Rab21 regulates caveolin-1-mediated endocytic trafficking to promote immature neurite pruning.

Transmembrane proteins are internalized by clathrin- and caveolin-dependent endocytosis. Both pathways converge on early endosomes and are thought to share the small GTPase Rab5 as common regulator. In contrast to this notion, we show here that the clathrin- and caveolin-mediated endocytic pathways are differentially regulated. Rab5 and Rab21 localize to distinct populations of early endosomes in cortical neurons and preferentially regulate clathrin- and caveolin-mediated pathways, respectively, suggesting heterogeneity in the early endosomes, rather than a converging point. Suppression of Rab21, but not Rab5, results in decreased plasma membrane localization and total protein levels of caveolin-1, which perturbs immature neurite pruning of cortical neurons, an in vivo-specific step of neuronal maturation. Taken together, our data indicate that clathrin- and caveolin-mediated endocytic pathways run in parallel in early endosomes, which show different molecular regulation and physiological function.

Caveolin-1 Promotes Early Neuronal Maturation via Caveolae-Independent Trafficking of N-Cadherin and L1.

Axon specification is morphologically reproducible in vitro, whereas dendrite formation differs in vitro and in vivo. Cortical neurons initially develop immature neurites, but in vivo these are eliminated concurrently with the formation of a leading process, the future dendrite. However, the molecular mechanisms underlying these neuronal maturation events remain unclear. Here we show that caveolin-1, a major component of caveolae that are never observed in neurons, regulates in vivo-specific steps of neuronal maturation. Caveolin-1 is predominantly expressed in immature cortical neurons and regulates clathrin-independent endocytosis. In vivo knockdown of caveolin-1 disturbs immature neurite pruning, leading process elongation, and subsequent neuronal migration. Importantly, N-cadherin and L1, which are required for immature neurite formation, undergo caveolin-1-mediated endocytosis to eliminate immature neurites. Collectively, our findings indicate that caveolin-1 regulates N-cadherin and L1 trafficking independent of caveolae, which contributes to spatiotemporally restricted cellular events; immature neurite pruning and leading process elongation during early neuronal maturation.

Rab family small GTPases-mediated regulation of intracellular logistics in neural development.

Rab family small GTPases play essential roles in various cellular events via the regulation of intracellular logistics comprising a large number of membrane traffic pathways. Emerging evidence reveals the physiological roles of Rab proteins in several tissues, including developing brains. Many Rab proteins, such as Rab5, Rab6, Rab7, Rab8, Rab10, Rab11, Rab17 and Rab18, are shown to regulate neurite outgrowth in PC12 cells and/or axon and dendrite formation in primary cultured neurons. Recent studies have also revealed in vivo roles of several Rab family small GTPases in brain development and its related neurological disorders. In this review, we introduce the physiological function of Rab family proteins in neural development with particular focus on neurite outgrowth and neuronal migration.

Drosophila Strip serves as a platform for early endosome organization during axon elongation.

Early endosomes are essential for regulating cell signalling and controlling the amount of cell surface molecules during neuronal morphogenesis. Early endosomes undergo retrograde transport (clustering) before their homotypic fusion. Small GTPase Rab5 is known to promote early endosomal fusion, but the mechanism linking the transport/clustering with Rab5 activity is unclear. Here we show that Drosophila Strip is a key regulator for neuronal morphogenesis. Strip knockdown disturbs the early endosome clustering, and Rab5-positive early endosomes become smaller and scattered. Strip genetically and biochemically interacts with both Glued (the regulator of dynein-dependent transport) and Sprint (the guanine nucleotide exchange factor for Rab5), suggesting that Strip is a molecular linker between retrograde transport and Rab5 activation. Overexpression of an active form of Rab5 in strip-mutant neurons suppresses the axon elongation defects. Thus, Strip acts as a molecular platform for the early endosome organization that has important roles in neuronal morphogenesis.

Cdk5 and its substrates, Dcx and p27kip1, regulate cytoplasmic dilation formation and nuclear elongation in migrating neurons.

Neuronal migration is crucial for development of the mammalian-specific six-layered cerebral cortex. Migrating neurons are known to exhibit distinct features; they form a cytoplasmic dilation, a structure specific to migrating neurons, at the proximal region of the leading process, followed by nuclear elongation and forward movement. However, the molecular mechanisms of dilation formation and nuclear elongation remain unclear. Using ex vivo chemical inhibitor experiments, we show here that rottlerin, which is widely used as a specific inhibitor for PKCδ, suppresses the formation of a cytoplasmic dilation and nuclear elongation in cortical migrating neurons. Although our previous study showed that cortical neuronal migration depends on Jnk, another downstream target of rottlerin, Jnk inhibition disturbs only the nuclear elongation and forward movement, but not the dilation formation. We found that an unconventional cyclin-dependent kinase, Cdk5, is a novel downstream target of rottlerin, and that pharmacological or knockdown-mediated inhibition of Cdk5 suppresses both the dilation formation and nuclear elongation. We also show that Cdk5 inhibition perturbs endocytic trafficking as well as microtubule organization, both of which have been shown to be required for dilation formation. Furthermore, knockdown of Dcx, a Cdk5 substrate involved in microtubule organization and membrane trafficking, or p27(kip1), another Cdk5 substrate involved in actin and microtubule organization, disturbs the dilation formation and nuclear elongation. These data suggest that Cdk5 and its substrates, Dcx and p27(kip1), characterize migrating neuron-specific features, cytoplasmic dilation formation and nuclear elongation in the mouse cerebral cortex, possibly through the regulation of microtubule organization and an endocytic pathway.

Extra-cell cycle regulatory functions of cyclin-dependent kinases (CDK) and CDK inhibitor proteins contribute to brain development and neurological disorders.

In developing brains, neural progenitors exhibit cell cycle-dependent nuclear movement within the ventricular zone [interkinetic nuclear migration (INM)] and actively proliferate to produce daughter progenitors and/or neurons, whereas newly generated neurons exit from the cell cycle and begin pial surface-directed migration and maturation. Dysregulation of the balance between the proliferation and the cell cycle exit in neural progenitors is one of the major causes of microcephaly (small brain). Recent studies indicate that cell cycle machinery influences not only the proliferation but also INM in neural progenitors. Furthermore, several cell cycle-related proteins, including p27(kip1) , p57(kip2) , Cdk5, and Rb, regulate the migration of neurons in the postmitotic state, suggesting that the growth arrest confers dual functions on cell cycle regulators. Consistently, several types of microcephaly occur in conjunction with neuronal migration disorders, such as periventricular heterotopia and lissencephaly. However, cell cycle re-entry by disturbance of growth arrest in mature neurons is thought to trigger neuronal cell death in Alzheimer's disease. In this review, we introduce the cell cycle protein-mediated regulation of two types of nuclear movement, INM and neuronal migration, during cerebral cortical development, and discuss the roles of growth arrest in cortical development and neurological disorders.

Induction of cancerous stem cells during embryonic stem cell differentiation.

Stem cell maintenance depends on their surrounding microenvironment, and aberrancies in the environment have been associated with tumorigenesis. However, it remains to be elucidated whether an environmental aberrancy can act as a carcinogenic stress for cellular transformation of differentiating stem cells into cancer stem cells. Here, utilizing mouse embryonic stem cells as a model, it was illustrated that environmental aberrancy during differentiation leads to the emergence of pluripotent cells showing cancerous characteristics. Analogous to precancerous stages, DNA lesions were spontaneously accumulated during embryonic stem cell differentiation under aberrational environments, which activates barrier responses such as senescence and apoptosis. However, overwhelming such barrier responses, piled-up spheres were subsequently induced from the previously senescent cells. The sphere cells exhibit aneuploidy and dysfunction of the Arf-p53 module as well as enhanced tumorigenicity and a strong self-renewal capacity, suggesting development of cancerous stem cells. Our current study suggests that stem cells differentiating in an aberrational environment are at risk of cellular transformation into malignant counterparts.

N-cadherin regulates radial glial fiber-dependent migration of cortical locomoting neurons.

During cerebral cortical development, post-mitotic neurons exhibit a multi-step migration. The locomotion mode covers most of the neuronal migration path. Although for many decades, locomoting neurons have been known to migrate along radial glial fibers, how the cortical locomoting neurons attach to and migrate along radial glial fibers was largely unknown. We recently reported that N-cadherin is required for cortical neuronal migration in vivo. Knockdown or dominant negative inhibition of N-cadherin results in severe neuronal migration defects. Furthermore, suppression of Rab5-dependent endocytosis increases cell surface levels of N-cadherin and perturbs neuronal migration. We showed here that N-cadherin overexpression, which would mimic Rab5 suppression, weakly suppressed neuronal migration, suggesting that excess N-cadherin also disturbs neuronal migration. Interestingly, however, N-cadherin knockdown and overexpression in neurons resulted in different morphologies. While N-cadherin-overexpressing cells closely attached to the radial glial fibers similar to control or Rab5-knockdown cells, N-cadherin knockdown weakened the attachment as the average distance between the soma and radial glial fibers was significantly increased. Taken together, these findings suggest that N-cadherin controls the neuronal attachment to radial glial fibers and that N-cadherin-mediated adhesion complexes are reconstituted through Rab GTPases-dependent endocytic pathways to maintain the proper surface N-cadherin level and to promote neuronal migration.

Onset of quiescence following p53 mediated down-regulation of H2AX in normal cells.

Normal cells, both in vivo and in vitro, become quiescent after serial cell proliferation. During this process, cells can develop immortality with genomic instability, although the mechanisms by which this is regulated are unclear. Here, we show that a growth-arrested cellular status is produced by the down-regulation of histone H2AX in normal cells. Normal mouse embryonic fibroblast cells preserve an H2AX diminished quiescent status through p53 regulation and stable-diploidy maintenance. However, such quiescence is abrogated under continuous growth stimulation, inducing DNA replication stress. Because DNA replication stress-associated lesions are cryptogenic and capable of mediating chromosome-bridge formation and cytokinesis failure, this results in tetraploidization. Arf/p53 module-mutation is induced during tetraploidization with the resulting H2AX recovery and immortality acquisition. Thus, although cellular homeostasis is preserved under quiescence with stable diploidy, tetraploidization induced under growth stimulation disrupts the homeostasis and triggers immortality acquisition.

Rab GTPases-dependent endocytic pathways regulate neuronal migration and maturation through N-cadherin trafficking.

Although membrane trafficking pathways are involved in basic cellular functions, the evolutionally expanded number of their related family proteins suggests additional roles for membrane trafficking in higher organisms. Here, we show that several Rab-dependent trafficking pathways differentially participate in neuronal migration, an essential step for the formation of the mammalian-specific six-layered brain structure. In vivo electroporation-mediated suppression of Rab5 or dynamin to block endocytosis caused a severe neuronal migration defect in mouse cerebral cortex. Among many downstream endocytic pathways, suppression of Rab11-dependent recycling pathways exhibited a similar migration disorder, whereas inhibition of Rab7-dependent lysosomal degradation pathways affected only the final phase of neuronal migration and dendrite morphology. Inhibition of Rab5 or Rab11 perturbed the trafficking of N-cadherin, whose suppression also disturbed neuronal migration. Taken together, our findings reveal physiological roles of endocytic pathways, each of which has specific functions in distinct steps of neuronal migration and maturation during mammalian brain formation.

A novel method of mouse ex utero transplantation of hepatic progenitor cells into the fetal liver.

Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or alpha-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells became mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.