We find that Loa mutant mice exhibit defects in cortical and hippocampal development. Using live cell imaging techniques, we found direct evidence for a migration delay of bipolar neurons. Our results further indicate that this effect is not associated with defects in dynein interactions with LIS, NudE, or dynactin, and is therefore likely to result directly from the inherent mechanochemical defects caused by the Loa mutation [23
The abnormal cortical lamination we observe in the Loa/Loa mouse brain is similar to that seen in LIS1 compound heterozygous mutant mice [5
]. LIS1 mice exhibit severe disorganization of pyramidal cells in the CA1, CA2, and CA3 regions, as well as a reduced density of granule cells in the dentate gyrus [9
]. We observe a clear delay in the migration of granule neurons in the Loa/Loa mouse to form the dentate gyrus. Although the formation of the dentate gyrus has not been examined in the LIS1 mice, a delay in granule cell migration could provide a potential explanation for the diminished size of the dentate gyrus in these mice.
We detected a decrease in axon extension in Loa/Loa neurons both in vivo
and in vitro
, as well as a decrease in axon length. Dynein as well as LIS1 are necessary for the reorganization and extension of the growth cone during axonogenesis [7
]. These proteins are required to help microtubules extending into the peripheral zone of the growth cone resist retrograde actin flow [36
]. A Loa mutant dynein detaches from microtubules about twice as frequently as the wild-type motor protein [23
]. Therefore, microtubule penetration into the peripheral zone could be compromised, providing an explanation for altered axon elongation.
Altered expression of LIS, cytoplasmic dynein, and NudE have each been found to affect mitotic index in the developing brain [6
]. These effects reflect direct roles for these proteins in mitosis [31
]. Furthermore, LIS1 and cytoplasmic dynein RNAi interfere with apical migration of radial glial nuclei to the ventricular surface, which has been found to be essential for mitotic entry [7
]. The similarity in mitotic index between Loa/Loa and wild-type mouse brains suggests that any effect on mitotic entry or progression must be relatively minor, though further live analysis of mitosis might still reveal altered mitotic behavior.
The current study indicates that the Loa mutation affects neuronal migration in a similar manner as reduced LIS1 expression [5
]. However, the Loa mutation affects dynein processivity [23
], whereas LIS1 contributes to dynein force production [44
]. Our results indicate, therefore, that different defects in dynein function can lead to a common developmental outcome. The mechanical basis for this result is uncertain, but is likely a consequence of the coordinate behavior of multiple dynein molecules during high load functions. In in vitro
studies, LIS1 was found to convert cytoplasmic dynein to a persistent force-producing state, though the level of force generation by individual dynein molecules was unaffected. However, force generation by multiple motors is summated, and this effect was further enhanced by LIS1 as revealed in in vitro
laser trap bead assays and in computational simulations [44
]. Thus, LIS1 appears to be ideally suited for a role in high load functions such as nuclear transport [12
]. The Loa mutation, in contrast, had a clear effect on motor processivity [23
]. How the Loa mutation might affect transport of high load structures is uncertain, but could also involve coordination of multiple dynein molecules. The Loa mutation results not only in decreased single molecule processivity in vitro
, but also in decreased run-lengths for lysosomes/late endosomes in vivo
, which are thought to be driven by an average of approximately five to seven dyneins [23
]. Computational modeling indicates that, although cargo run length increases with motor number, the Loa processivity defect extends to the multimotor condition [23
]. Thus, defects in neuronal migration could conceivably be explained by a reduction in run length during nuclear transport. We reason, however, that Loa should also affect aggregate force production by multiple motors, which is a reflection of the number of individual motors simultaneously 'engaged' with microtubules at any given time. This value should be reduced for Loa as a simple consequence of its increased microtubule detachment rate. Thus, the defect in neuronal migration we observe in the Loa/Loa mouse could result from shorter nuclear run lengths, decreased force, or both. The prolonged state of somal distortion we observe during neuronal migration in the Loa/Loa neurons could be indicative, in particular, of a reduction in nuclear translocation forces, but further work will be needed to directly determine the effects of the Loa mutation at the multiple motor level.
The defects we observe in the mutant neurons may explain the perilethality of the Loa/Loa mice. The Loa homozygotes die within 24 hours of birth due to an inability to move their mouths, and therefore feed. A 2-day delay in the migration of facial motor neurons to their destination may be responsible for this facial paralysis. Correspondingly, a delay in axon extension could also contribute to facial paralysis due to a lack of muscle innervation.