Kazuhiko Nozaki

Secreted Klotho protein in sera and CSF: implication for post-translational cleavage in release of Klotho protein from cell membrane

Klotho mutant mice exhibit a set of phenotypes resembling human ageing. Although the function of Klotho remains unclear, mediation of its pleiotropic functions by putative humoral factor(s) has been presumed. Newly established antibodies against Klotho allowed the detection of secreted Klotho, a candidate for the putative humoral factor, in sera and cerebrospinal fluid. Surprisingly the secreted Klotho was 130 kDa, in contrast to the 70 kDa predicted form from klotho gene transcripts. The secreted as well as the membrane‐bound Klotho proteins were suggested to form oligomerized complex. These results delineate post‐translation processing of Klotho and possible regulatory mechanisms for secretion of Klotho in vivo.

Alpha-Klotho as a regulator of calcium homeostasis

α-klotho was identified as a gene associated with premature aging–like phenotypes characterized by short lifespan. In mice, we found the molecular association of α-Klotho (α-Kl) and Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) and provide evidence for an increase of abundance of Na+,K+-ATPase at the plasma membrane. Low concentrations of extracellular free calcium ([Ca2+]e) rapidly induce regulated parathyroid hormone (PTH) secretion in an α-Kl- and Na+,K+-ATPase–dependent manner. The increased Na+ gradient created by Na+,K+-ATPase activity might drive the transepithelial transport of Ca2+ in cooperation with ion channels and transporters in the choroid plexus and the kidney. Our findings reveal fundamental roles of α-Kl in the regulation of calcium metabolism.

Proliferation of neuronal precursor cells in the dentate gyrus is accelerated after transient forebrain ischemia in mice

We investigated the proliferation of neuronal progenitor cells by labeling dividing cells by systemic application of the thymidine analog 5-bromodeoxyuridine (BrdU) during transient forebrain ischemia in mice. At 3 (n=6), 7 (n=6), 10 (n=6), and 17 days (n=6) after reperfusion, BrdU-labeled cells were detected in the dentate gyrus and paraventricle lesion. After ischemia-reperfusion, BrdU-labeled cells in the dentate gyrus significantly increased in number but not in the paraventricle lesion. These observations may help to clarify the mechanism of functional recovery after stroke.

Prevention of Rat Cerebral Aneurysm Formation by Inhibition of Nitric Oxide Synthase

BACKGROUND Cerebral saccular aneurysm is a major cause of subarachnoid hemorrhage, one of the cerebrovascular diseases with the highest mortality. The mechanisms underlying the development of aneurysms, however, still remain unclear. We have made a series of reports on an animal model of experimentally induced cerebral aneurysms that resemble human cerebral aneurysms in their location and morphology, suggesting that the arterial wall degeneration associated with aneurysm formation develops near the apex of arterial bifurcation as a result of an increase in wall shear stress. Using the animal model and human specimens, we examined the role of nitric oxide (NO) in the degenerative changes and cerebral aneurysm formation. METHODS AND RESULTS Inducible NO synthase (iNOS) was immunohistochemically located at the orifice of human and rat aneurysms. Nitrotyrosine distribution was also seen in the human aneurysm. Although no iNOS immunostaining was found in normal arteries, iNOS immunoreactivity was observed in parallel with the development of early aneurysmal changes in rats. In contrast, during the early development of aneurysm, endothelial NOS immunostaining in the endothelium was weakened compared with that in the control arteries. An NOS inhibitor, aminoguanidine, attenuated both early aneurysmal changes and the incidence of induced aneurysms. A defibrinogenic agent, batroxobin, which may diminish shear stress by reduction of blood viscosity, prevented iNOS induction as well as early aneurysmal changes. CONCLUSIONS The evidence suggests that NO, particularly that derived from iNOS, is a key requirement for the development of cerebral aneurysm. The iNOS induction may be caused by an increase in shear stress near the apex.

Macrophage-Derived Matrix Metalloproteinase-2 and -9 Promote the Progression of Cerebral Aneurysms in Rats

Background and Purpose— Mechanisms of initiation, progression and rupture of cerebral aneurysms have not yet been fully understood despite its clinical significance. Matrix metalloproteinases (MMPs) are a family of proteinases which are involved in the remodeling of vascular walls. In the present study, we investigated the significance of MMPs in the progression of cerebral aneurysms. Methods— Cerebral aneurysms were experimentally induced in 7-week-old male Sprague-Dawley rats. MMP-2 and MMP-9 expression was examined by immunohistochemistry and RT-PCR. Gelatinase activity in aneurysmal walls was assessed by in situ zymography. A selective inhibitor for MMP-2, -9 and -12, tolylsam, was used to examine the effect of inhibition of MMP-2 and MMP-9. Results— Macrophages infiltrated in arterial walls of experimentally induced rat cerebral aneurysms and expressed MMP-2 and -9. Macrophage infiltration and MMP expression was increased with the progression of aneurysms. Gelatinase activity attributable to MMP-2 and MMP-9 increased in arterial walls of rat cerebral aneurysms. Furthermore, tolylsam reduced the ratio of advanced aneurysms in our rat model. Conclusions— These data suggest that macrophage-derived MMP-2 and -9 may play an important role in the progression of cerebral aneurysms. The findings of this study will shed a new light into the pathogenesis of cerebral aneurysms and highlight the importance of inflammatory response causing the degeneration of extracellular matrix in the process of this disease.

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.

NF-κB Is a Key Mediator of Cerebral Aneurysm Formation

Background— Subarachnoid hemorrhage caused by the rupture of cerebral aneurysm (CA) remains a life-threatening disease despite recent diagnostic and therapeutic advancements. Recent studies strongly suggest the active participation of macrophage-mediated chronic inflammatory response in the pathogenesis of CA. We examined the role of nuclear factor-&kgr;B (NF-&kgr;B) in the pathogenesis of CA formation in this study. Methods and Results— In experimentally induced CAs in rats, NF-&kgr;B was activated in cerebral arterial walls in the early stage of aneurysm formation with upregulated expression of downstream genes. NF-&kgr;B p50 subunit–deficient mice showed a decreased incidence of CA formation with less macrophage infiltration into the arterial wall. NF-&kgr;B decoy oligodeoxynucleotide also prevented CA formation when it was administered at the early stage of aneurysm formation in rats. Macrophage infiltration and expression of downstream genes were dramatically inhibited by NF-&kgr;B decoy oligodeoxynucleotide. In human CA walls, NF-&kgr;B also was activated, especially in the intima. Conclusions— Our data indicate that NF-&kgr;B plays a crucial role as a key regulator in the initiation of CA development by inducing some inflammatory genes related to macrophage recruitment and activation. NF-&kgr;B may represent a therapeutic target of a novel medical treatment for CA.

Risk factors for subsequent hemorrhage in patients with cerebral arteriovenous malformations

OBJECT The aim of this study was to identify the natural history of untreated cerebral arteriovenous malformations (AVMs) and the risk factors for subsequent hemorrhage after an initial AVM diagnosis. METHODS The authors studied 305 consecutive patients with AVMs at the Kyoto University Hospital between 1983 and 2005. These patients were followed up until the first subsequent hemorrhage, the start of any treatment, or the end of 2005. Possible risk factors that were investigated included age at initial diagnosis, sex, type of initial presentation, size and location of the AVM nidus, and the venous drainage pattern. Subsequent hemorrhage occurred in 26 patients from the hemorrhagic group during 380 patient-years, and in 16 patients from the nonhemorrhagic group during 512 patient-years. RESULTS The annual bleeding rate in the hemorrhagic group was 6.84% after the initial hemorrhage; however, that rate decreased in the first 5 years (15.42% in the first year, 5.32% in the subsequent 4 years, and 1.72% in more than 5 years). In the nonhemorrhagic group (annual bleeding rate of 3.12%), the patients initially presenting with headaches (annual bleeding rate of 6.48%) or asymptomatic presentations (annual bleeding rate of 6.44%) had a higher risk for subsequent hemorrhage. Conversely, those patients presenting with seizures (annual bleeding rate of 2.20%) or neurological deficits (annual bleeding rate of 1.73%) had a lower risk. A significantly increased risk (p < 0.05) of rebleeding was found among children (hazard ratio [HR] = 2.69), females (HR = 2.93), or patients with deep-seated AVMs (HR = 3.07). CONCLUSIONS Children, females, and patients with deep-seated AVMs had a threefold increased risk of rebleeding after an initial cerebral AVM. This increased risk was highest in the first year after the initial hemorrhage, and thereafter gradually decreased.

Impact of Monocyte Chemoattractant Protein-1 Deficiency on Cerebral Aneurysm Formation

Background and Purpose— Recent studies have suggested that chronic inflammation actively participates in cerebral aneurysm (CA) formation. Macrophages accumulate in CA walls and express proinflammatory genes promoting CA progression, but the molecular mechanisms of monocyte/macrophage recruitment into CA walls remain to be elucidated. Methods— Monocyte chemoattractant protein-1 (MCP-1) expression in experimentally induced CAs was assessed by immunohistochemistry and Western blotting. The role of MCP-1 in CA formation was examined by MCP-1−/− mice and a plasmid DNA encoding a dominant negative mutant of MCP-1 (7ND). MCP-1 expression in human CAs was examined by immunohistochemistry. Results— MCP-1 expression was upregulated in aneurysmal walls at the early stage of CA formation. MCP-1−/− mice exhibited a significant decrease of CA formation and macrophage accumulation with decreased expression of matrix metalloproteinase-2, -9, and inducible nitric oxide synthase. Immunohistochemistry for the DNA binding form of nuclear factor-kappa B showed nuclear factor-kappa B activation in MCP-1-expressing cells. Blockade of MCP-1 activity by 7ND resulted in the inhibition of CA progression in rats. In human CAs, MCP-1 was also expressed in CA walls. Conclusions— These data suggest that MCP-1 plays a crucial role in CA formation as a major chemoattractant for monocyte/macrophage. MCP-1 expression in CA walls is induced through nuclear factor-kappa B activation. MCP-1 may be a novel therapeutic target of medical treatment preventing CA progression.