Investigation of the effect of RHO-associated protein kinase (Rock) on microtubule stability in SMN deficiency

Spinal muscular atrophy (SMA) is one of the rare genetic diseases with an autosomal recessive inheritance. SMA is involved in both neurodegenerative and neuromuscular disease groups due to the loss of α motor neurons in the spinal cord and progressive muscle atrophy. Mutations on Survival of motor neuron 1 (SMN1) gene cause SMA. SMN1 encodes Survival motor neuron (SMN) protein, which can not be synthesized in full length due to mutations. The molecular mechanisms underlying microtubule alterations in SMN deficiency remains unclear. Previous studies performed at our Department have revealed reduced microtubule stability in the SMN deficiency. One of the signaling pathways that regulates microtubule stability is RhoA/ROCK pathway, which has been shown to be upregulated in SMA models, but its effect on microtubule stability has not been investigated. This thesis aimed to investigate the effects of ROCK and its downstream target, Collapsin response mediator 2 (CRMP2), a microtubule-associated protein, on microtubule stability. Studies were performed by using motor neuron-like NSC34 cell line. Western blot analysis revealed no alteration in the protein level of CRMP2 in siRNA-mediated SMN depleted cells compared to control. However, there was a tendency for a decrease in phosphorylated form of CRMP2. When ROCK activity was inhibited by the compound named fasudil, no significant change was detected on the level of detyrosinated α tubulin, a marker of stabile microtubule, in the control cells. However, an upward trend in detyrosination of α-tubulin in SMN depleted cells was found when compared to the control cells, although it was not statistically significant. In this thesis, the relationship between ROCK, CRMP2 and microtubule stability was investigated for the first time in an in vitro SMA model. These findings may indicate the involvement of ROCK in the loss of microtubule stability and will guide future studies focusing on the loss of microtubule stability in SMA.