Hiroaki Matsubayashi

Microglial α7 nicotinic acetylcholine receptors drive a phospholipase C/IP3 pathway and modulate the cell activation toward a neuroprotective role

Microglia perform both neuroprotective and neurotoxic functions in the brain, with this depending on their state of activation and their release of mediators. Upon P2X7 receptor stimulation, for example, microglia release small amounts of TNF, which protect neurons, whereas LPS causes massive TNF release leading to neuroinflammation. Here we report that, in rat primary cultured microglia, nicotine enhances P2X7 receptor‐mediated TNF release, whilst suppressing LPS‐induced TNF release but without affecting TNF mRNA expression via activation of α7 nicotinic acetylcholine receptors (α7 nAChRs). In microglia, nicotine elicited a transient increase in intracellular Ca2+ levels, which was abolished by specific blockers of α7 nAChRs. However, this response was independent of extracellular Ca2+ and blocked by U73122, an inhibitor of phospholipase C (PLC), and xestospongin C, a blocker of the IP3 receptor. Repeated experiments showed that currents were not detected in nicotine‐stimulated microglia. Moreover, nicotine modulation of LPS‐induced TNF release was also blocked by xestospongin C. Upon LPS stimulation, inhibition of TNF release by nicotine was associated with the suppression of JNK and p38 MAP kinase activation, which regulate the post‐transcriptional steps of TNF synthesis. In contrast, nicotine did not alter any MAP kinase activation, but enhanced Ca2+ response in P2X7 receptor‐activated microglia. In conclusion, microglial α7 nAChRs might drive a signaling process involving the activation of PLC and Ca2+ release from intracellular Ca2+ stores, rather than function as conventional ion channels. This novel α7 nAChR signal may be involved in the nicotine modification of microglia activation towards a neuroprotective role by suppressing the inflammatory state and strengthening the protective function.

Mutant protein kinase Cγ found in spinocerebellar ataxia type 14 is susceptible to aggregation and causes cell death

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease characterized by various symptoms including cerebellar ataxia. Recently, several missense mutations in the protein kinase Cγ (γPKC) gene have been found in different SCA14 families. To elucidate how the mutant γPKC causes SCA14, we examined the molecular properties of seven mutant (H101Y, G118D, S119P, S119F, Q127R, G128D, and F643L) γPKCs fused with green fluorescent protein (γPKC-GFP). Wild-type γPKC-GFP was expressed ubiquitously in the cytoplasm of CHO cells, whereas mutant γPKC-GFP tended to aggregate in the cytoplasm. The insolubility of mutant γPKC-GFP to Triton X-100 was increased and correlated with the extent of aggregation. γPKC-GFP in the Triton-insoluble fraction was rarely phosphorylated at Thr514, whereas γPKC-GFP in the Triton-soluble fraction was phosphorylated. Furthermore, the stimulation of the P2Y receptor triggered the rapid aggregation of mutant γPKC-GFP within 10 min after transient translocation to the plasma membrane. Overexpression of the mutant γPKC-GFP caused cell death that was more prominent than wild type. The cytotoxicity was exacerbated in parallel with the expression level of the mutant. These results indicate that SCA14 mutations make γPKC form cytoplasmic aggregates, suggesting the involvement of this property in the etiology of SCA14.

Epileptic seizures induced by N-acetyl-l-aspartate in rats: in vivo and in vitro studies

Tremor rat (tm/tm), the parent strain of spontaneously epileptic rat (SER: zi/zi, tm/tm), exhibits absence-like seizures characterized by 5-7 Hz spike-wave-like complexes on cortical and hippocampal electroencephalograms (EEG) after 10 weeks of age, prior to development of convulsive seizures. Recently, this animal model has been demonstrated to display a genomic microdeletion within the critical region of tm, where aspartoacylase hydrolyzing N-acetyl-L aspartate (NAA) is located, besides showing the ability to accumulate NAA in the brain. Therefore, the present study was performed to determine the involvement of NAA in the induction of epileptic seizures. When NAA (4 micromol) was applied intracerebroventricularly (i.c.v.) to normal Wistar rats, 4-10 Hz polyspikes and/or spike-wave-like complexes followed by absence-like seizure before persistent 1-5 Hz waxing high-voltage after-discharges were observed on cortical and hippocampal EEG. At a higher dose (8 micromol), NAA induced convulsive seizures. The absence-like seizures with polyspikes and/or spike-wave-like complexes on the EEG were also observed with i.c.v. NAA in premature tremor rats without seizures. The NAA-induced seizures in normal rats were antagonized by i.c.v. glutamic acid diethyl ester, a non-selective glutamate receptor antagonist. In addition, NAA applied to the bath rapidly induced a long-lasting depolarization concomitantly with repetitive firings in hippocampal CA3 neurons of normal rat brain slice preparations. These findings suggest that NAA is involved in the induction of absence-like seizures and/or convulsion, probably via glutamate receptors.

Dopamine-induced protection of striatal neurons against kainate receptor-mediated glutamate cytotoxicity in vitro

The effects of dopamine on glutamate-induced cytotoxicity were examined using the primary cultures of rat striatal neurons. Cell viability was significantly reduced by exposure of cultures to glutamate or kainate for 24 h. In contrast, similar application of N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) did not induce cytotoxicity. Kainate-induced cytotoxicity was significantly inhibited by kynurenate but not by MK-801. Dopamine at concentrations of 1-100 microM dose-dependently reduced kainate-induced cytotoxicity. Forskolin also significantly reduced kainate cytotoxicity. The neuroprotective effect of dopamine was antagonized by SCH 23390, a D1 receptor antagonist, but not by domperidone, a D2 receptor antagonist. Moreover, kainate-induced cytotoxicity was prevented by SKF 38393, a D1 receptor agonist, or forskolin but not by quinpirole, a D2 receptor agonist. The patch clamp study revealed that the same striatal neurons responded to both kainate and NMDA. During voltage clamp recording, neither kainate-induced currents nor NMDA-induced currents were affected by dopamine. Moreover, dopamine did not affect glutamate- or kainate-induced Ca2+ influx measured with fura-2. These findings indicate that dopamine prevents kainate receptor-mediated cytotoxicity without affecting the kainate receptor activities and intracellular Ca2+ movement. Dopamine-induced neuroprotection may be mediated by an increased intracellular cAMP formed following activation of D1 receptors.

Ontogeny of muscarinic receptors in the rat brain with emphasis on the differentiation of M1- and M2-subtypes— semi-quantitative in vitro autoradiography

The ontogeny of muscarinic acetylcholine receptors (mAChR) in the rat brain was studied with emphasis on the differentiation of M1- and M2-receptor subtypes through semi-quantitative in vitro autoradiography. [3H]Quinuclidinyl benzilate (QNB) and [3H]pirenzepine (PZ) were used for labeling total mAChR and M1-receptors, respectively. In the cerebral cortex of adult rats, [3H]QNB binding sites were more richly present in the superficial and deeper layers than in the middle layer, while M1-receptors were diffusely observed in all the layers. This means that M2-receptors are highly concentrated in the superficial and deeper layers. The ontogenetical differentiation of the laminar distribution between M1- and M2-receptors first appeared at 14 days of postnatal age. In the hippocampus and striatum whose mAChR were predominantly of the M1-type in the adult rat brain, ontogenic patterns of M1-receptors were almost identical to those of total mAChR. On the other hand, mAChR in the cerebellar cortex and lower brainstem of the adult rat were mainly of the M2-subtype. In these areas, the ontogeny of total mAChR was apparently observed. However, M1-receptors were not observed at any stage of the ontogeny. The above-mentioned results indicate that M1- and M2-receptors show distinct developmental behaviors in the rat brain.

Orphanin FQ-induced outward current in rat hippocampus

Orphanin FQ (OFQ) is a heptadecapeptide that structurally resembles opioid peptides. It has been demonstrated that the moderate density of binding sites of OFQ were localized in the hippocampus and that the expression of OFQ receptor in the hippocampus have an important role in learning and memory. This study was designed to investigate whether activation of the OFQ receptor could induced hyperpolarization in the cultured hippocampus neurons in rats. In the current clamp mode, the application of OFQ (10(-8)-10(-5) M) hyperpolarized the membranes in cultured hippocampus neurons in a concentration-dependent manner. Moreover, in the voltage clamp mode, application of OFQ (10(-6) M) induced outward current in hippocampus CA3 pyramidal neurons. In the presence of TTX (3x10(-7) M), the average maximal amplitude of the outward current deflection induced by OFQ (10(-6) M) at -60 mV of a holding potential was 24.7+/-0.54 pA. The OFQ-induced current reversed at -99.06+/-3.80 mV (3 mM), which was quite close to the K(+) equilibrium potential as calculated by the Nernst equation (E(k)=-96.08 mV, 3 mM) for K(+) in our standard solution. This suggests that OFQ-induced current was mediated by K(+) ion. It has been demonstrated that [Phe(1)psi(CH(2)-NH)Gly(2)]Nociceptin(1-13)NH(2)) (a pseudopeptide analog of nociceptin), and nocistatin are selective antagonists of OFQ. OFQ (10(-6) M)-induced outward current was antagonized by application of [Phe(1)psi(CH(2)-NH)Gly(2)]Nociceptin(1-13)NH(2) (10(-5) M). In contrast, OFQ-induced outward current was not antagonized by application of nocistatin (10(-5) M). These results indicates that there is the physiological functioning receptor of OFQ in the hippocampus.

Phosphorylation of PKC activation loop plays an important role in receptor-mediated translocation of PKC.

Protein kinase C (PKC) is translocated to various cellular regions in a subtype and stimulation‐dependent manner. Thereafter, the activated PKC phosphorylates its substrate and causes subsequent cellular responses (PKC targeting). The 3‐phosphoinositide‐dependent protein kinase‐1 (PDK1) has an essential role in the maturation of PKC by phosphorylating a threonine residue in the PKC activation loop. To elucidate the role of PDK1 in PKC targeting, we expressed mutant γ‐ or δ‐PKC fused with GFP (γ‐ or δ‐PKC‐ALM (activation loop mutant)‐GFP), whose threonine residue in the activation loop was replaced with alanine, and compared their P2Y receptor‐mediated translocation with wild‐type PKC‐GFP in CHO cells. ATP (1 mm) induced the transient translocation of wild‐type γ‐ or δ‐PKC‐GFP from cytoplasm to plasma membrane and following retranslocation from membrane to the cytoplasm. γ‐ or δ‐PKC‐ALM‐GFP was also translocated to plasma membrane, which was, however, retained at the membrane for a longer period than wild type. Similar results were observed in kinase‐negative PKC mutants, indicating that the phosphorylation by PDK1 affects the retranslocation step of PKC by regulating the kinase activity. The simultaneous monitoring of [Ca2+]i and diacylglycerol (DG) levels with the translocation of PKC demonstrated that PKC‐ALM induced the prolonged accumulation of DG, resulting in the prolonged retention of PKC‐ALM at the plasma membrane. It is possible that PKC‐ALM with decreased kinase activity could delay the conversion of DG at the plasma membrane. Our present study suggests that the activation loop phosphorylation plays an important role in receptor‐mediated PKC targeting.

Enhanced Calcium Influx in Hippocampal CA3 Neurons of Spontaneously Epileptic Rats

Summary:  Purpose: The spontaneously epileptic rat (SER: tm/tm, zi/zi) shows both absence‐like seizures and tonic convulsions. Our previous electrophysiologic studies have demonstrated that SER has abnormal excitability of hippocampal CA3 neurons, which shows a long‐lasting depolarization shift by a single stimulation of mossy fibers, probably resulting from the Ca2+ channel abnormalities. The present study was performed to determine whether Ca2+ influx is actually enhanced in the CA3 area of SER.

Role of C-terminal region in the functional regulation of rat serotonin transporter (SERT).

Previously, we revealed that the state of the actin cytoskeleton affects the uptake activity of the serotonin transporter (SERT). Recently, it was reported that the C-terminus of SERT interacts with MacMARCKS, a substrate of PKC that can bind to the actin cytoskeleton. To elucidate the importance of the C-terminal region in the regulation of SERT activity and the interaction with the actin cytoskeleton, we examined whether the overexpression of the C-terminus affects the transport activity of SERT. To this end, we overexpressed a GFP-fused 30-amino acid construct of the SERT C-terminus (GFP-SERT-CT) in HEK293 cells stably expressing FLAG-tagged SERT (FL-SERT-HEK293 cells). The SERT uptake activity and transporter current were attenuated in GFP-SERT-CT-expressing FL-SERT-HEK293 cells, as compared with GFP-expressing FL-SERT-HEK293 cells. Eadie-Hofstee analysis revealed that GFP-SERT-CT overexpression attenuated the SERT uptake activity by reducing the Vmax, but not changing the Km, which was consistent with the results of experiments on the cell-surface expression of SET using biotinylation/immunoblot analysis. Immunocytochemical analysis demonstrated that GFP-SERT-CT was co-localized with FLAG-SERT and cortical actin at the plasma membrane. In addition, the SERT C-terminus did not affect dopamine transporter activity. These findings showed the significance of the C-terminal region to the functional regulation of SERT, suggesting that GFP-SERT-CT acts as a molecular decoy to disrupt the interaction between SERT and the actin cytoskeleton.

Adenoviral gene transfer of aspartoacylase ameliorates tonic convulsions of spontaneously epileptic rats

The spontaneously epileptic rat (SER: tm/tm, zi/zi) shows both absence-like seizures and tonic convulsions. Our previous studies have demonstrated that absence-like seizures of the tremor rat (tm/tm), one of the parent strains of SER, were inhibited by adenoviral transfer of the aspartoacylase (ASPA) gene, a deleted gene in the tremor rat. In the present study, we examined whether the adenoviral gene transfer of ASPA inhibited the tonic convulsions of SER. Replication-defective recombinant adenoviral vectors carrying the rat ASPA gene (AxASPA) or Escherichia coli beta-galactosidase gene (AxLacZ), as a control, were constructed. After it was confirmed that AxASPA-infected HeLa cells expressed ASPA in vitro, AxASPA or AxLacZ was administered into the left lateral ventricle of 11-week-old SER. The occurrence and duration of tonic convulsions in SER were evaluated before and after the administration of adenoviral vector. Intracerebroventricular administration of AxASPA (5 x 10(7) plaque forming units) transiently, but significantly, inhibited the occurrence of tonic convulsions in SER without affecting the duration per single convulsion 7 days after the administration. No inhibitory effects were observed 10 and 14 days after AxASPA administration. In contrast, the administration of AxLacZ did not have any effect on tonic convulsions in SER. Survival rates did not differ between AxASPA- and AxLacZ-treated SERs. Adenoviral gene transfer of ASPA, one of the deleted genes of SER, transiently rescued SERs from tonic convulsion, although it did not improve their survival time.