Kenji Mandai

Ischemic damage and subsequent proliferation of oligodendrocytes in focal cerebral ischemia

In order to achieve a better understanding of the pathophysiology of ischemic white matter lesions, oligodendrocytic degeneration and subsequent proliferation were examined in the mouse model of middle cerebral artery occlusion. In situ hybridization histochemistry for proteolipid protein messenger RNA was employed as a sensitive and specific marker of oligodendrocytes, and immunohistochemistry for myelin basic protein was used as a compact myelin marker. Immunohistochemistry for microtubule-associated protein 2 and albumin was employed to monitor neuronal degeneration and the breakdown of the blood brain barrier, respectively. In the ischemic core of the caudoputamen, the immunoreactivity for microtubule-associated protein 2 disappeared and massive albumin extravasation occurred several hours after vessel occlusion, while proteolipid protein messenger RNA signals remained relatively strong at this time. The messenger RNA signals began to attenuate 12 h after ischemia and were hardly detectable 24 h after ischemia in the whole ischemic lesion. In situ end-labeling of fragmented DNA showed some cells with proteolipid protein messenger RNAs to have DNA fragmentation at this period. In contrast to proteolipid protein messenger RNA signals, the immunoreactivity for myelin basic protein was detected as long as five days after ischemia. An apparent increase in the cells possessing strong proteolipid protein messenger RNA signals was found five days after ischemia, mainly in the corpus callosum and the cortex bordering the infarcted areas. A double simultaneous procedure with in situ hybridization for proteolipid protein messenger RNA and immunohistochemistry for glial fibrillary acid protein or lectin histochemistry for macrophages/microglia showed proliferating oligodendrocytes to be co-localized with reactive astrocytes and macrophages/microglia. These findings show that oligodendrocytic damage occurred following ischemic neuronal damage and the breakdown of the blood brain barrier, but preceded the breakdown of myelin proteins in the ischemic lesion, that an apoptosis-like process was involved in ischemic oligodendrocytic death, and that surviving oligodendrocytes responded and proliferated in the outer border of the infarcted area.

Effect of systemic zinc administration on delayed neuronal death in the gerbil hippocampus

The divalent cation zinc has been reported to possess several physiological properties such as blocking apoptotic cell death through an inhibitory effect on Ca(2+)-Mg2+ endonuclease activity, or modulating the neurotoxicity via glutamate receptor subtypes. In the present study, we investigated the effect of peripherally injected zinc on delayed neuronal death seen in the hippocampus after transient global ischemia, in order to elucidate a possible beneficial role on zinc in ischemic neuronal cell death. Forty-five adult Mongolian gerbils of both sexes underwent transient bilateral clipping of the common carotid arteries for 3 min. In the pretreated animals, ZnCl2 (20 mg/kg) was injected subcutaneously once, 1 h before ischemia (superacute group; n = 6) or twice at 24 and 48 h before ischemia (subacute group; n = 14). Histological survey was carried out 3 days later by in situ DNA fragmentation method and 4 days later by hematoxylin-eosin staining by semiquantatively counting dead neurons in the CA1 sector. Subacute zinc pre-administration significantly reduced the nuclear damage and subsequent neuronal death; however, superacutely pre-administered zinc did not protect hippocampal neurons against ischemia but it did not aggravate the effect of ischemia, either. The present study suggested that transfer of exogenous zinc into the intracellular space is required for neuroprotection, presumably via the anti-endonuclease activity.

Frabin, a Novel FGD1-related Actin Filament-binding Protein Capable of Changing Cell Shape and Activating c-Jun N-terminal Kinase

We purified from rat brain a novel F-actin-binding protein with a M r of about 105,000 (p105), which was estimated by SDS-polyacrylamide gel electrophoresis. We cloned its cDNA from a rat brain cDNA library and characterized it. p105 was a protein of 766 amino acids and showed a calculated M r of 86,449. p105 consisted of one F-actin-binding domain at the N-terminal region, one Dbl homology domain and one pleckstrin homology domain at the middle region, and one cysteine-rich domain at the C-terminal region. This domain organization of p105 was similar to that of FGD1, which has been determined to be the genetic locus responsible for faciogenital dysplasia or Aarskog-Scott syndrome. We therefore named p105 frabin (FGD1-related F-actin-binding protein). Frabin bound along the sides of F-actin and showed F-actin-cross-linking activity. Overexpression of frabin in Swiss 3T3 cells and COS7 cells induced cell shape change and c-Jun N-terminal kinase activation, respectively, as described for FGD1. Because FGD1 has been shown to serve as a GDP/GTP exchange protein for Cdc42 small G protein, it is likely that frabin is a direct linker between Cdc42 and the actin cytoskeleton.

Induction of cyclooxygenase-2 mRNA in gerbil hippocampal neurons after transient forebrain ischemia

We examined the effect of brain ischemia on neuronal expression of cyclooxygenase-2 gene in the hippocampus. Transient forebrain ischemia was produced by occluding bilateral carotid arteries for 5 min in Mongolian gerbil. Northern blotting and in situ hybridization demonstrated that expression of cyclooxygenase-2 mRNA was transiently induced in the hippocampal neurons. Although future studies will be needed to clarify if induced cyclooxygenase-2 following ischemia is involved in neuronal damage or neuronal protection, selective cyclooxygenase-2 inhibitors may be a new therapeutical approach for the treatment of stroke.

Two actions of frabin: direct activation of Cdc42 and indirect activation of Rac

Frabin is an actin filament-binding protein which shows GDP/GTP exchange activity specific for Cdc42 small G protein and induces filopodium-like microspike formation and c-Jun N-terminal kinase (JNK) activation presumably through the activation of Cdc42. Frabin has one actin filament-binding (FAB) domain, one Dbl homology (DH) domain, first pleckstrin homology (PH) domain adjacent to the DH domain, one cysteine-rich FYVE domain, and second PH domain from the N-terminus to the C-terminus in this order. Different domains of frabin are involved in the microspike formation and the JNK activation, and the association of frabin with the actin cytoskeleton through the FAB domain is necessary for the microspike formation, but not for the JNK activation. We have found here that frabin induces the formation of not only filopodium-like microspikes but also lamellipodium-like structures in NIH3T3 and L fibroblasts. We have analysed the mechanism of frabin in these two actions and found that frabin induces filopodium-like microspike formation through the direct activation of Cdc42 and lamellipodium-like structure formation through the Cdc42-independent indirect activation of Rac small G protein. The FAB domain of frabin in addition to the DH domain and the first PH domain is necessary for the filopodium-like microspike formation, but not for the lamellipodium-like structure formation. The FYVE domain and the second PH domain in addition to the DH domain and the first PH domain are necessary for the lamellipodium-like structure formation. We show here these two actions of frabin in the regulation of cell morphology.

Localization of l-afadin at puncta adhaerentia-like junctions between the mossy fiber terminals and the dendritic trunks of pyramidal cells in the adult mouse hippocampus†

We have recently found a novel cell–cell adhesion system at cadherin‐based adherens junctions. This system consists of at least two components: nectin, an immunoglobulin‐like cell adhesion molecule with Ca2+‐independent homophilic binding activity, and l‐afadin, an actin filament‐binding protein that connects nectin to the actin cytoskeleton. In the present study, we investigated immunocytochemically the localization of l‐afadin in the mouse hippocampus. At the light microscopic level, l‐afadin immunoreactivity was demonstrated as flattened disks in the stratum lucidum of the CA3 area. By immunoelectron microscopy, signals for l‐afadin were highly concentrated in a symmetrical manner at the puncta adhaerentia‐like junctions between the mossy fiber terminals and the dendritic trunks of pyramidal cells. We furthermore immunostained the hippocampus with antibodies recognizing both l‐afadin and s‐afadin, a small splicing variant of l‐afadin that is identical to AF‐6. Immunoreactivity for l‐ and s‐afadins was demonstrated not only as the flattened disks similar to that for l‐afadin, but also as numerous fine dots widely distributed in all synaptic layers of the CA1 and CA3 areas. The latter finding may correspond with the recent report by Buchert et al. ( 1999 , J. Cell. Biol. 144:361–371), who found that s‐afadin (AF‐6) and/or l‐afadin was localized at the postsynaptic membranes of asymmetric synaptic junctions. Our present results indicate that l‐ and s‐afadins are differentially distributed in the hippocampus and suggest that l‐afadin localized at the puncta adhaerentia‐like junctions in the mossy fiber terminals may regulate the structural and functional organization of these complex synaptic structures. J. Comp. Neurol. 424:297–306, 2000.

Ischemic tolerance in hippocampal CA1 neurons studied using contralateral controls

We induced ischemic tolerance unilaterally in gerbil hippocampus using the contralateral hippocampus as control. Ischemia for 2 min of right common carotid occlusion was reversible but sufficient to cause heat-shock protein 70 production in CA1 neurons. This pretreatment given four days prior to occlusion of both common carotids for 5 min, but not at longer preceding intervals, induced tolerance in right CA1 neurons. Neuroprotection was still evident two months after the 5 min occlusion. Adenosine triphosphate content and immunoreactive microtubule associate protein 2 in the hippocampus showed that the 5 min ischemic insult was essentially equal in both hemispheres. Repetitive pretreatments at two day intervals caused almost complete protection of CA1 neurons against subsequent 5 min ischemia, while a single pretreatment showed 80% protection. However, the increase in heat-shock protein 70 with repeated pretreatments was not significantly more than with one pretreatment. We concluded that true ischemic tolerance was induced by ischemic stress itself, was long-lasting, was not due to mitigation of subsequent ischemia, and was augmented by repetition without further increase of heat-shock protein 70.

Ischemic tolerance in moderately symptomatic gerbils after unilateral carotid occlusion

Ischemic tolerance following transient global cerebral ischemia has drawn considerable attention because of the putative cell defense mechanism which may be inducible by ischemic stress. The purpose of this study is to investigate the inducibility of ischemic tolerance in moderately symptomatic gerbils after unilateral carotid occlusion. Adult Mongolian gerbils were used. Under ether inhalation, the right common carotid artery was occluded for up to 30 min with an aneurysmal clip. Immediately after occlusion, neurological signs and motor function were evaluated and gerbils with moderate signs were selected for investigation of ischemic tolerance. Ischemia for 30 min to gerbils with moderate signs constantly caused neuronal death in the caudoputamen, but it was prevented by pretreatment with 10 min ischemia which was reversible but strong enough to produce heat shock protein 70. The results show that ischemic tolerance can be induced after hemispheric cerebral ischemia as in the case of global cerebral ischemia and suggest that ischemic tolerance may be relevant in human stroke.

Localization of mLin-7 at nectin-based cell-cell junctions

In C. elegans, lin-7 as well as lin-2/lin-10 is involved in the proper localization of the LET-23 receptor tyrosine kinase that regulates vulval induction. The mammalian homologue, mLin-7, forms a ternary complex with the mammalian homologues of LIN-2 and LIN-10 and localizes at cell–cell junctions in epithelial cells, but the mechanism of this localization of mLin-7 is unknown. Nectin is an immunoglobulin-like cell–cell adhesion molecule that is involved in organization of adherens and tight junctions in epithelial cells. Nectin is indirectly associated with the cadherin–catenin system and the actin cytoskeleton through afadin, an actin filament-binding protein. We showed here that mLin-7 localized at the nectin-based cell–cell junctions. This localization of mLin-7 required the interaction of nectin with afadin, but not the cadherin–catenin system or the actin cytoskeleton. mLin-7 did not directly interact with nectin or afadin. The results indicate that mLin-7 localizes at cell–cell junctions through the nectin–afadin system.

Restoration of E-cadherin-based cell-cell adhesion by overexpression of nectin in HSC-39 cells, a human signet ring cell gastric cancer cell line.

Nectin is an immunoglobulin-like adhesion molecule that comprises a family consisting of four members, nectin-1, -2, -3, and -4. Nectin is associated with the actin cytoskeleton through afadin, a nectin- and actin filament-binding protein. The nectin-afadin and cadherin-catenin systems are associated with each other and cooperatively form cell–cell adherens junctions in intact epithelial cells. HSC-39 cells, a human signet ring cell gastric cancer cell line, express E-cadherin but do not form cell–cell adhesion. The β-catenin gene has been shown to be truncated at the N-terminal region including the α-catenin-binding domain in HSC-39 cells, but overexpression of normal β-catenin failed to form cell–cell adhesion. HSC-39 cells expressed nectin-1, -2, and afadin, but not nectin-3. Overexpression of nectin-3 or -2 formed cell–cell adhesion and accumulation of E-cadherin, but not actin filaments, at the cell–cell adhesion sites. Overexpression of a truncated form of nectin-2 incapable of interacting with afadin failed to form cell–cell adhesion. However, the nectin-formed cell–cell adhesion was not so strong as that observed in epithelial cells, such as CaCo-2 cells. Co-expression of nectin-2 and normal β-catenin did not form strong cell–cell adhesion. These results suggest that an unidentified mechanism, by which nectin and E-cadherin form the actin cytoskeleton-associated adherens junctions to form strong cell–cell adhesion, is impaired in HSC-39 cells.