Regulation of tau on septin3 in neuronal branching

Cytoskeleton is a network which consists of an interconnected filamentous proteins and regulatory motor proteins. In addition, cytoskeleton has important cellular activity roles for surviving and functioning such as determining cell shape and cell movement. Rearrangement of cytoskeleton is responsible for environmental changes and adaptation. Neurons distinguish from other cell types in the nature with their morphology. Their unique morphology allows them to perform their specific tasks. All neurons contain common structural backbone which is microtubule. Neurons have capacity to reconfigure their morphology and it is related to microtubule scaffolding. Neuronal cells undergo major developmental changes. Complex nervous systems require cytoskeleton-based processes that coordinate neuronal activites. Neurons are highly polarized cells and the establishment and maintenance of neuronal polarization is crucial for correct development of nervous system. They develop two types of cytoplasmic extensions; axons and dendrites. In response to electrical signals from the cell body, axons release neurotransmitters at the axon terminals. Dendrites, especially dendritic spines contain receptors for neurotransmitters and important function in signaling. The motility capacities of microtubules are directly related to their length. Mostly, the parent axon is dominated by very long microtubules chopped into short microtubules for direction of axon outgrowth. Microtubule severing is an important mechanism that take role in formation of specific neuron morphology. Neurons are rich in an enzyme called p60-katanin that makes breaks in a microtubule array. p60-katanin severes microtubules from the centrosome of neurons and later develops short microtubules that move more avidly than longer microtubules. There is a model called 'cut and run', in which the longer microtubules are mobilized by enzymes that sever them into shorter polymers. p60-katanin is not phosphorylated itself, yet its severing activity is regulated by phosporylation of microtubule binding proteins. Also, it is indicated that phosphorylation of MAP4 has regulatory effect on p60-katanin microtubule-severing activity in non-neuronal cells. Neuronal cells does not contain MAP4 but in place of MAP4, it has been shown that tau protects microtubules in the axon from severing by p60-katanin. According to this model, microtubule-associated proteins (MAPs) especially the axonal MAP tau bind along the axonal microtubules and has protective activity from severing proteins on microtubules. Tau deattaches from microtubules by phosphorylation and this controls levels of short microtubules in specific axonal regions. Short microtubules can then be transported by motor proteins into collateral branches to promote their growth and stabilization. Over the last several decades, Septin protein family has been recognized as fourth cytoskeleton compenent because of their filamentous appereance and their association between other cytoskeleton elements such as microtubules. Septins are evolutionarily conserved family classified under the GTPase superclass of P-loop NTPases. Septins have important roles in maintenance of cell shape and microtubule-dependent processes. For instance, it has been demonstrated that Septin2 and MAP4 intract and this interaction results in microtubule destabilization. Septin3 (Sept3) specifically associates with neurons and nerve terminals, which is found as a neuronal spesific protein after examination of 12 different tissues. Septin3 is found enriched in synaptosomes and presynaptic terminals. Here we sought to determine Septin3 - Tau interaction from the point of neuronal process formation and branching and how this interaction would effect p60-katanin activity on Septin3 filament formation. Initially, physical interaction of Tau & Septin3 and Tau & p60-katanin proteins were determined co-immunoprecipitation (co-IP) with rat brain lysate. co-IP results indicated that Septin3 and tau physically interacted, however tau and p60-katanin did not physically interact. The studies on RFL6 fibroblasts sought seek to test Septin3 – Tau – p60-katanin interaction in non-neuronal cell which do not express Septin3 and Tau endogenously. RFL6 cells were over-expressed both with tau and Septin3 and co-localization of tau then were confirmed. Septin3 over-expression resulted in filament formation, however after over-expression of both Septin3 and p60-katanin, Septin3 filament formation was damaged. This effect of p60-katanin is considered as similar to its effect on microtubules. In addition, RFL6 cells nucleofected with Septin3, tau and p60-katanin and Septin3 filament formation was investigated. According to these results, Septin3 protein formed filamentous appearance, in spite of p60-katanin over-expression. Several studies showed that septins increase spine morphogenesis and dendritic spine formation. For instance, Septin7 is found to localize at dendritic protrusions and dendritic branch points in cultured hippocampal neurons. Primary cortical neuron studies were performed to determine Septin3 – Tau – p60 - katanin interaction from the point of neuronal processes formation. Primary rat cortical neurons over-expression studies were carried out by nucleofection and gene silencing was carried out by Accell siRNA transfection. Septin3 over-expression increased dendritic branch formation and expedited neuronal development. Also, over-expression of Septin3 resulted in long and thick bundles in microtubules. Upon down regulation of Septin3, dendritic spine morphology and exon elongation were impaired. Following over-expression of both Septin3 and p60-katanin in the same cell, Septin3 was found to be interrupted along the axon in the presence of p60-katanin. Moreover, microtubules remained partially uninterrupted. Septin3 and tau were localized predominantly along the axon and resulted in even thicker bundles of Septin3 and microtubules. Besides Tau' s localization in mainly axons, Septin3 over-expressed and Tau silenced cells still lost their axon elongation. Observation in this study showed that, in addition to other known septin' s, Septin3 forms filaments in non-neuronal cells. Also, Septin3 has a role on neuronal polarization. Similar to some septins, Septin3 interacts with Tau and this interaction results in long and thicker microtubule bundles on neuronal microtubules. Although Further experiments needs to be carried out. tau may have a protective effect on Septin3 filaments against severing, like it does on the microtubules.