Investigation of the effects of phosphorylation of spastin on neuronal morphology

Abstract

Cytoskeleton is an essential structure within a cell that takes part in cell signaling, cell movement, cell division and internal molecule transportation. Components of the cytoskeleton consist of three different polymeric structures which are actin filaments, intermediate filaments, and microtubules. Actin filaments play roles in cell movement and conservation of cell shape while intermediate filaments take part in intercellular junctions and supporting cell structure. The third cytoskeletal element, microtubules (MT), take part in organelle transportation within cells, mitotic cell division and neuronal development. Ranging between several centimeters with a diameter of 25 nm, microtubules consist of α-tubulin ve β-tubulin protein subunits which create heterodimers by making covalent bonds with each other. Another subunit, γ-tubulin, ensures the proper assembly of α-tubulin and β-tubulin by serving as a template. Microtubules have a dynamic structure which enables them to grow and shrink or reorganize within a cell via microtubule severing enzymes. Spastin is an enzyme which belongs to ATPase Associated with diverse cellular Activities (AAA) family and takes a role in microtubule severing. Spastin activity and proper functioning are essential for neuronal morphogenesis. Spastin protein encoded by SPG4 gene consists of 616 amino acids. Previous studies suggest a relationship between mutations in SPG4 gene and Hereditary Spastic Paraplegia (HSP) which is a disease causing spasticity in lower extremities. Spastin, which plays a role in the degeneration of motor pathways in HSP, is especially involved in the regulation of synapse development, axonal arborization, axonal transport, neurite outgrowth. Regulation of these cellular processes is dynamic, so it is crucial to understand regulation of spastin protein at the post-translational level. Recent study has shown that HIPK2-mediated phosphorylation of spastin on serine 268 residue is required for increased stability of spastin by inhibiting its degradation. In this study, we aimed to investigate the effect of post-translational modifications of spastin on neuronal morphology in hippocampal neurons. Within the scope of this thesis, non-phosphorylated (S268A) and phosphomimetic (S268D) mutant forms of spastin were overexpressed in primary rat hippocampal neurons, and changes in neuronal morphology were investigated via immunocytochemistry (ICC) analysis. Firstly, the effect of S268 phosphorylation of spastin on length of axon was examined. There was no significant difference between the groups at 24 hours and 48 hours in all stage 3 neurons. Then, the number of axonal branches was counted and statistically analyzed in early stage 3 neurons (axonal length of 40-100 µm) and stage 3 neurons (axon length of +100 µm) in which spastin mutants were overexpressed. In early stage 3 neurons, there was a statistical difference only between the S268A and control groups at 24 hours. At 48 hours, there was no significant difference between the groups. In stage 3 neurons expressing the S268A form of spastin, the number of axonal branches was statistically significantly decreased compared to control neurons and neurons expressing S268D spastin. There was no statistically significant difference between S268D spastin and the control group. These results show that the change in the S268 phosphorylation of spastin has an effect on axonal branching in hippocampal neurons in axon length of +100 µm. Then, minor processes in stage 2 and stage 3 neurons were counted and statistically analyzed to understand whether the altered S268 phosphorylation of spastin would affect the number of minor processes. In stage 2 neurons expressing S268A or S268D, no difference was observed between S268A and S268D spastins at 24 and 48 hours, whereas both forms of spastin increased the number of minor processes compared to the control group. For early stage 3 neurons, there was no statistical significance between the groups at 24 hours, while the number of minor processes was increased in S268A or S268D expressing neurons at 48 hours compared to the control group. This statistical increase is higher in S268D spastin than in S268A spastin, but there is no significant difference between S268A and S268D expressing neurons. Statistical analysis could not be performed for stage 3 neurons due to insufficient cell numbers at 24 hours. For these neurons at 48 hours, the number of minor processes was increased in groups S268A spastin and S268D spastin compared to the control group. While there is no statistical significance between S268A and S268D, the statistical increase in S268D is higher than in S268A compared to the control group. In addition to these statistical analyzes, the number of stage 1, stage 2 and stage 3 neurons in S268A expressing neurons and S268D expressing neurons were determined. Although stage 2 and stage 3 neuron numbers were similar for S268A and S268D during the transition from 24 hours to 48 hours, the number of neurons remaining in stage 1 in the S268D group was higher than S268A during the transition from 24 hours to 48 hours. As a conclusion, these results suggest that phosphorylation status of S268 position affects axonal branching by regulating spastin function. Considering the observed changes in the neuronal morphology of spastin due to phosphorylation, it was thought that the altered phosphorylation status of spastin might be caused by proteins involved in the function and regulation. Based on this, mass spectrometry analyzes were performed using primary rat hippocampal neurons to identify interaction partners that may change depending on S268 phosphorylation. Given these preliminary data, two candidate proteins were considered for further analysis for their interaction with spastin depending on spastin's phosphorylation status. One of these proteins is septin3, the neuron-specific protein previously studied in our laboratory and known to play a role in neuronal morphology, while another candidate protein is SQSTM1/p62, which is known to play a role in proteosomal degradation. Considering that in a previous study, S268 phosphorylation increased spastin stability by preventing proteosomal degradation, analysis of p62 protein as a candidate protein is important for this study. In this study, "septin3 & spastin", or "p62 & spastin" interactions were defined for the first time. However, the results showed that the interaction of septin3 or p62 proteins with spastin was not altered due to by S268 phosphorylation of spastin.