Taizo Taniguchi

Expression of apolipoprotein E mRNA in rat microglia

Apolipoprotein E (apoE) is a major risk factor for Alzheimer disease (AD), which is the most common cause of progressive dementing illness. ApoE has been postulated to be synthesized by astrocytes and taken up by microglia and neuronal cells. However, it remains unknown whether apoE is also produced by microglia in the brain. We analyzed apoE mRNA expression of microglia using a rat primary culture system. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed expression of apoE mRNA in cultured rat microglia. By RT-in situ-PCR, microglia showed positive staining for the PCR product of apoE mRNA. These results indicated that apoE was biosynthesized in rat microglia. We suggest that microglia might be one of the sources of apoE in the brain, and that apoE synthesized in microglia might be closely related to the pathogenesis of AD.

Cloning and characterization of gastrin receptor from ECL carcinoid tumor of Mastomys natalensis

We report here the cDNA cloning of a putative gastrin receptor from enterochromaffin-like (ECL) carcinoid tumor of Mastomys natalensis. For this study, we used the polymerase chain reaction technique to amplify transmembrane domain sequences related to rat pancreatic cholecystokinin (CCK)-A receptor from the ECL tumor cDNA library. The amino acid sequence deduced from the cloned cDNA showed 85.7% and 49.0% identity to canine parietal cell gastrin receptor and rat pancreatic CCK-A receptor, respectively. Ligand binding studies using COS-7 cells transfected with the cDNA showed the same binding specificity for gastrin and CCK-8 as the gastrin receptor on the Mastomys carcinoid tumor membrane. Both gastrin and CCK-8 elevated free cytosolic calcium concentration in COS-7 cells expressing the cloned receptor. RNA blot analysis revealed the expression of the gastrin receptor in both Mastomys stomach and brain.

Transgenic mice expressing mutant (N279K) human tau show mutation dependent cognitive deficits without neurofibrillary tangle formation

Mutations in the tau gene, which is located on chromosome 17, were found causative for autosomal dominantly inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17). To determine if cognitive deficits could be caused by tau mutations, two transgenic mouse lines were generated expressing a four‐repeat isoform of human tau or its mutant, containing one of the FTDP‐17 mutations (WILD mice and N279K mice). In open field test, N279K mice showed hyperactivity in locomotion and rearing. In prepulse inhibition test, N279K mice but not Wild mice showed significant deficits. Both transgenic mice, especially N279K mice, showed impairment in acquisition of spatial learning in Morris water maze. Although both N279K mice and Wild mice acquired passive avoidance as well as non‐transgenic mice, N279K mice but not Wild mice showed severe deficits in acquisition of active avoidance. Histological analysis of the present mutant mice did not show any signs of neurofibrillary tangle formations in the brain, and cognitive dysfunction seemed to precede such neuropathological changes or occur independently from them. The behavioral phenotype of N279K mice mimics features of human FTDP‐17 and provides a basic model for elucidating mechanisms underlying cognitive deficits in not only FTDP‐17, but also diverse tauopathies.

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.

Different inhibitory response of cyanidin and methylene blue for filament formation of tau microtubule-binding domain.

One of the priorities in Alzheimer research is to develop a compound that inhibits the filament formation of tau protein. Since the three- or four-repeat microtubule-binding domain (MBD) in tau protein plays an essential role in filament formation, the inhibitory behavior of cyanidin (Cy) and methylene blue (MB) with respect to heparin-induced filament formation of MBD in a neutral solution (pH 7.6) was characterized by fluorescence, circular dichroism, and electron microscopy measurements. The planar aromatic ring of Cy and the N-unsubstituted phenothiazine ring of MB were shown to be necessary for the inhibition. However, the inhibitory responses with respect to heparin-induced filament formation to the second and third repeat peptides of MBD were different: Cy suppresses the formation and MB does not prevent the formation. This suggests the importance of the first and fourth repeat peptides in the inhibitory activity of MB for MBD filament formation. In this study, we showed that the decrease of thioflavin S fluorescence intensity is not always linked to inhibition of filament formation.

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.

Amphipathic helical behavior of the third repeat fragment in the tau microtubule-binding domain, studied by 1H NMR spectroscopy

The third repeat fragment (3MBD, 31 residues) in the four-repeat microtubule-binding domain of water-soluble tau protein has been considered to be responsible for the formation of the neuropathological filament. To clarify the structural requisite of 3MBD for the filamentous assembly, the solution structures in water and trifluoroethanol (TFE) were investigated by a combination of two-dimensional (1)H-NMR measurements and molecular modeling calculations. All protons were assigned by various 2D NMR spectral measurements. The NOE patterns characteristic to the typical helical structure were observed in TFE solution, as was expected from the CD spectra. Using 273 NOE and 23 (3)J(NHC(alpha)H) data, possible 3D structures were generated by the dynamical simulated annealing method. The constructed NMR conformers showed that the N-terminal Val1-Lys6 and Leu10-Leu20 fragments form the well-refined extended and alpha-helical structures, respectively, whereas the C-terminal moiety is highly flexible. Interestingly, the helical structure showed amphipathic distribution of the respective side chains. This amphipathic behavior of the 3MBD structure would be necessary for self-associating into a helical filament of the tau MBD domain, because such a filament is stabilized by the alternating hydrophilic and hydrophobic interactions between the 3MBD fragments.

Effects of different anti-tau antibodies on tau fibrillogenesis: RTA-1 and RTA-2 counteract tau aggregation.

Tau is the major antigenic component of neurofibrillary pathology in tauopathy, including Alzheimers disease. Although conversion of soluble tau to an insoluble polymerized fibrillar form is a key factor in the pathogenesis of tauopathy, the mechanism of the change is unclear and no inhibitors of fibril formation are available. Monoclonal antibodies against the 1st or 2nd repeat of the microtubule binding domain, but not the C‐terminal 16 residues, completely inhibited tau aggregation into PHF. Furthermore, they did not inhibit tau‐induced tubulin assembly. Thus, they are useful to investigate tau protein conversion and will be useful therapeutic lead materials.