Epstein-Barr virus-based plasmid enables inheritable transgene expression in mouse cerebral cortex.

Continuous development of the cerebral cortex from the prenatal to postnatal period depends on neurons and glial cells, both of which are generated from neural progenitor cells (NPCs). Owing to technical limitations regarding the transfer of genes into mouse brain, the mechanisms behind the long-term development of the cerebral cortex have not been well studied. Plasmid transfection into NPCs in embryonic mouse brains by in utero electroporation (IUE) is a widely used technique aimed at expressing transgenes in NPCs and their recent progeny neurons. Because the plasmids in NPCs are attenuated with each cell division, the transgene is not expressed in their descendants, including glial cells. The present study shows that an Epstein-Barr virus-based plasmid (EB-oriP plasmid) is helpful for studying long-term cerebral cortex development. The use of the EB-oriP plasmid for IUE allowed transgene expression even in the descendant progeny cells of adult mouse brains. Combining the EB-oriP plasmid with the shRNA expression cassette allowed examination of the genes of interest in the continuous development of the cerebral cortex. Furthermore, preferential transgene expression was achieved in combination with cell type-specific promoter-driven transgene expression. Meanwhile, introducing the EB-oriP plasmid twice into the same individual embryos during separate embryonic development stages suggested heterogeneity of NPCs. In summary, IUE using the EB-oriP plasmid is a novel option to study the long-term development of the cerebral cortex in mice.

Molecular basis of the microtubule-regulating activity of microtubule crosslinking factor 1.

The variety of microtubule arrays observed across different cell types should require a diverse group of proteins that control microtubule organization. Nevertheless, mainly because of the intrinsic propensity of microtubules to easily form bundles upon stabilization, only a small number of microtubule crosslinking proteins have been identified, especially in postmitotic cells. Among them is microtubule crosslinking factor 1 (MTCL1) that not only interconnects microtubules via its N-terminal microtubule-binding domain (N-MTBD), but also stabilizes microtubules via its C-terminal microtubule-binding domain (C-MTBD). Here, we comprehensively analyzed the assembly structure of MTCL1 to elucidate the molecular basis of this dual activity in microtubule regulation. Our results indicate that MTCL1 forms a parallel dimer not only through multiple homo-interactions of the central coiled-coil motifs, but also the most C-terminal non-coiled-coil region immediately downstream of the C-MTBD. Among these homo-interaction regions, the first coiled-coil motif adjacent to N-MTBD is sufficient for the MTCL1 function to crosslink microtubules without affecting the dynamic property, and disruption of this motif drastically transformed MTCL1-induced microtubule assembly from tight to network-like bundles. Notably, suppression of the homo-interaction of this motif inhibited the endogenous MTCL1 function to stabilize Golgi-associated microtubules that are essential for Golgi-ribbon formation. Because the microtubule-stabilizing activity of MTCL1 is completely attributed to C-MTBD, the present study suggests possible interplay between N-MTBD and C-MTBD, in which normal crosslinking and accumulation of microtubules by N-MTBD is essential for microtubule stabilization by C-MTBD.

Regulatory mechanisms and cellular functions of non-centrosomal microtubules.

Proper organization of microtubule (MT) arrays is essential for numerous cellular functions, including intracellular transport and cell migration. Although the centrosome generally serves as the primary MT-organizing centre in proliferating animal cells, MTs are also organized at the Golgi apparatus in a wide range of cell types to regulate Golgi ribbon formation that is required for polarized cell migration. Furthermore, differentiated epithelial cells and neurons possess organized non-centrosomal MTs predominantly at the apical cortical regions and the axonal and dendritic neurites, respectively, to establish and maintain their highly polarized morphology. Unlike radial arrays of centrosomal MTs, non-centrosomal MTs are organized into non-radial asymmetric network, which facilitates polarized transport and cell polarization. In this review, we will focus on recent advances in our understanding of the regulatory mechanisms and cellular functions of non-centrosomal MTs.

MTCL1 plays an essential role in maintaining Purkinje neuron axon initial segment.

The axon initial segment (AIS) is a specialized domain essential for neuronal function, the formation of which begins with localization of an ankyrin-G (AnkG) scaffold. However, the mechanism directing and maintaining AnkG localization is largely unknown. In this study, we demonstrate that in vivo knockdown of microtubule cross-linking factor 1 (MTCL1) in cerebellar Purkinje cells causes loss of axonal polarity coupled with AnkG mislocalization. MTCL1 lacking MT-stabilizing activity failed to restore these defects, and stable MT bundles spanning the AIS were disorganized in knockdown cells. Interestingly, during early postnatal development, colocalization of MTCL1 with these stable MT bundles was observed prominently in the axon hillock and proximal axon. These results indicate that MTCL1-mediated formation of stable MT bundles is crucial for maintenance of AnkG localization. We also demonstrate that Mtcl1 gene disruption results in abnormal motor coordination with Purkinje cell degeneration, and provide evidence suggesting possible involvement of MTCL1 dysfunction in the pathogenesis of spinocerebellar ataxia.

The interaction of Kinesin-1 with its adaptor protein JIP1 can be regulated via proteins binding to the JIP1-PTB domain.

BACKGROUND: The regulatory mechanisms of motor protein-dependent intracellular transport are still not fully understood. The kinesin-1-binding protein, JIP1, can function as an adaptor protein that links kinesin-1 and other JIP1-binding "cargo" proteins. However, it is unknown whether these "cargo" proteins influence the JIP1-kinesin-1 binding. RESULTS: We show here that JIP1-kinesin-1 binding in Neuro2a cells was dependent on conserved amino acid residues in the JIP1-phosphotyrosine binding (PTB) domain, including F687. In addition, mutation of F687 severely affected the neurite tip localization of JIP1. Proteomic analysis revealed another kinesin-1 binding protein, JIP3, as a major JIP1 binding protein. The association between JIP1 and JIP3 was dependent on the F687 residue in JIP1, and this association induced the formation of a stable ternary complex with kinesin-1. On the other hand, the binding of JIP1 and JIP3 was independent of kinesin-1 binding. We also show that other PTB binding proteins can interrupt the formation of the ternary complex. CONCLUSIONS: The formation of the JIP1-kinesin-1 complex depends on the protein binding-status of the JIP1 PTB domain. This may imply a regulatory mechanism of kinesin-1-dependent intracellular transport.

Axon formation in neocortical neurons depends on stage-specific regulation of microtubule stability by the dual leucine zipper kinase-c-Jun N-terminal kinase pathway.

Studies using cultured neurons have established the critical role of microtubule regulators in neuronal polarization. The c-Jun N-terminal kinase (JNK) pathway is one of the candidate signaling pathways driving microtubule regulation during neuronal polarization. However, the significance of the JNK pathway in axon formation, a fundamental step in neuronal polarization, in vivo, remains unclear. Here, we provide evidence supporting the notion that the JNK pathway contributes to axon formation, in vivo, by identifying the genetic interactions between mouse JNK1 and dual leucine zipper kinase (DLK). Double mutants exhibited severe defects in axon formation in the cerebral neocortex. Moreover, RNA interference rescue experiments, in vitro, showed that DLK and JNK1 function in a common pathway to support neuronal polarization by promoting short-neurite and axon formation. Defects in axon formation caused by perturbations of the DLK-JNK pathway were significantly improved by Taxol. However, defects in short-neurite formation caused by perturbations of the DLK-JNK pathway were enhanced by Taxol. Together, these in vivo and in vitro observations indicate that the DLK-JNK pathway facilitates axon formation in neocortical neurons via stage-specific regulation of microtubule stability.

Identification of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible and -suppressive genes in the rat placenta: induction of interferon-regulated genes with possible inhibitory roles for angiogenesis in the placenta.

Exposure to a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in a variety of toxic manifestations, including fetal death. In order to evaluate the effects of low dose TCDD on placental function, pregnant Holtzman rats were given a single oral dose of 1600 ng TCDD/kg body wt or an equivalent volume of vehicle (control) on gestation day 15 (GD15), and changes in the gene expression in the placenta on GD20 were analyzed by two comprehensive methods, representational difference analysis (RDA) and DNA microarray technology. Candidates of TCDD-inducible and -suppressive genes were selected. Quantitative real-time PCR analysis was then performed to verify the induction or suppression levels of the candidate genes. Finally, we identified 81 TCDD-inducible and 21 TCDD-suppressive genes from the placenta of TCDD-treated Holtzman rats on GD20. One of the remarkable profiles of the gene expression was that glucose transporters were strongly up-regulated by the TCDD treatment. Furthermore, many interferon-inducible genes were also up-regulated by the treatment. They included several cytokines such as IP-10 known as a potent angiogenesis inhibitor. In addition, interferon molecules are known to suppress angiogenesis. The above observations suggest that activation of the interferon signaling pathway and the induction of anti-angiogenic factors by TCDD might have a role in causing the inhibition of neovascularization, resulting in the hypoxic state of placenta and increased incidence of fetal death.