| SAD-A and SAD-B are mammalian orthologues of SAD-1, a C. elegans kinase needed for synaptogenesis and axon outgrowth. To see if mammalian SADs are important in neural development, I analyzed SAD-A and -B mutant mice. Single mutants of each gene are viable and fertile, but SAD-A, -B double mutants (AB-/-) are immobile at birth and die within a few hours.; Histological analysis showed that AB-/- animals have small forebrains with an abnormally thin neocortex. All of the expected cell types in the cortex are present in the mutant, but they are more diffusely distributed, compared to the well defined laminae in controls. As laminae in the cortex are formed by ordered neuronal migration, this phenotype suggests SAD may be involved in neuronal migration.; Next, I asked whether AB-/- neurons differentiate properly. In the cerebral cortex most neurons have radially oriented processes, with dendrites pointing to the pia and axons pointing to the ventricle. Yet when I labeled individual cells in the AB-/- cortex, I saw that many cells had randomly oriented processes, and long axons that normally connect the cortex and sub-cortical areas were mostly lost. To see if these effects were cell autonomous, embryonic neurons were cultured to observe their polarization in vitro. Control neurons become polarized, forming a single long dephospho-tau-rich axon and multiple shorter MAP2-rich dendrites. The AB-/- neurons, in contrast, formed multiple processes that bore both axonal and dendritic markers. Thus, SAD-A and B are required for neuronal polarization in a cell autonomous manner.; Overexpression of SAD-A in cultured cells increased phosphorylation of tau, a protein that controls the assembly of microtubules. Additionally, in AB-/- cortex, levels of phospho-tau were decreased and levels of dephospho-tau increased. In both cases, overall tau levels stayed the same. These results suggest that SAD kinases act by locally regulating phosphorylation of tau, and that consequent alterations in microtubule organization are critical for neuronal migration and polarization.; Based on the phenotype of the C. elegans SAD mutant, SAD-A and -B may be required for synaptic differentiation. I have generated a SAD-A conditional mutant to bypass polarity defects and lethality, thereby allowing me to test this possibility. |
