Tomohiro Torii

Evaluation of drug toxicity with hepatocytes cultured in a micro-space cell culture system.

A micro-space cell culture system was recently developed in which cells such as hepatocytes can be cultured and formed into a multicellular three-dimensional (3D) architecture. In this study, we assessed the performance of HepG2 cells cultured in this micro-space cell culture system in a drug toxicity test, and evaluated the effects of micro-space culture on their hepatocyte-specific functions. The micro-space cell culture facilitated the formation of 3D HepG2 cell architecture. HepG2 cells cultured in a micro-space culture plate exhibited increased albumin secretion and enhanced mRNA expression levels of cytochrome P450 (CYP) enzyme compared to those cultured in a monolayer culture. When the cells were exposed to acetaminophen, a hepatotoxic drug, the damage to the HepG2 cells grown in micro-space culture was greater than the damage to the HepG2 cells grown in monolayer culture. In addition, human primary hepatocytes grown in micro-space culture also exhibited increased albumin secretion, enhanced CYP mRNA expression levels and increased sensitivity to acetaminophen compared to those grown in monolayer culture. These results suggest that this micro-space culture method enhances the hepatocyte-specific functions of hepatocytes, including drug-metabolizing enzyme activities, making hepatocytes grown in the micro-space culture system a useful tool for evaluating drug toxicity in vitro.

Cytohesin-2/ARNO, through Its Interaction with Focal Adhesion Adaptor Protein Paxillin, Regulates Preadipocyte Migration via the Downstream Activation of Arf6

The formation of primitive adipose tissue is the initial process in adipose tissue development followed by the migration of preadipocytes into adipocyte clusters. Comparatively little is known about the molecular mechanism controlling preadipocyte migration. Here, we show that cytohesin-2, the guanine-nucleotide exchange factor for the Arf family GTP-binding proteins, regulates migration of mouse preadipocyte 3T3-L1 cells through Arf6. SecinH3, a specific inhibitor of the cytohesin family, markedly inhibits migration of 3T3-L1 cells. 3T3-L1 cells express cytohesin-2 and cytohesin-3, and knockdown of cytohesin-2 with its small interfering RNA effectively decreases cell migration. Cytohesin-2 preferentially acts upstream of Arf6 in this signaling pathway. Furthermore, we find that the focal adhesion protein paxillin forms a complex with cytohesin-2. Paxillin colocalizes with cytohesin-2 at the leading edges of migrating cells. This interaction is mediated by the LIM2 domain of paxillin and the isolated polybasic region of cytohesin-2. Importantly, migration is inhibited by expression of the constructs containing these regions. These results suggest that cytohesin-2, through a previously unexplored complex formation with paxillin, regulates preadipocyte migration and that paxillin plays a previously unknown role as a scaffold protein of Arf guanine-nucleotide exchange factor.

Akt and PP2A Reciprocally Regulate the Guanine Nucleotide Exchange Factor Dock6 to Control Axon Growth of Sensory Neurons

The phosphorylation status of Dock6 causes sensory neuron axons to switch from extension to branching. Switching from Axon Extension to Branching Understanding the signaling pathways that enable neurons, such as dorsal root ganglion neurons, to extend axons into the periphery can identify potential therapeutic targets to promote nerve regeneration after injury. Miyamoto et al. found that the guanine nucleotide exchange factor Dock6, which regulates the cytoskeleton, was required for axonal extension during development and after nerve crush injury. Phosphorylation of Dock6 by the kinase Akt inhibited its activity; dephosphorylation by the phosphatase PP2A promoted Dock6 activity. During development, peripheral sensory neurons extend axons until the axons contact their targets, at which point they branch. During developmental stages when axons were growing, extending periods were associated with the interaction of PP2A with Dock6 and low Dock6 phosphorylation, whereas periods of branching were associated with the interaction of Akt with Dock6 and high Dock6 phosphorylation. Rescue experiments indicated that the phosphorylation status of Dock6 switched dorsal root ganglion neurons from axon extension to branching. Thus, regulation of the phosphorylation status of Dock6 by Akt and PP2A determines whether it promotes axon extension or branching in dorsal root ganglion neurons, and timing of treatments targeting this pathway for nerve regeneration is essential to proper reinnervation. During neuronal development, axons navigate long distances, eventually forming precise connections with such targets as peripheral tissues. Dock6 is a guanine nucleotide exchange factor (GEF) that activates the Rho family guanosine triphosphatases Rac1 and Cdc42 to regulate the actin cytoskeleton. We found that phosphorylation of Ser1194 in Dock6 inhibited its GEF activity and suppressed axonal growth of embryonic sensory neurons and axon regeneration of postnatal sensory neurons in vitro and in vivo. At early developmental stages, when axons are growing, the protein phosphatase PP2A interacted with and dephosphorylated Dock6, thereby increasing the activity of Dock6. At later developmental stages, the abundance of the kinase Akt increased, resulting in the binding of Akt to Dock6 and the phosphorylation of Dock6 at Ser1194. In dorsal root ganglion neurons from mice lacking Dock6, reintroduction of Dock6 with a nonphosphorylatable S1194A mutation rescued axon extension but not branch number, whereas reintroduction of Dock6 with a phosphomimetic S1194E mutation resulted in premature branching. Thus, the phosphorylation status of Dock6 at Ser1194 determines whether it promotes axon extension or branching in sensory neurons, revealing interplay between kinase and phosphatase action on a Rho-GEF during axon growth.

Phosphorylation of cytohesin-1 by Fyn is required for initiation of myelination and the extent of myelination during development.

Transgenic and knockout mice reveal a pathway that controls the extent of myelination by Schwann cells. Promoting Myelination Through Phosphorylation In response to signals from axons in the peripheral nervous system, Schwann cells remodel their plasma membrane and wrap it around the length of the axon to produce myelin, which increases the efficiency with which axons conduct action potentials. Yamauchi et al. investigated signaling within Schwann cells and found that the guanine nucleotide exchange factor cytohesin-1 was required for efficient myelination. The tyrosine kinase Fyn phosphorylated and activated cytohesin-1, leading to activation of the guanosine triphosphatase Arf6 and changes in cellular morphology. Mice expressing a mutant cytohesin-1 devoid of the Fyn target site in Schwann cells had axons with decreased myelin thickness compared to those in their wild-type counterparts. This work identifies critical steps in the pathway leading from signals produced by neurons to myelination by Schwann cells, which could have implications for demyelinating diseases. Schwann cells respond to cues from axons by transforming their cellular morphology and forming myelin. We demonstrated that the guanine nucleotide exchange factor (GEF) cytohesin-1 promoted myelination by activating the small guanosine triphosphatase (GTPase) Arf6. In mice, ablating cytohesin-1 delayed myelination and diminished the amount of myelin produced. We determined that the Src-family kinase Fyn phosphorylated tyrosine 382 (Y382) of cytohesin-1, and we generated transgenic mice that expressed a Schwann cell–specific phosphorylation mutant of cytohesin-1 (Y382F) that could not be targeted by Fyn. During development, these transgenic mice displayed delayed myelination compared to that of wild-type mice, as well as a decrease in the amount of myelin produced, similar to that observed in cytohesin-1−/− mice. These findings demonstrate that phosphorylation of cytohesin-1 by Fyn is required for full myelination and suggest that tyrosine phosphorylation of GEFs may be a mechanism to activate small GTPases engaged in cell morphogenesis.

The mood stabilizer valproic acid improves defective neurite formation caused by charcot‐marie‐tooth disease‐associated mutant Rab7 through the JNK signaling pathway

Charcot‐Marie‐Tooth (CMT) disease is the most frequent peripheral neuropathy affecting the Schwann cells and neurons. CMT disease type 2 (CMT2) neuropathies are characterized by peripheral nerve aberrance. Four missense mutations of Rab7, a small GTPase of the Rab family involved in intracellular vesicular trafficking, are associated with the CMT2B phenotype. Despite a growing body of evidence concerning the gene structures responsible for genetically heterogenous CMT2B and other CMT2 neuropathies, little is known about the in vitro neuropathy model and how CMT2B‐associated mutation‐caused aberrant neuritogenesis is properly reversed. Here, we show that valproic acid (VPA), a classical mood‐stabilizing drug, improves defective neurite formation in N1E‐115 neuroblastoma cells regardless of which CMT2B‐associated Rab7 mutant protein is expressed. The effect is mediated by c‐Jun N‐terminal kinase (JNK) signaling, but not by deacetylase inhibition activity of VPA itself. Furthermore, VPA has similar effects in dorsal root ganglion (DRG) neurons expressing any of the four mutant Rab7 proteins. Thus, VPA has a previously unknown potential to improve defective neuritogenesis associated with CMT2B in vitro, indicating that JNK should be a potential therapeutic target for treatments aimed at improving neuritogenesis.

The Atypical Guanine-Nucleotide Exchange Factor, Dock7, Negatively Regulates Schwann Cell Differentiation and Myelination

In development of the peripheral nervous system, Schwann cells proliferate, migrate, and ultimately differentiate to form myelin sheath. In all of the myelination stages, Schwann cells continuously undergo morphological changes; however, little is known about their underlying molecular mechanisms. We previously cloned the dock7 gene encoding the atypical Rho family guanine-nucleotide exchange factor (GEF) and reported the positive role of Dock7, the target Rho GTPases Rac/Cdc42, and the downstream c-Jun N-terminal kinase in Schwann cell migration (Yamauchi et al., 2008). We investigated the role of Dock7 in Schwann cell differentiation and myelination. Knockdown of Dock7 by the specific small interfering (si)RNA in primary Schwann cells promotes dibutyryl cAMP-induced morphological differentiation, indicating the negative role of Dock7 in Schwann cell differentiation. It also results in a shorter duration of activation of Rac/Cdc42 and JNK, which is the negative regulator of myelination, and the earlier activation of Rho and Rho-kinase, which is the positive regulator of myelination. To obtain the in vivo evidence, we generated Dock7 short hairpin (sh)RNA transgenic mice. They exhibited a decreased expression of Dock7 in the sciatic nerves and enhanced myelin thickness, consistent with in vitro observation. The effects of the in vivo knockdown on the signals to Rho GTPases are similar to those of the in vitro knockdown. Collectively, the signaling through Dock7 negatively regulates Schwann cell differentiation and the onset of myelination, demonstrating the unexpected role of Dock7 in the interplay between Schwann cell migration and myelination.

Valproic acid-inducible Arl4D and cytohesin-2/ARNO, acting through the downstream Arf6, regulate neurite outgrowth in N1E-115 cells.

The mood-stabilizing agent valproic acid (VPA) potently promotes neuronal differentiation. As yet, however, little is known about the underlying molecular mechanism. Here, we show that VPA upregulates cytohesin-2 and mediates neurite outgrowth in N1E-115 neuroblastoma cells. Cytohesin-2 is the guanine-nucleotide exchange factor (GEF) for small GTPases of the Arf family; it regulates many aspects of cellular functions including morphological changes. Treatment with the specific cytohesin family inhibitor SecinH3 or knockdown of cytohesin-2 with its siRNA results in blunted induction of neurite outgrowth in N1E-115 cells. The outgrowth is specifically inhibited by siRNA knockdown of Arf6, but not by that of Arf1. Furthermore, VPA upregulates Arl4D, an Arf-like small GTPase that has recently been identified as the regulator that binds to cytohesin-2. Arl4D knockdown displays an inhibitory effect on neurite outgrowth resulting from VPA, while expression of constitutively active Arl4D induces outgrowth. We also demonstrate that the addition of cell-permeable peptide, coupling the cytohesin-2-binding region of Arl4D into cells, reduces the effect of VPA. Thus, Arl4D is a previously unknown regulator of neurite formation through cytohesin-2 and Arf6, providing another example that the functional interaction of two different small GTPases controls an important cellular function.

Rab35, acting through ACAP2 switching off Arf6, negatively regulates oligodendrocyte differentiation and myelination

Oligodendrocyte precursor cells differentiate into oligodendrocytes to form myelin sheaths. Rab35/ACAP2 and cytohesin-2 antagonistically control oligodendrocyte differentiation and myelination through Arf6 on/off regulation, presenting a unique way of regulating oligodendrocyte differentiation and myelination by a small GTPase network.

Sorting nexin 3, a protein upregulated by lithium, contains a novel phosphatidylinositol-binding sequence and mediates neurite outgrowth in N1E-115 cells.

Lithium, a drug in the treatment of bipolar disorder, modulates many aspects of neuronal developmental processes such as neurogenesis, survival, and neuritogenesis. However, the underlying mechanism still remains to be understood. Here, we show that lithium upregulates the expression of sorting nexin 3 (SNX3), one of the Phox (PX) domain-containing proteins involved in endosomal sorting, and regulates neurite outgrowth in mouse N1E-115 neuroblastoma cells. The inhibition of SNX3 function by its knockdown decreases lithium-induced outgrowth of neurites. Transfection of the full-length SNX3 construct into cells facilitates the outgrowth. We also find that the C-terminus, as well as the PX domain, of SNX3 has a functional binding sequence with phosphatidylinositol monophosphates. Transfection of the C-terminal deletion mutant or only the C-terminus does not have an effect on the outgrowth. These results suggest that SNX3, a protein upregulated by lithium, is an as yet unknown regulator of neurite formation and that it contains another functional phosphatidylinositol phosphate-binding region at the C-terminus.

Pelizaeus–Merzbacher disease: Cellular pathogenesis and pharmacologic therapy

Pelizaeus–Merzbacher disease (PMD) is a rare leukodystrophy that causes severe dysmyelination in the central nervous system in infancy and early childhood. Many previous studies showed that various proteolipid protein 1 (plp1) mutations, including duplications, point mutations, and deletions, lead to oligodendrocyte dysfunction in patients with PMD. PMD onset and clinical severity range widely, depending on the type of plp1 mutation. Patients with PMD exhibit a delayed mental and physical development phenotype, but specific pharmacological therapy and clinical treatment for PMD are not yet well established. This review describes PMD pathology and establishment of new clinical treatment for PMD. These findings support the development of a new therapy for PMD and these treatments may improve the quality of life in patients with PMD.