliosis and Glutamate Transporter Protein GLT1 Expression in 6-OHDA induced Parkinson's Disease Model

Abstract

Although the mechanisms that cause neuronal death are not fully understood in Parkinson's disease, which is characterized by the loss of dopamine neurons located in the substantia nigra pars compacta, some evidence suggests that the glutamatergic system plays a role in the pathogenesis of the disease. The increase in the concentration of glutamate, the main excitatory neurotransmitter of the CNS, in the synaptic gap causes excitotoxicity. The main mechanism that protects neurons from glutamate-induced toxicity is the removal of synaptic glutamate through the uptake system mediated by plasma membrane proteins known as excitatory amino acid transporters. Dysfunction of these transporters has been shown to be associated with some neurodegenerative diseases. In this study, it was aimed to demonstrate the possible changes in astrocytes and GLT1 expression in the 6-hydroxy dopamine (6-OHDA) induced Parkinson’s Disease model by dual immunohistochemical method. The rats were randomly divided into two groups and injected stereotactically with intranigral saline or 6-OHDA. Rotational tests were performed 15 days after the injections and then the rats were sacrificed. Free floating sections were labeled with glial acidic fibrillar protein (GFAP) - GLT1 and tyrosine hydroxylase (TH) - GFAP antibodies using dual immunofluorescence and dual indirect immunoperoxidase methods, respectively. While intranigral 6-OHDA injection caused loss of dopaminergic neurons, enlargement of the glial cell bodies and an increase in the number and diameter of the astroglial extensions (glial reaction) were assessed. Furthermore, the glial reactivity was accompanied by upregulation of the synthesis of glial fibrillary acidic protein which is intermediate filament protein of astrocytic cytoskeleton. The fact that no changes in the density of GLT1 expression was found in SN in despite of glial activation in the rats injected with 6-OHDA was evaluated as down-regulation of GLT1. After dopaminergic neuron damage induced by 6-OHDA in SNpc, the fact that astrogliosis and despite of astrocytic activation GLT1 protein did not increase suggest that astrocytes and/or glutamate transporter GLT1 may play a role in the etiopathology of Parkinson's disease, which is characterized by dopaminergic neuron death in SNpc. This also suggests that the survival and function of astrocytes may be targeted approaches for studies to seek new therapeutic agents for the treatment of CNS diseases characterized by neuronal loss.

Keywords

• Substantia nigra
• GLT1
• astrogliosis
• dopaminergic neurons
• excitotoxicity

Project Number

106S029 [SBAG 3307]

6-OHDA ile Oluşturulan Parkinson Hastalığı Modelinde Astrogliozis ve Glutamat Taşıyıcı Protein GLT1 Ekspresyonu

Abstract

Substansiya nigra pars kompakta yerleşik dopamin nöronlarının kaybı ile karakterize Parkinson hastalığında nöron ölümüne neden olan mekanizmalar tam olarak anlaşılamamış olsa da, bazı kanıtlar hastalığın patogenezinde glutamaterjik sistemin rol oynadığını göstermektedir. MSS’nin ana eksitatör nörotransmitteri olan glutamatın sinaptik aralıktaki konsantrasyonunun yükselmesi eksitotoksisiteye neden olmaktadır. Nöronları glutamat kaynaklı toksisiteden koruyan ana mekanizma, eksitatör amino asit taşıyıcıları olarak bilinen plazma membran proteinlerinin aracılık ettiği alım sistemi yoluyla sinaptik glutamatın ortamdan uzaklaştırılmasıdır. Bu taşıyıcıların disfonksiyonunun bazı nörodejeneratif hastalıklarla ilişkili olduğu gösterilmiştir. Bu çalışmada, 6-hidroksi dopamin (6-OHDA) ile oluşturulan deneysel Parkinson modelinde astrositlerde ve GLT1 ekspresyonundaki olası değişikliklerin ikili immünohistokimyasal yöntem ile gösterilmesi amaçlandı. Denekler rasgele iki gruba ayrıldı ve streotaksik olarak intranigral serum fizyolojik ya da 6-OHDA enjeksiyonu yapıldı. 15 gün sonra yapılan rotasyonel testlerin ardından denekler sakrifiye edildi ve çıkarılan beyinlerden alınan yüzen kesitler ikili immünofloresans ve ikili indirekt immünoperoksidaz yöntemleri kullanılarak sırasıyla glial asitik fibriler protein (GFAP) - GLT1 ve tirozin hidroksilaz (TH) - GFAP antikorları ile işaretlendi. İntranigral 6-OHDA enjeksiyonu dopaminerjik nöron kaybına neden olurken, glial hücre gövdelerinde genişleme astrogilial uzantılarda sayı ve çap artışı gözlendi (glial reaksiyon). Glial reaksiyona klasik intermediyet filament belirteci olan GFAP up-regülasyonu eşlik ediyordu. 6-OHDA uygulanan grupta astrositik aktivasyona karşın GLT1 ekspresyon yoğunluğunun değişmemesi, GLT1 down-regülasyonu olarak değerlendirildi. Sonuç olarak; SNpc’da 6-OHDA ile oluşturulan dopaminerjik nöron hasarı sonrası immünohistokimyasal yöntemlerle belirlediğimiz astrogliozis ve astrositik aktivasyona karşın GLT1 proteininin artış göstermemesi, astrositlerin ve/veya glutamat taşıyıcısı GLT1’in, SNpc’da dopaminerjik nöron ölümü ile karakterize Parkinson hastalığının etyopatolojinde rol oynayabileceğini ve ayrıca astrositlerin sağkalımı ve fonksiyonlarının korunmasının, nöron kaybı ile karakterize MSS hastalıklarının sağaltımı için yeni terapötik ajan arayışına yönelik çalışmalar için hedef yaklaşımlar olabileceğini düşündürmüştür.

Keywords

• Substansiya nigra
• GLT1
• astrogliozis
• dopaminerjik nöronlar
• eksisitotoksisite

Supporting Institution

TÜBİTAK

Project Number

106S029 [SBAG 3307]

Thanks

Bu çalışma TÜBİTAK tarafından desteklenmiştir (106S029 [SBAG 3307]).

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