Akira Terashima

Isoform-Specific Redistribution of Calcineurin Aα and Aβ in the Hippocampal CA1 Region of Gerbils After Transient Ischemia

Abstract: To investigate isoform‐specific roles of Ca2+/calmodulin‐dependent phosphatase [calcineurin (CaN)] in ischemia‐induced cell death, we raised antibodies specific to CaN Aα and CaN Aβ and localized the CaN isoforms in the hippocampal CA1 region of Mongolian gerbils subjected to a 5‐min occlusion of carotid arteries. In the nonischemic gerbil, immunoreactions of both isoforms were highly enriched in CA1 regions, especially in the cytoplasm and apical dendrites of CA1 pyramidal neurons. At 4–7 days after the induced ischemia, immunoreactivities of the CaN Aα isoform in CA1 pyramidal cells were markedly reduced, whereas they were enhanced in the CA1 radiatum and oriens layers. In contrast, CaN Aβ immunoreactivities were reduced in all layers of the ischemic CA1 region, whereas they were enhanced in activated astrocytes, colocalizing with glial fibrillary acidic protein. These findings suggest that up‐regulation of CaN Aα in afferent fibers in CA1 and up‐regulation of CaN Aβ in reactive astrocytes may be involved in neuronal reorganization after ischemic injury.

PKC and tyrosine kinase involvement in amyloid β (25-35)-induced chemotaxis of microglia

MICROGLIA are activated by amyloid β (Aβ) in vivo and in vitro, and Aβ-activated microglia may be involved in the pathogenesis of Alzheimers disease (AD). We investigated the mechanism of microglial chemotaxis induced by Aβ (25–35), an active fragment of Aβ. Aβ (25–35) 0.1 and 1 nM stimulated microglial chemotaxis. The protein kinase C (PKC) inhibitors chelerythrine (0.5 and 2 μM), calphostin C (1 μM) and staurospine (10 nM) significantly inhibited the microglial chemotaxis induced by Aβ (25–35) (1 nM). The chemotactic effect of Aβ (25–35) on microglia was desensitized by pretreatment of microglia with 1 ng/ml 12-O-tetrade-canoylphorbol 13-acetate (TPA). Pretreatment of cells with Aβ (25–35) (1 nM) also desensitized the chemotactic effect by Aβ (25–35) (1 nM). The desensitization by TPA or Aβ (25–35) was inhibited when staurosporine was present in the pretreatment media. The tyrosine kinase inhibitor herbimycin A (0.1 and 1 μM) significantly inhibited the microglial chemotaxis induced by Aβ (25–35) (1 nM). Based on these observations, it seems likely that PKC and tyrosine kinase are involved in the Aβ-induced chemotaxis of microglia.

Single-channel activity of the Ca2+-dependent K+ channel is modulated by FK506 and rapamycin

Single-channel patch clamp recordings were performed in primary cultured neurons from rat dorsal hippocampi. Ca2+-dependent and TEA-sensitive K+ current was recorded from the neurons. Application of immunosuppressants FK506 and rapamycin to the channel inside the plasma membrane of the neurons significantly prolonged the mean open time of the channel. Calcineurin autoinhibitory fragment and W-7 induced no significant alteration in the mean open time of the channel. These results suggest that modulation of the activity of the Ca2+-dependent K+ channel by FK506 and rapamycin is directly through association of immunosuppressants with FKBP12.

Amyloid β Protein (25‐35) Phosphorylates MARCKS Through Tyrosine Kinase‐Activated Protein Kinase C Signaling Pathway in Microglia

Abstract : Myristoylated alanine‐rich C kinase substrate (MARCKS) is a widely distributed specific protein kinase C (PKC) substrate and has been implicated in membrane trafficking, cell motility, secretion, cell cycle, and transformation. We found that amyloid β protein (Aβ) (25‐35) and Aβ (1‐40) phosphorylate MARCKS in primary cultured rat microglia. Treatment of microglia with Aβ (25‐35) at 10 nM or 12‐O‐tetradecanoylphorbol 13‐acetate (1.6 nM) led to phosphorylation of MARCKS, an event inhibited by PKC inhibitors, staurosporine, calphostin C, and chelerythrine. The Aβ (25‐35)‐induced phosphorylation of MARCKS was inhibited by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A, but not with pertussis toxin. PKC isoforms α, δ, and £ were identified in microglia by immunocytochemistry and western blots using isoform‐specific antibodies. PKC‐δ was tyrosine‐phosphorylated by the treatment of microglia for 10 min with Aβ (25‐35) at 10 nM. Other PKC isoforms α and £ were tyrosine‐phosphorylated by Aβ (25‐35), but only to a small extent. We propose that a tyrosine kinase‐activated PKC pathway is involved in the Aβ (25‐35)‐induced phosphorylation of MARCKS in rat microglia.

Amyloid β protein activates PKC-δ and induces translocation of myristoylated alanine-rich C kinase substrate (MARCKS) in microglia

Abstract The increased accumulation of activated microglia containing amyloid β protein (Aβ) around senile plaques is a common pathological feature in subjects with Alzheimers disease (AD). Much less is known, however, of intracellular signal transduction pathways for microglial activation in response to Aβ. We investigated intracellular signaling in response to Aβ stimulation in primary cultured rat microglia. We found that the kinase activity of PKC-δ but not that of PKC-α or -e is increased by stimulation of microglia with Aβ, with a striking tyrosine phosphorylation of PKC-δ. In microglia stimulated with Aβ, tyrosine phosphorylation of PKC-δ was evident at the membrane fraction without an overt translocation of PKC-δ. PKC-δ co-immunoprecipitated with MARCKS from microglia stimulated with Aβ. Aβ induced translocation of MARCKS from the membrane fraction to the cytosolic fraction. Immunocytochemical analysis revealed that phosphorylated MARCKS accumulated in the cytoplasm, particularly at the perinuclear region in microglia treated with Aβ. Taken together with our previous observations that Aβ-induced phosphorylation of MARCKS and chemotaxis of microglia are inhibited by either tyrosine kinase or PKC inhibitors, our results provide evidence that Aβ induces phosphorylation and translocation of MARCKS through the tyrosine kinase-PKC-δ signaling pathway in microglia.

Rapamycin and FK506 induce long-term potentiation by pairing stimulation via an intracellular Ca2+ signaling mechanism in rat hippocampal CA1 neurons

Immunophilin-CsA and -FK506 complexes bind to calcineurin (CaN) and inhibit its phosphatase activity leading to enhancement of neuronal activities. However, inhibition of CaN activity is not the mediator of modulatory activity for IP3 and ryanodine receptors and does not mediate the neurotrophic actions of FK506. FK506 binding protein (FKBP)-12 also binds rapamycin, another immunosuppressant which does not affect CaN activity. Using whole-cell patch clamp techniques, excitatory postsynaptic currents (EPSCs) were recorded and we analyzed the effect of immunosuppressants on the synaptic potentiation induced by pairing weak presynaptic stimulation with postsynaptic depolarization in CA1 neurons of rat hippocampal slices. We found that postsynaptic application of rapamycin or FK506, at low concentrations, but not cyclosporin A, in conjunction with weak pairing stimulation, induced NMDA-dependent long-term potentiation (LTP). The rapamycin-induced LTP was blocked by chelating intracellular Ca(2+) or by inhibiting the intracellular Ca(2+) release. Thus, Ca(2+) release from intracellular Ca(2+) stores is required for the induction of LTP by weak pairing stimulation in the presence of rapamycin or FK506 at postsynaptic sites. We propose that postsynaptic FKBP-12 regulates synaptic transmission by stabilizing the postsynaptic Ca(2+) signaling mechanism in rat hippocampal CA1 neurons.

Myocardial scintigraphy may predict the conversion to probable dementia with Lewy bodies.

Objective: To compare the usefulness of brain perfusion SPECT and 123I-metaiodobenzylguanidine (123I-MIBG) in predicting the conversion of possible dementia with Lewy bodies (DLB) to probable DLB. Methods: We examined 94 patients with possible DLB based on the Consensus Criteria for the Clinical Diagnosis of DLB by N-Isopropyl-p-123I-iodoamphetamine (123I-IMP) brain perfusion SPECT and 123I-MIBG myocardial scintigraphy. After 1 year of follow-up, 33 of 94 patients met the criteria for probable DLB. 123I-IMP brain perfusion SPECT and 123I-MIBG myocardial scintigraphy were tested as predictors of the conversion from possible DLB to probable DLB. A receiver operating characteristic (ROC) analysis was performed. Results: The areas under the ROC curves for SPECT for predicting the conversion to probable DLB from possible DLB based on the occipital/cerebellum and occipital/striatum cortex ratios of blood flow counts were 0.591 and 0.585, respectively. The areas under the ROC curves for 123I-MIBG based on the early heart to mediastinum (H/M) ratio, delayed H/M ratio, and washout rate were 0.935, 0.936, and 0.884, respectively. Conclusion: 123I-MIBG myocardial scintigraphy is a good predictor of the future conversion of possible DLB to probable DLB.

Effects of amino acids and chirality for molecular folding of desoxazoline‐ascidiacyclamide derivatives: X‐ray crystal structures of four cyclic octapeptides including unusual amino acids, cyclo(–Ile–aThr–D‐Val–Thz–)2, cyclo(–Ala–aThr–D‐Val–Thz–Ile–aThr–D‐Val–Thz–), cyclo(–Val–aThr–D‐Val–Thz–Ile–aThr–D‐Val–Thz–), and cyclo(–Ile–aThr–Val–Thz–Ile–aThr–D‐Val–Thz–)

Desoxazoline derivative of ascidiacyclamide (1), cyclo(-L-Ile-L-allo-threonine-D-Val-thiazole-)(2), was modified to disturb the C(2)-symmetry. An Ile(1) residue of 1 was replaced for Ala (2) or Val (3), and the D-Val(3) residue was replaced for Val (4). The crystal structures of 1-4 were analyzed by x-ray diffraction methods. The molecules of all compounds were folded and this type of structure was not observed in x-ray structures of ascidiacyclamide derivatives so far except for patellamide D. The folding patterns of 1-4 were similar to each other and resembled that of patellamide. The asymmetric modifications at position 1 caused the conformational changes at local area, and these were related with the peptide-peptide and peptide-solvent interactions. Despite the diverse backbone conformation by the epimeric modification at position 3, the entire molecule of 4 was folded. These results mean that (1) the desoxazoline-ascidiacyclamides favored the folded structures and (2) the modifications of the side chain size at position 1 and the chirality at position 3 brought the local conformational changes to derivatives, suggesting that (3) the lack of the oxazoline block leads to conformational flexibility of 1-4, which accepts the conformational change with no drastic change on the entire structure.

Cloning of the cDNA encoding rat Presenilin-1

We isolated rat presenilin-1 (PS-1; also called S182 previously) cDNA from total brain RNA by using a reverse transcription-polymerase chain reaction (RT-PCR) technique with primers homologous to the conserved sequences of human and mouse PS-1. Rat PS-1 cDNA encoded 468 amino acids (aa) and the deduced aa sequence was highly homologous to those of the human (88.4%) and mouse (92.7%). Northern blot analysis of the rat PS-1 cDNA revealed two mRNA species in rat neurotypic pheochromocytoma and glioma cell lines (PC-12 and C6, respectively) that migrated at rates corresponding to approximately 3.0 and 7.5 kb.

Microglial signaling by amyloid β protein through mitogen-activated protein kinase mediating phosphorylation of Marcks

Myristoylated alanine-rich C kinase substrate (MARCKS), an acidic protein associated with cell motility and phagocytosis, is activated upon phosphorylation by protein kinase C (PKC) and proline-directed protein kinases. In Alzheimer disease (AD), activated microglia expressing MARCKS migrates around senile plaques. We reported that amyloid β protein (Aβ), a major component of senile plaques, activated MARCKS through a tyrosine kinase and PKC-δ. We have now identified another Aβ signaling pathway through a mitogen-activated protein kinase (MAPK) involved in the phosphorylation of MARCKS and analysed cross-talk between PKC and MAPK pathways in primary cultured rat microglia. A selective inhibitor for MAPK kinase, PD098059, significantly inhibited the phosphorylation of MARCKS induced by Aβ. Extracellulary regulated kinases, the activities of which were induced by Aβ, directly phosphorylated a recombinant MARCKS in vitro. The MAPK pathway was sensitive to wortmannin, but not to a PKC inhibitor or to tyrosine kinase inhibitors. The activation of PKC by Aβ was not sensitive to wortmannin. Our findings suggest involvement of the MAPK pathway through phosphoinositol 3-kinase in the phosphorylation of MARCKS in rat cultured microglia, an event may be associated with mechanisms activating microglia in AD.