Yoshiki Arakawa

ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts

The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632–induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632–induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.

Control of axon elongation via an SDF-1α/Rho/mDia pathway in cultured cerebellar granule neurons

Rho–GTPase has been implicated in axon outgrowth. However, not all of the critical steps controlled by Rho have been well characterized. Using cultured cerebellar granule neurons, we show here that stromal cell–derived factor (SDF)-1α, a neural chemokine, is a physiological ligand that can turn on two distinct Rho-dependent pathways with opposite consequences. A low concentration of the ligand stimulated a Rho-dependent pathway that mediated facilitation of axon elongation. In contrast, Rho/ROCK activation achieved by a higher concentration of SDF-1α caused repression of axon formation and induced no more increase in axon length. However, even at this higher concentration a Rho-dependent axon elongating activity could be recovered upon removal of ROCK activity using Y-27632. SDF-1α–induced axon elongating activity under ROCK inhibition was replicated by the dominant-active form of the mammalian homologue of the Drosophila gene Diaphanous (mDia)1 and counteracted by its dominant-negative form. Furthermore, RNAi knockdown of mDia1 abolished SDF-1α–induced axon elongation. Together, our results support a critical role for an SDF-1α/Rho/mDia1 pathway in mediating axon elongation.

The Rho-mDia1 Pathway Regulates Cell Polarity and Focal Adhesion Turnover in Migrating Cells through Mobilizing Apc and c-Src†

ABSTRACT Directed cell migration requires cell polarization and adhesion turnover, in which the actin cytoskeleton and microtubules work critically. The Rho GTPases induce specific types of actin cytoskeleton and regulate microtubule dynamics. In migrating cells, Cdc42 regulates cell polarity and Rac works in membrane protrusion. However, the role of Rho in migration is little known. Rho acts on two major effectors, ROCK and mDia1, among which mDia1 produces straight actin filaments and aligns microtubules. Here we depleted mDia1 by RNA interference and found that mDia1 depletion impaired directed migration of rat C6 glioma cells by inhibiting both cell polarization and adhesion turnover. Apc and active Cdc42, which work together for cell polarization, localized in the front of migrating cells, while active c-Src, which regulates adhesion turnover, localized in focal adhesions. mDia1 depletion impaired localization of these molecules at their respective sites. Conversely, expression of active mDia1 facilitated microtubule-dependent accumulation of Apc and active Cdc42 in the polar ends of the cells and actin-dependent recruitment of c-Src in adhesions. Thus, the Rho-mDia1 pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and delivering Apc/Cdc42 and c-Src to their respective sites of action.

Inhibition of the Rho/ROCK pathway reduces apoptosis during transplantation of embryonic stem cell-derived neural precursors.

Rho‐GTPase has been implicated in the apoptosis of many cell types, including neurons, but the mechanism by which it acts is not fully understood. Here, we investigate the roles of Rho and ROCK in apoptosis during transplantation of embryonic stem cell‐derived neural precursor cells. We find that dissociation of neural precursors activates Rho and induces apoptosis. Treatment with the Rho inhibitor C3 exoenzyme and/or the ROCK inhibitor Y‐27632 decreases the amount of dissociation‐induced apoptosis (anoikis) by 20–30%. Membrane blebbing, which is an early morphological sign of apoptosis; cleavage of caspase‐3; and release of cytochrome c from the mitochondria are also reduced by ROCK inhibition. These results suggest that dissociation of neural precursor cells elicits an intrinsic pathway of cell death that is at least partially mediated through the Rho/ROCK pathway. Moreover, in an animal transplantation model, inhibition of Rho and/or ROCK suppresses acute apoptosis of grafted cells. After transplantation, tumor necrosis factor‐α and pro‐nerve growth factor are strongly expressed around the graft. ROCK inhibition also suppresses apoptosis enhanced by these inflammatory cytokines. Taken together, these results indicate that inhibition of Rho/ROCK signaling may improve survival of grafted cells in cell replacement therapy.

Milrinone for the treatment of cerebral vasospasm after subarachnoid hemorrhage: report of seven cases.

OBJECTIVEThe intra-arterial infusion of papaverine has been used for dilation of spastic cerebral vessels after aneurysmal subarachnoid hemorrhage, although its efficacy is controversial. Milrinone is an inotropic drug that dilates vessels by phosphodiesterase inhibition in a mechanism similar to that of papaverine. We examined the effects of intra-arterial and subsequent intravenous administration of milrinone on patients with symptomatic cerebral vasospasm. METHODSSeven patients with cerebral vasospasm were enrolled in this study. Milrinone was delivered intra-arterially via catheter at a rate of 0.25 mg/min. The total delivered dose was between 2.5 and 15 mg. Radiological measurement of the middle cerebral artery diameter and cerebral blood flow was carried out before and after arterial infusion. Intravenous treatment followed at 0.50 or 0.75 &mgr;g/kg/min for up to 2 weeks from the onset of subarachnoid hemorrhage. RESULTSDilation of the vasospastic vessels occurred in all patients. The rate of cerebral blood flow was calculated in six patients and was increased in all. Subsequent intravenous infusion was effective in preventing a recurrence of symptomatic vasospasm in four of the seven patients. CONCLUSIONIt is suggested that milrinone was effective and safe for the treatment of cerebral vasospasm after subarachnoid hemorrhage in the patients in this series. Intra-arterial infusion with adjunctive intravenous infusion holds promise as a clinically advantageous treatment regimen.

Multiple spatiotemporal modes of actin reorganization by NMDA receptors and voltage-gated Ca2+ channels

Cytoskeleton is believed to contribute to activity-dependent processes underlying neuronal plasticity, such as regulations of cellular morphology and localization of signaling proteins. However, how neuronal activity controls actin cytoskeleton remains obscure. Taking advantage of confocal imaging of enhanced GFP-actin in the primary culture of hippocampal neurons, we show that synaptic activity induces multiple types of actin reorganization, both at the spines and at the somatic periphery. Activation of N-methyl-d-aspartate receptors, accompanied with a local rise in [Ca2+]i, was sufficient to trigger a slow and sustained recruitment of actin into dendritic spines. In contrast, opening of voltage-gated Ca2+ channels rapidly and reversibly enhanced cortical actin at the somatic periphery but not in the spines, in keeping with a high transient rise in somatic [Ca2+]i. These data suggest that spatiotemporal dynamics of [Ca2+]i, triggered by activation of N-methyl-d-aspartate receptors and voltage-gated Ca2+ channels, provides the molecular basis for activity-dependent actin remodeling.

In vivo fluorescence resonance energy transfer imaging reveals differential activation of Rho-family gtpases in glioblastoma cell invasion

Two-photon excitation microscopy was used to visualized two different modes of invasion at perivascular and intraparenchymal regions of rat C6 glioblastoma cells that were orthotopically implanted into rat brains. Probes based on the principle of Förster resonance energy transfer (FRET) further revealed that glioblastoma cells penetrating the brain parenchyma showed higher Rac1 and Cdc42 activities and lower RhoA activity than those advancing in the perivascular regions. This spatial regulation of Rho-family GTPase activities was recapitulated in three-dimensional spheroid invasion assays with rat and human glioblastoma cells, in which multipod glioblastoma cells that invaded the gels and led the other glioblastoma cells exhibited higher Rac1 and Cdc42 activities than the trailing glioblastoma cells. We also studied the Cdc42-specific guanine nucleotide exchange factor Zizimin1 (also known as DOCK9) as a possible contributor to this spatially controlled activation of Rho-family GTPases, because it is known to play an essential role in the extension of neurites. We found that shRNA-mediated knockdown of Zizimin1 inhibited formation of pseudopodia and concomitant invasion of glioblastoma cells both under a 3D culture condition and in vivo. Our results suggest that the difference in the activity balance of Rac1 and Cdc42 versus RhoA determines the mode of glioblastoma invasion and that Zizimin1 contributes to the invasiveness of glioblastoma cells with high Rac1 and Cdc42 activities.

Administration of ex vivo-expanded bone marrow-derived endothelial progenitor cells attenuates focal cerebral ischemia-reperfusion injury in rats.

OBJECTIVE:This study aimed to examine early effects of ex vivo-expanded bone marrow-derived endothelial progenitor cells (EPCs) on focal cerebral ischemia-reperfusion injury. METHODS:EPCs were obtained from mononuclear cells of autologous bone marrow of a rat. After culture on fibronectin-coated dishes for 10 to 14 days, 2.5 × 105 cells of EPCs were administered transarterially after 90 minute occlusion of the middle cerebral artery. RESULTS:Administration of EPCs significantly reduced both the infarct volume and the scores of neurological deficits at 24 and 48 hours. EPCs administered 2 hours after insult did not reduce infarct volume, but attenuated neurological deficits at 24 hours. Administration of EPCs significantly reduced the number of myeloperoxidase-immunoreactive cells in the ischemic lesion at 24 hours and increased regional cortical blood flow at 48 hours. EPCs were observed in the ischemic hemisphere and around the endothelial layer of the pial arteries. Most of them expressed endothelial nitric oxide synthase. CONCLUSION:Administration of ex vivo-expanded bone marrow-derived EPCs reduced infarct volume and neurological deficits in acute focal brain ischemia-reperfusion injury caused, at least in part, by attenuation of endothelial dysfunction.

Molecular Cloning and Characterization of CLICK-III/CaMKIγ, a Novel Membrane-anchored Neuronal Ca2+/Calmodulin-dependent Protein Kinase (CaMK)

During a screen for novel putative Ca2+/calmodulin-dependent protein kinase (CaMK)-like CREB kinases (CLICKs), we have cloned a full-length cDNA for CLICK-III/CaMKIγ, an isoform of the CaMKI family with an extended C-terminal domain ending with CAAX motif (whereAA is aliphatic acid). As expected from the similarity of its kinase domain with the other CaMKI isoforms, full activation of CLICK-III/CaMKIγ required both Ca2+/CaM and phosphorylation by CaMKK. We also found that Ca2+/cAMP-response element-binding protein (CREB) was a good substrate for CLICK-III/CaMKIγ, at least in vitro. Interestingly enough, CLICK-III/CaMKIγ transcripts were most abundant in neurons, with the highest levels in limited nuclei such as the central nucleus of the amygdala (CeA) and the ventromedial hypothalamus. Consistent with the presence of the CAAXmotif, CLICK-III/CaMKIγ was found to be anchored to various membrane compartments, especially to Golgi and plasma membranes. Both point mutation in the CAAX motif and treatment with compactin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, disrupted such membrane localization, suggesting that membrane localization of CLICK-III/CaMKIγ occurred in a prenylation-dependent way. These findings provide a novel mechanism by which neuronal CaMK activity could be targeted to specific membrane compartments.

Intraoperative dorsal language network mapping by using single‐pulse electrical stimulation

The preservation of language function during brain surgery still poses a challenge. No intraoperative methods have been established to monitor the language network reliably. We aimed to establish intraoperative language network monitoring by means of cortico‐cortical evoked potentials (CCEPs). Subjects were six patients with tumors located close to the arcuate fasciculus (AF) in the language‐dominant left hemisphere. Under general anesthesia, the anterior perisylvian language area (AL) was first defined by the CCEP connectivity patterns between the ventrolateral frontal and temporoparietal area, and also by presurgical neuroimaging findings. We then monitored the integrity of the language network by stimulating AL and by recording CCEPs from the posterior perisylvian language area (PL) consecutively during both general anesthesia and awake condition. High‐frequency electrical stimulation (ES) performed during awake craniotomy confirmed language function at AL in all six patients. Despite an amplitude decline (≤32%) in two patients, CCEP monitoring successfully prevented persistent language impairment. After tumor removal, single‐pulse ES was applied to the white matter tract beneath the floor of the removal cavity in five patients, in order to trace its connections into the language cortices. In three patients in whom high‐frequency ES of the white matter produced naming impairment, this “eloquent” subcortical site directly connected AL and PL, judging from the latencies and distributions of cortico‐ and subcortico‐cortical evoked potentials. In conclusion, this study provided the direct evidence that AL, PL, and AF constitute the dorsal language network. Intraoperative CCEP monitoring is clinically useful for evaluating the integrity of the language network. Hum Brain Mapp 35:4345–4361, 2014.