ALS Sinek Modelinde Nörodejenerasyon Mekanizmasının Proteomik ve Genetik Analizi

Year 2016, Volume: 6 Issue: 12, 49 - 59, 01.12.2016

Hilal Kalkan , Fabian Feiguin Raffaella Klima

Abstract

Amaç : TDP-43; RNA bağlanma motifleri içeren ve pre-mRNA ekleme, transkripsiyon, mRNA stabilitesi

ve mRNA transferinde
yer aldığı rapor edilen oldukça korunmuş ve her yerde
eksprese edilen

nükleer proteindir. Son zamanlarda TDP-43 yapılan çalışmalar ile Amyotrofik Lateral Skleroz (ALS)

hastalarının etkilenmiş beyin bölgelerinde gözlenen hücre içi inklüzyonların ana protein bileşeni olarak

tanımlandı. Bu çalışmada Drosophila
melanogaster’de TDP-43’ün analoğunun (TBPH’in) ALS modelindeki

sinekler üzerinde proteomik ve genetik mekanizmaları içerisindeki rolünün araştırılması amaçlanmıştır.

Materyal-Metod : WIII8,UAS GFP, GMR,GMR TBPH genotiplerinde bakire dişi sinekler ve erkekler

uygun besiyeri ortamında çiftleştirilerek farklı genotip ve
fenotiplerde ALS için etki gösterebilen

transgenik model
sinekler üretildi. Transgenik
sineklere TBPH entegre edildi. PCR , Jel Elektroforezi ve

2D kütle spektrofotometri yöntemleri kullanılarak TBPH mutantlarının analizi yapıldı.

Bulgular : TDP-43’ün analoğu olan TBPH’i eksprese eden farklı transgenik sinekler
kullanılarak ,

TBPH’in
ekspresyonunun kontrol gruplarına kıyasla nörodejenerasyona neden
olup olmadığı kontrol

edildi. TPBH’nin ekspresyonunun sineklerin gözlerinde siyah lekelerin (nekrozun) gelişmesine neden

olduğu ve gözlerde nörodejenerasyon oluşturduğu görüldü. Dahası, mutasyon geçirmiş sinekler üzerinde

2D jel analizi ile
protein agregasyonunda TBPH’in rolünü düşündüren sinek türlerine kıyasla diğer

sineklerin gözlerinde azalmış yağ vücut proteinleri 1
(Fbp1) seviyeleri gösterildi.

Sonuçlar : Sonuç olarak TBPH’in Fbp1’nın üretimini artırarak nörodejeneratif sürece karıştığı görüldü.

Keywords

ALS, Drosophila, TDP-43, Fbp1

Proteomic and Genetic Analysıs of the Mechanism of Neurodegeneration in a Fly Model of ALS

Abstract

Objective : TDP-43 is a highly
conserved and ubiquitously expressed nuclear protein containing

RNA binding motives and reported to be
involved in pre-mRNA splicing, transcription, mRNA stability

and mRNA transport. Recently, TDP-43 was
identified as the main protein component of the intracellular

inclusions observed in affected brain areas
of patients suffering from Amyotrophic Lateral Sclerosis

(ALS).

Material and Methods : In WIII8, UAS GFP, GMR, GMR TBPH genotypes, virgin and male
transgenic

model flies were produced which were able
to act for ALS in different genotypes and phenotypes by

mating them in the appropriate medium. TBPH
was integrated into the transgenic flies. TBPH mutants

were analyzed using PCR, Gel
Electrophoresis and 2D mass spectrophotometry methods.

Results : We have used different
transgenic flies expressing TDP-43 analogue TBPH and checked

whether the expression of TBPH is causing
the neurodegeneration as compared to the control groups.

Expression of TPBH caused development of
black spot (necrosis) in the eyes of the flies clearly suggested

neurodegeneration in the eye. Moreover, 2D
gel analysis on TBPH mutated flies showed reduced

fat body proteins 1 (Fbp1) levels in the
eyes of flies as compared to wild type, suggesting the role of

TBPH in the protein aggregation.

Conclusion : Our date showed TBPH
is involved in the neurodegenerative process by enhancing the

production of Fbp11.

Keywords

ALS, Drosophila, TDP-43, TBPH, Fbp1

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