Masamichi Nakai

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.

The extracellular calcium-sensing receptor is expressed in rat microglia and modulates an outward K+ channel.

Abstract: The calcium‐sensing receptor (CaR) is a G protein‐coupled receptor that “senses” extracellular calcium ions (Ca2+o) as an extracellular first messenger. In this report, we have shown that the CaR is expressed in primary cultures of microglial cells derived from rat brain as assessed by RT‐PCR using four CaR‐specific primer pairs followed by sequencing of the amplified products, by northern blot analysis using a CaR‐specific probe, as well as by immunocytochemistry and western analysis utilizing a specific polyclonal anti‐CaR antiserum. In addition, raising Ca2+, from 0.75 to 3.0 mM or addition of the polycationic CaR agonist neomycin or a “calcimimetic” CaR activator (R‐467; NPS Pharmaceuticals) increased the open state probability (Po) of a Ca+‐activated Kf channel having a unitary conductance of 84 ± 4 pS, indicating that the channel is modulated by the CaR. Therefore, our data strongly suggest that a functional CaR is expressed in cultured rat microglia, similar to that in parathyroid gland and kidney, which could potentially play an important role(s) in regulating microglial function.

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.

Possible different mechanism between amyloid-beta (25-35)-and substance P-induced chemotaxis of murine microglia.

The mechanism of murine microglial chemotaxis induced by amyloid-beta protein (A beta (25-35)) was investigated. A beta (25-35) dose-dependently stimulated microglial chemotaxis at concentrations between 100 pM and 10 nM. Substance P, a NK-1 agonist, stimulated chemotaxis at concentrations of 10 nM or more. GR-64349, a NK-2 agonist, and senktide, a NK-3 agonist, did not stimulate microglial chemotaxis. We examined whether homologous desensitization of chemotaxis would occur by A beta (25-35). The chemotactic effect of microglia was homologously desensitized by 10 nM A beta (25-35). On the other hand, substance P at 10 nM did not desensitize the A beta (25-35)-induced chemotaxis. These data show that A beta (25-35) induces the chemotaxis of microglia probably through a receptor other than the NK-1 receptor.

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.

Phorbol esters inhibit phosphate uptake in opossum kidney cells: A model of proximal renal tubular cells

The effects of phorbol esters and diacylglycerol on phosphate uptake in opossum kidney (OK) cells were investigated to assess the possible role of Ca2+-activated, phospholipid dependent protein kinase (protein kinase C) on renal phosphate handling. OK cells are widely used as a model of proximal renal tubular cells and are reported to possess a Na+-dependent phosphate transport system. Phorbol-12,13-dibutyrate (PDBu) inhibited phosphate uptake. This inhibitory effect was synergistically enhanced with A23187. 4 beta-phorbol 12,13-didecanoate inhibited phosphate uptake, while 4 alpha-phorbol 12,13-didecanoate did not. 1-oleoyl-2-acetyl-glycerol (OAG), a synthetic diacylglycerol, also exhibited an inhibitory effect on phosphate uptake. These data suggest the possible involvement of protein kinase C in proximal renal tubular phosphate transport.