Spastin and P60-katanin are two distinct microtubule-severing proteins. Autosomal dominant mutations in the SPG4 locus corresponding to spastin are the most common cause of hereditary spastic paraplegia (HSP), a neurodegenerative disease that afflicts the adult corticospinal tracts. Here we sought to evaluate whether SPG4-based HSP is best understood as a "loss-of-function" disease. Using various rat tissues, we found that P60-katanin levels are much higher than spastin levels during development. In the adult, P60-katanin levels plunge dramatically but spastin levels decline only slightly. Quantitative data of spastin expression in specific regions of the nervous system failed to reveal any obvious explanation for the selective sensitivity of adult corticospinal tracts to loss of spastin activity. An alternative explanation relates to the fact that the mammalian spastin gene has two start codons, resulting in a 616 amino acid protein called M1 and a slightly shorter protein called M85. We found that M1 is almost absent from developing neurons and most adult neurons but comprises 20 -25% of the spastin in the adult spinal cord, the location of the axons that degenerate during HSP. Experimental expression in cultured neurons of a short dysfunctional M1 polypeptide (but not a short dysfunctional M85 peptide) is deleterious to normal axonal growth. In squid axoplasm, the M1 peptide dramatically inhibits fast axonal transport, whereas the M85 peptide does not. These results are consistent with a "gain-of-function" mechanism underlying HSP wherein spastin mutations produce a cytotoxic protein in the case of M1 but not M85.

Kinesin-5 is traditionally considered a mitotic motor protein. This article presents evidence that kinesin-5 is also critically influential in the process of neuronal migration, wherein terminally postmitotic neurons undergo orderly movement from their sites of birth to their final destinations.

Collateral branches from axons are key components of functional neural circuits that allow neurons to connect with multiple synaptic targets. Like axon growth and guidance, formation of collateral branches depends on the regulation of microtubules, but how such regulation is coordinated to ensure proper circuit development is not known. Based on microarray analysis, we have identified a role for microtubule-associated protein 7 (MAP7) during collateral branch development of dorsal root ganglion (DRG) sensory neurons. We show that MAP7 is expressed at the onset of collateral branch formation. Perturbation of its expression by overexpression or shRNA knockdown alters axon branching in cultured DRG neurons. Localization and time-lapse imaging analysis reveals that MAP7 is enriched at branch points and colocalizes with stable microtubules, but enters the new branch with a delay, suggesting a role in branch maturation. We have also investigated a spontaneous mutant mouse that expresses a truncated MAP7 and found a gain-of-function phenotype both in vitro and in vivo. Further domain analysis suggests that the amino half of MAP7 is responsible for branch formation, suggesting a mechanism thatisindependentofitsknowninteractionwithkinesin.Moreover,thismouseexhibitsincreasedpainsensitivity,aphenotypethatisconsistent with increased collateral branch formation. Therefore, our study not only uncovers the first neuronal function of MAP7, but also demonstrates the importance of proper microtubule regulation in neural circuit development. Furthermore, our data provide new insights into microtubule regulation during axonal morphogenesis and may shed light on MAP7 function in neurological disorders.

Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy for spinal cord injury (SCI) that has not been extensively explored, despite the integral roles played by astrocytes in the central nervous system (CNS). Induced pluripotent stem (iPS) cells are a clinically-relevant source of pluripotent cells that both avoid ethical issues of embryonic stem cells and allow for homogeneous derivation of mature cell types in large quantities, potentially in an autologous fashion. Despite their promise, the iPS cell field is in its infancy with respect to evaluating in vivo graft integration and therapeutic efficacy in SCI models. Astrocytes express the major glutamate transporter, GLT1, which is responsible for the vast majority of glutamate uptake in spinal cord. Following SCI, compromised GLT1 expression/function can increase susceptibility to excitotoxicity. We therefore evaluated intraspinal transplantation of human iPS cell-derived astrocytes (hIPSAs) following cervical contusion SCI as a novel strategy for reconstituting GLT1 expression and for protecting diaphragmatic respiratory neural circuitry. Transplant-derived cells showed robust long-term survival post-injection and efficiently differentiated into astrocytes in injured spinal cord of both immunesuppressed mice and rats. However, the majority of transplant-derived astrocytes did not express high levels of GLT1, particularly at early times post-injection. To enhance their ability to modulate extracellular glutamate levels, we engineered hIPSAs with lentivirus to constitutively express GLT1. Overexpression significantly increased GLT1 protein and functional GLT1-mediated glutamate uptake levels in hIPSAs both in vitro and in vivo post-transplantation. Compared to human fibroblast control and unmodified hIPSA transplantation, GLT1-overexpressing hIPSAs reduced (1) lesion size within the injured cervical spinal cord, (2) morphological denervation by respiratory phrenic motor neurons at the diaphragm neuromuscular junction, and (3) functional diaphragm denervation as measured by recording of spontaneous EMGs and evoked compound muscle action potentials. Our findings demonstrate that hiPSA transplantation is a therapeutically-powerful approach for SCI.

The structural and biochemical study of Kif15 provides insight into this potential drug target and allows comparison with Eg5, a kinesin that partially shares the functions of Kif15.