"Survival Motor Nöron" Proteini Eksikliğinin Mikrotübül Pozitif Uç Proteinleri Üzerindeki Etkisinin Araştırılması

Abstract

Spinal muscular atrophy (SMA) is one of the hereditary neuromuscular disease, which is caused by mutations in the Survival of motor neuron 1 (SMN1) gene. SMN deficiency primarily affects motor neurons and regulation of microtubule dynamics and its stability is important for differentiation and functions of neuronal cells. Loss of SMN causes alterations in microtubule polymerization, however, the molecular mechanisms are largely unknown. Microtubule cytoskeleton plays role in establishment of dendrite and axon morphology as well as mRNA, protein and organelle transport to axonal terminals in neurons. Microtubule stability is regulated by both microtubule-associated proteins (MAPs) and plus-end tracking proteins (+ TIPs), which localizes plus-end of microtubules. Among + TIPs, End Binding Protein 1 (EB1) and EB3 proteins bind to tubulins in plus ends and form a molecular platform for the interation of other + TIP proteins. MAP1B, a microtubule-associated protein, binds to EB proteins and prevents their binding to microtubule plus ends by keeping them into cytoplasm. Previously we showed that SMN deficiency causes MAP1B upregulation. Therefore, in this study we investigated whether expressions and/or localizations of EB1 and EB3 proteins are altered in motor neuron like NSC34 cell line. To establish in vitro SMA model, SMN was knocked-down by siRNA, then EB1 and EB3 protein levels were analyzed by Western blot. We found that EB1 is not endogenously expressed in NSC34 cells. In addition, we determined 44% downregulation in EB3 protein level in SMN depleted cells. To analyze localization of EB3 proteins at the begining and tip of neurites, immunofluorescence stainings were performed in SMN knock-down cells. Increased number of comets, as well as decreased comet area was found at the begining of neurites in SMN depleted cells compared to controls. We did not find significant alterations neither in total comet area nor floresean intensities of comets and also total EB3 proteins. Our results suggested that microtubules at the begining of neurites are more dynamic in the absence of SMN and EB3 proteins may be involved in SMA pathomechanisms. Further studies will help to explain the role of SMN protein in microtubule dynamics.