Phd Degree / Doktora
Permanent URI for this collectionhttps://hdl.handle.net/11147/2869
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Doctoral Thesis Tay-sachs Hastalığı Fare Modelinde AAVrh10-hNeu3 Vektörünün İntratekal Uygulamasının Terapötik Etkisi(2025) Basırlı, Hatice Hande; Seyrantepe, VolkanTay-Sachs hastalığı, HEXA genindeki mutasyonlardan sonucu olan nörodejeneratif bir bozukluktur. Bu gen, GM2 gangliositinin yıkımını katalize eden β-hekzosaminidaz A (HexA) enziminin α-alt birimini kodlamaktadır. HexA eksikliği, Tay-Sachs hastalarında GM2'nin birikmesine ve ilerleyici nörodejenerasyona sebep olmaktadır. İlginç bir şekilde, Hexa-/- farelerinde, hastalığın hafif düzeyde seyretmesi hastalarda gözlemlenen nöropatolojik bulguların ortaya çıkmasını sınırlamaktadır. Erken başlangıçlı Tay-Sachs hastalığı fare modeli, Hexa-/-Neu3-/-, Tay-Sachs hastalarına benzeyen nöropatolojik ve davranışsal bozukluklarla birlikte 20 haftalık kısa bir yaşam süresi göstermektedir. Farelerde, sialidaz Neu3 enziminin GM2'nin yıkımı bir 'bypass' yolağı ile kolaylaştırdığı gösterilmişken, insan Neu3'ün (hNeu3) bu bağlamdaki potansiyel rolü henüz keşfedilmemiştir. Ayrıca, bu çalışmada, anti-inflamatuar bir ilaç olan İstradefilinin Hexa-/-Neu3-/- farelerinde nöroinflamasyonu azaltma potansiyeli incelenmiştir. İstradefilin ile birlikte hNeu3'ün terapötik etkinliğini moleküler biyolojik, immunohistokimyasal ve davranışsal analizler kullanarak açıklamak amacıyla AAVrh10-hNeu3, 8 haftalık Hexa-/-Neu3-/- farelerine intratekal olarak uygulanmıştır. Bunun neticesinde, Hexa-/-Neu3-/- farelerinde hNeu3 ve İstradefilinin yaşam süresini 28 haftaya kadar uzattığı ve farelerin vücut ağırlığında tespit edilebilir iyileşmelere yol açtığını bulduk. Özellikle sadece AAV veya İstradefilin ile birlikte AAV verilen 20 haftalık farelerde, GM2 birikiminde, lizozomal LAMP1 ve TUNEL pozitif hücrelerde azalma ve CNPase seviyelerinde artış gözlemlendik. Bunun yanı sıra, Rotarod ve ayak izi analizleri, tedavi edilen farelerde 20.haftada iyileşme olduğunu göstermektedir. Bulgularımız, insan Neu3 gen iletimi ve İstradefilinin, Hexa-/-Neu3-/- farelerinde görülen hastalık ilerlemesini hafifletme ve nöropatolojik ve motor eksikliklerini iyileştirme konusundaki terapötik potansiyeline ilişkin ilk in vivo kanıtı ortaya koymaktadır.Doctoral Thesis Therapeutic Efficacy Of Intrathecal Administration Of AAVrh10-mHEXA Vector In A Mouse Model Of Tay-Sachs Disease(01. Izmir Institute of Technology, 2025) Can Özgür, Melike; Seyrantepe, VolkanTay-Sachs hastalığı, bir lizozomal depolama hastalığıdır ve HEXA genindeki mutasyonlar sonucu oluşmaktadrr. HEXA geni, GM2 gangliosidini parçalamaktan sorumlu β-hekzosaminidaz A enziminin α-alt birimini kodlar. Son zamanlarda, Hexa ve Neu3 genlerinin birleşik eksikliklerine sahip Tay-Sachs hastalığı fare modeli (DKO fareleri), şiddetli nöropatolojik semptomlar ve nöroinflamasyon sergileyerek 20 haftaya kadar hayatta kalmıştır. Birçok lizozomal depolama hastalığı için tedavi stratejileri değerlendirilmiş olsa da, Tay-Sachs hastalığı için etkin tedaviler henüz geliştirilememiştir. Bu çalışmada, AAVrh10-mHexa (AAV) vektörünün intratekal uygulanmasının DKO farelerinde terapötik etkinliğini araştırmayı amaçladık. Ayrıca, AAV ilişkili gen tedavisi ile bir anti-inflamatuar ajanı olan istradefylline tedavisinin, DKO farelerinde nöroinflamasyonu hafifletme potansiyelini değerlendirdik. Moleküler biyolojik, immünohistokimyasal ve davranışsal analizler yapıldı. AAV tek başına ve istradefylline ile kombinasyon halinde uygulandıktan sonra DKO farelerinin yaşam süresinin 30 haftaya kadar uzadığını gösterdik. Moleküler biyolojik analizler, AAV tedavisi uygulanan ve istradefylline ile kombinasyon halinde tedavi edilen DKO farelerinin korteks, beyincik ve böbrek, karaciğer ve dalak gibi organlarında Hexa aktivitesinin arttığını ve lizozomal markerlar ile pro-inflamatuar sitokinler olan Ccl2 ve Ccl3 seviyelerinin azaldığını ortaya koydu. İmmünohistokimyasal veriler, AAV ve AAV-istradefylline kombinasyonu ile tedavi edilen farelerin beyinlerinde GM2 birikintisinin temizlendiğini, lizozom sayılarının azaldığını, aktif astrosit düzeylerinin düştüğünü ve nöronlar ile oligodendrositlerde iyileşmeler olduğunu gösterdi. Ayrıca, bu farelerde motor aktivitesi de iyileşti. Bu sonuçlar, AAVrh10 ilişkili intratekal uygulamanın, tek başına veya istradefylline ile kombinasyon halinde, Tay-Sachs hastalığını tedavi etmek için umut verici bir terapötik yaklaşım olduğunu göstermektedir.Doctoral Thesis Investigation of the Effects of Ketogenic Diet Therapy in a Mouse Model of Gm2 Gangliosidosis(01. Izmir Institute of Technology, 2024) İnci, Orhan Kerim; Seyrantepe, VolkanGM2 gangliosidosis is an autosomal recessive lysosomal storage disorder in which GM2 ganglioside is accumulated, especially in the brain. GM2 gangliosidosis is divided into three different variants: Tay Sachs (B-variant or type I), Sandhoff (O-variant or type 2), and GM2AP deficiency (AB-variant). Accumulation of the GM2 ganglioside in brain causes disease pathology as neurodegeneration and neuroinflammation. Our lab first displayed the novel GM2 gangliosidosis type-I mouse model (Hexa-/-Neu3-/-). Compared to control, this model could survive a maximum of five-months due to severe pathologies, neurodegeneration, and neuroinflammation. The ketogenic diet is high-fat and low-carbohydrate/protein diet that triggers burning fat instead of carbohydrates. The ketogenic diet is used comprehensively in neurodegenerative disorders such as Sandhoff, Alzheimer’s, and Parkinson’s to regulate inflammation, neurodegeneration, and autophagy. In addition, elevation of CCL2 expression in Hexa-/-Neu3-/- mice resulted in increased amounts of active microglia and astrocytes. Therefore, the CCL2/CCR2 signaling inhibitor of propagermanium was used in addition to ketogenic diet. Thus, reducing neuroinflammation is aimed to be more effective as a combined therapy. In my Ph.D. thesis, expression and protein levels of autophagy and inflammation-associated genes were analyzed in the mouse brain to exhibit whether beneficial effects on autophagic flux and neuroinflammation are found after the ketogenic diet and propagermanium treatment. The pathology of the GM2 gangliosidosis mouse type-I brain was illustrated by thin-layer chromatography and immunofluorescence staining to display whether the ketogenic diet affects the ganglioside metabolism. Briefly, ketogenic diet therapy and its anti-inflammatory and neuroprotective effects were explored in the GM2 gangliosidosis mouse model.Doctoral Thesis Investigation of the Effects of Antiinflammatory in the Tissues of Gm2 Gangliosidosis Mouse Model(01. Izmir Institute of Technology, 2023) Ateş, Nurselin; Seyrantepe, VolkanTay- Sachs disease is an autosomal recessively inherited lysosomal storage disorder caused by mutations on the HEXA gene encoding α-subunit of β- Hexosaminidase A enzyme. The enzyme catalyzes GM2 to GM3 conversion but when it is deficient the GM2 degradation is interrupted and GM2 ganglioside is progressively accumulated especially in neurons. Progressive accumulation of GM2 causes increasing death of neurons, disruption in mental and motor functions and eventually death at 2-4 years of age. The Hexa-/- Tay-Sachs model was normal thanks to a bypass mechanism mediated by Neurominidase3. It was determined that Hexa-/-Neu3-/- mice mimicked the neuropathologic and clinical phenotype of the Tay-Sachs disease. Previously we showed GM2 accumulation in Hexa-/-Neu3-/- Tay Sachs disease mouse model triggers release of proinflammatory cytokines, microgliosis, astrogliosis consequently activation of inflammatory cascades as well as oxidative stress. These inflammatory events contribute to neurodegeneration observed in the disease pathology. In Sandhoff Disease mouse model it was shown that astrocytes express adenosine A2A receptors which induces ccl2 chemokine overexpression. A2A receptor antagonist istradefylline treatment reduces microglial activation and ccl2 expression in Sandhoff mice. In this study; A2A receptor antagonist istradefylline treatment was applied to Tay Sachs disease mouse model and whether this treatment would alleviate the neuroinflammation and redox imbalance; and prolong the lifespan was investigated by molecular biological and behavioural analyses. Modulation of ccl2 expression by istradefylline was used as potential therapeutic target to slow down Tay Sachs disease mouse model.Doctoral Thesis Understanding the Biological Role of Sialidase Neu3 in Tay-Sachs Disease Mouse Model(Izmir Institute of Technology, 2019) Akyıldız Demir, Seçil; Seyrantepe, VolkanTay-Sachs disease is a severe lysosomal storage disorder characterized by mutations in the lysosomal ß-Hexosaminidase A (HEXA) enzyme which converts GM2 to GM3 ganglioside. The GM2 ganglioside accumulation is observed predominantly in the neurons. The infants appear normal in their inborn time, but the progressive accumulation of undegraded GM2 results with death. Hexa-/- mice were created. However, they have a normal lifespan with no obvious neurological impairment until one year. It was thought that stored GM2 catabolized to GA2 using sialidase(s), which is further processed by HEXB. To determine the contribution of sialidase NEU3 to degradation of GM2, a mouse with combined deficiencies of Hexa and Neu3 genes was generated. The Hexa-/-Neu3-/- mice were healthy at birth, but they died between 1.5 and 5 months of age. Thin-layer chromatography and mass spectrometric analysis of the brains of Hexa-/-Neu3-/- mice revealed the abnormal accumulation of GM2. The progressive GM2 accumulation was also verified on testes, liver, and kidney of Hexa-/- Neu3-/- mice. GM2 accumulation in the brain leads to increased lysosomes with membranous cytoplasmic bodies, Purkinje cell depletion, cytoplasmic vacuolization, astrogliosis, and age-dependent lessening in neurons and oligodendrocytes. These mice have prominent disorders such as growth impairment, skeletal bones abnormalities, slow movement, tremors, anxiety and age-dependent loss in both memory and muscle strength. Consequently, the Hexa-/-Neu3-/- mice mimic the pathological, biochemical and clinical abnormalities of the Tay-Sachs patients, and useful model for the future understanding of cellular pathologies that drive the progression of the disease. They are a suitable model for the future pre-clinical testing of possible treatments.Doctoral Thesis Immunohistochemical, Biochemical and Imaging-Mass Spectrometric Analysis of Brain Tissues of Mice With Combined Deficiencies of Ss-Hexosaminidase A, Sialidase Neu4 and Gm2-Ap(Izmir Institute of Technology, 2017) Timur, Zehra Kevser; Seyrantepe, VolkanGangliosides are complex glycosphingolipids derived from glucosylceramide or galactosylceramide and contain sialic acid in their carbohydrate chain. Sialidases, known also as neurominidases, are a family of glycohydrolytic enzymes functioning in the catabolism of sialoglycoconjugates by removing α-glycosidically linked sialic acid residues. Sialidase Neu4 is the lysosomal sialidase and GM2 activator protein is the cofactor of β-Hexosaminidase a enzyme to degrade the GM2 ganglioside. The activity of sialidase Neu4 activity against GD1a and GM2 gangliosides were significantly increased by the co-transfection with GM2 activator protein to the cells transfected with sialidase Neu4. This in vitro study revealed that lipid-binding proteins can facilitate the glycolipid degradation rendered by sialidase Neu4 in the lysosomes. In the concept of this study, a systematic comparison of the storage levels of gangliosides, gene expression ratios and behavioral features of new double (Hexa-/-GM2AP-/- and GM2AP-/-Neu4-/-) and triple (Hexa-/-GM2AP-/-Neu4-/-) knockout mice models with the existing double (Neu4-/-Hexa-/-) and single (Hexa-/-) knockout models revealed the possible involvement of the GM2 activator protein as a cofactor of sialidase Neu4 in the bypass mechanism in the Tay-Sachs mice, in vivo. Based on the increased GM2 ganglioside level in brain and cerebellum detected by the immunohistochemical and imaging mass spectrometric analysis, we speculate that the sialidase Neu4 functions on the degradation of GM2 ganglioside with GM2 activator protein, in vivo.