Abstract
The cerebellum is the region of central nervous system responsible for motor coordination and balance. It is an excellent model to study nervous system development as it gathers important cellular events such as proliferation, migration and differentiation of neuronal precursors. A mouse lineage presenting lack of motor coordination was selected in an ENU mutagenesis project. The mouse, named equilibrium (eqlb), shows increased proliferation of cerebellar neuronal precursors during early postnatal development (up to 15 days after birth), as well as disorganized Purkinje cell layer. Recently, our laboratory identified the mutation causing the phenotype as an A>T transversion at position 190 (transcribed sequence) in Nox3 gene, located on mouse chromosome 17, with consequent substitution of Asp by Tyr at position 64 of the protein. Using in silico analysis, we found that the aminoacid substitution changes both primary and secondary protein structure in eqlb when compared to BALB/c. In eqlb mouse, we predict that Nox3 will have only a beta strand formed by aminoacids 58-70 rather than two in the region 59-61 and 65-70 as it occurs in BALB/c besides, the additional Tyr, could generate a possible new phosphorylation site. Furthermore, eqlb mouse has high expression of Nox1, indicating a possible compensation caused by the expression of mutated Nox3. Nox3 is a member of the NADPH oxidases family, that are transmembrane proteins which main function is to reduce molecular oxygen to form reactive oxygen species (ROS). ROS produced by Nox can modulate cell signaling in various physiological processes including proliferation. SHH (Sonic Hedhog), secreted by Purkinje cells, is the main mitogen for cerebellar neuronal precursors during early postnatal development. Disruption of SHH signaling is associated with abnormal development of the cerebellum and continuous SHH signaling influences directly the proliferation of cerebellar neurons causing, in many cases, medulloblastoma. Besides this source, medulloblastoma can be formed by dysregulation of cerebellar neural stem cells that are dependent on SHH. The formation of cerebellar tumors has also been related to the action of ROS generated by Nox. Due to the described phenotype and mutation of eqlb mouse it becomes an excellent animal model for studying the control of proliferation of stem cells/neural precursors. Because SHH is the main mitogen for neuronal cell precursors in the developing cerebellum, our hypothesis for this project is that ROS produced by Nox3 can act in the SHH pathway influencing the activity of this mitogen. Our goal is to study the role of Nox and ROS in the control of proliferation of granule cell precursors and cerebellar neural stem cells by SHH signalling pathway. (AU)
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