Title: Distinct Roles for Dynein Regulatory Proteins NudE and NudEL in Brain Development
Description: The development of the mammalian neocortex requires the careful balancing of proliferation, migration, and differentiation. The cellular machinery coordinating these events includes molecular motor proteins such as dynein. Regulation of dynein activity is particularly important, since it is the major microtubule minus-end directed motor in cells. Dynein is a large, complex structure comprising several subunits and binding partners. Its function is critical for multiple stages of brain development. The dynein regulatory proteins NudE and NudEL have been implicated in several aspects of dynein function, including brain development. Originally identified as nuclear distribution (nud) factors in the dynein pathway, NudE and NudEL are now known to have diverse roles in mitosis, cell migration, and intracellular trafficking. Mice null for Nde1, the gene encoding NudE, have microcephaly, whereas mice null for Ndel1, which encodes NudEL, are embryonic lethal. Additionally, Nde1 mutations have recently been shown to result in microcephaly and lissencephaly in human patients.

NudE and NudEL are functionally related paralogs that are more than 70% similar. Both bind to dynein and LIS1, another dynein regulatory protein involved in brain development. In addition to serving as recruitment factors, NudE and NudEL impact dynein force production and allow dynein to serve as a persistent motor under high load. This would be particularly important during the proliferation of neural progenitors, which undergo cell cycle-linked nuclear oscillations. These oscillations, termed interkinetic nuclear migration (INM), require forces acting upon the nucleus to drive upward (basal) and downward (apical) movement in the proliferative ventricular zone (VZ) of the brain. Research from our lab has identified dynein, along with LIS1, as being responsible for apical movement, and the unconventional kinesin Kif1a as the driving force behind basal movement. The aim of this thesis has been to understand the mechanisms by which NudE and NudEL regulate dynein function in brain development.

We identify a role for NudE, but not NudEL, in INM and radial progenitor mitosis. Additionally, we find that both NudE and NudEL are involved in the multipolar-to-bipolar transition of neurons, and that NudEL has a role in bipolar neuronal migration. Our results provide an additional molecular explanation for microcephaly resulting from Nde1 mutations, implicating a block in INM as a cause for reduced proliferation, since cells are unable to reach the ventricular surface where they normally undergo mitosis. NudEL has previously been implicated in having a role in neurite extension and axon elongation. We found that NudE/EL localized to a single neurite of a Stage 2 hippocampal neuron as well as the axon tip of a Stage 3 neuron. In addition, the Stage 2 localization was coincident with the appearance of established early markers of neuronal polarity. We studied the role of NudE/EL in establishing neuronal polarity and found that Nde1 and Ndel1 RNAi inhibited axon formation. Overexpression of NudEL did not result in noticeable changes in axon formation.

We conclude that in addition to the role of NudEL in axon extension and outgrowth, NudE/EL serve as early markers of neuronal polarity and are required, though not necessarily sufficient, for axon specification.