Taku Amano

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.

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.

Mutant γPKC found in spinocerebellar ataxia type 14 induces aggregate-independent maldevelopment of dendrites in primary cultured Purkinje cells

Missense mutations in protein kinase Cgamma (gammaPKC) gene have been found in spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that mutant gammaPKC found in SCA14 is susceptible to aggregation and induces apoptosis in cultured cell lines. In the present study, we investigated whether mutant gammaPKC formed aggregates and how mutant gammaPKC affects the morphology and survival of cerebellar Purkinje cells (PCs), which are degenerated in SCA14 patients. Adenovirus-transfected primary cultured PCs expressing mutant gammaPKC-GFP also had aggregates and underwent apoptosis. Long-term time-lapse observation revealed that PCs have a potential to eliminate aggregates of mutant gammaPKC-GFP. Mutant gammaPKC-GFP disturbed the development of PC dendrites and reduced synapse formation, regardless of the presence or absence of its aggregates. In PCs without aggregates, mutant gammaPKC-GFP formed soluble oligomers, resulting in reduced mobility and attenuated translocation of mutant gammaPKC-GFP upon stimulation. These molecular properties of mutant gammaPKC might affect the dendritic morphology in PCs, and be involved in the pathogenesis of SCA14.

Chronic pain-induced astrocyte activation in the cingulate cortex with no change in neural or glial differentiation from neural stem cells in mice

Pain pathways terminate in discrete brain areas that monitor the sensory and affective qualities of the initiating stimulus and show remarkable plasticity. Here, we found that chronic pain by sciatic nerve ligation caused a dramatic increase in glial fibrillary acidic protein (GFAP)-like immunoreactivity (IR), which is located in the dendritic astrocytes, with its expanding distribution in the cingulate cortex (CG) of mice. The branched GFAP-like IR in the CG of nerve-ligated mice was overlapped with S100beta-like IR, which is highly limited to the cell body of astrocytes, whereas there was no difference of S100beta-like IR between sham-operated and nerve-ligated mice. The number of BrdU-positive cells on the CG was not changed by sciatic nerve ligation. Furthermore, subventricular zone (SVZ)-derived neural stem cells marked by pEGFP-C1 did not migrate toward the CG after sciatic nerve ligation. In the behavioral assay, the thermal hyperalgesia observed on the ipsirateral side in nerve-ligated mice was significantly suppressed by a single pre-microinjection of a glial-modulating agent propentofylline into the CG 24 h before nerve ligation. These results suggest that chronic painful stimuli induces astrocyte activation in the CG, whereas they do not affect the cell proliferation/differentiation from neural stem cells in the CG and the migration of neural stem cells from the SVZ area. The astrocyte activation in the CG may, at least in part, contribute to the development of a chronic pain-like state following sciatic nerve ligation in mice.

Role of Glucocorticoid in Vestibular Compensation in Relation to Activation of Vestibular Nucleus Neurons

It is still not established whether or not glucocorticoids are effective in the treatment of vestibular disorders such as dizziness and imbalance, although these drugs in combination with several others are used to treat dizziness and imbalance in some diseases. This study was undertaken to investigate the effects of a glucocorticoid, dexamethasone, on vestibular disorder following unilateral labyrinthectomy in pigmented rabbits. Neuronal activities of the medial vestibular nucleus (MVN) in alpha-chloralose-anesthetized cats were also investigated. Systemic injection of dexamethasone decreased the frequency of nystagmus and head deviation dose-dependently following hemilabyrinthectomy, and the rate of decrease was faster than that obtained by saline. In contrast, RU38486 (a glucocorticoid receptor antagonist) delayed the reduction of nystagmus and head deviation. Micro-iontophoretic application of dexamethasone rapidly enhanced the spontaneous firing of MVN neurons in a dose-dependent manner. These increases were blocked by RU38486, but not by GDEE (a glutamate receptor antagonist) or Co2+ (a Ca2+ channel blocker). These results suggest that dexamethasone directly activates the MVN neurons, thereby accelerating vestibular compensation.

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.

Inhibition by a putative antipsychotic quinolinone derivative (OPC-14597) of dopaminergic neurons in the ventral tegmental area

The effects of the newly synthesized quinolinone derivative, OPC-14597 (7-{4-[4-(2,3-dichlorophenyl)-1-piperazinyl]butyloxy}-3, 4-dihydro-2(1 H)-quinolinone), on dopaminergic neuronal activity in the ventral tegmental area were examined using both in vivo microiontophoretic methods in chloral hydrate-anesthetized rats and the tight-seal whole-cell patch-clamp technique in thin-slice preparations of the rat brain. Neurons in the ventral tegmental area were classified as type I or type II according to their responses to antidromic stimulation of the nucleus accumbens, probably corresponding to dopaminergic and non-dopaminergic neurons, respectively. Antidromic spikes elicited by nucleus accumbens stimulation were inhibited by microiontophoretic application of dopamine and OPC-14597 in type I, but not in type II neurons. Although the OPC-14597-induced inhibition was antagonized by simultaneous application of domperidone (5-chloro-1-[1-[3-(2,3-dihydro-2-oxo-1 H-benzimidazo-1-yl)-propy]-4-piperidinyl]-1,3-dihydro-2H- benzimidazol-2-one; dopamine D2 receptor antagonist), SCH 23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4, 5-tetrahydro-1 H-3-benzazepine hydrochloride; dopamine D1 receptor antagonist) had no such effect. Spontaneous firing of type I neurons was also inhibited by iontophoretically applied OPC-14597 and dopamine, whereas that of type II neurons was unaffected. The inhibitory effect of OPC-14597 on the spontaneous firing of type I neurons was antagonized by domperidone, but not by SCH 23390. In a whole-cell patch-clamp study using a thin-slice preparation of the rat brain, bath application of OPC-14597 induced hyperpolarization accompanied by inhibition of spontaneously occurring action potentials in the large neurons (> 20 microns in diameter) in a concentration-dependent manner. These results suggest that OPC-14597 acts on dopaminergic neurons in the ventral tegmental area as a dopamine D2 receptor agonist to inhibit neuronal activities, probably by increasing membrane potassium conductance.

Post-synaptic action of morphine on glutamatergic neuronal transmission related to the descending antinociceptive pathway in the rat thalamus.

Morphine is a prototypical μ‐opioid receptor (MOR) agonist, and can directly inhibit pain transmission at both spinal and supraspinal levels. In the present study, we investigated the properties of thalamic neurons in an opioid‐sensitive pain‐modulating circuit. Application of morphine to cultured thalamic neurons evoked a potentiation of glutamate‐induced peak currents, which was blocked by the MOR antagonist. Application of the protein kinase C inhibitor chelerythrine significantly inhibited the morphine‐evoked enhancement of glutamate‐induced currents. Immunoreactivity for MOR was observed with high density in the habenular nucleus (Hb) of the thalamus in rats, which was clearly co‐localized with NMDA receptor subunit 1 (NRI). In this study, we show that microinjection of morphine into the Hb produced a dose‐dependent increase in the tail‐flick latency and enhanced the antinociceptive effect induced by the intra‐Hb injection of glutamate. When fluoro‐gold (FG) was used as a retrograde tracer, we found that FG‐labeled neurons in the Hb after the microinjection of FG into the periaqueductal gray expressed both MOR and NR1. The present data suggest that the stimulation of MOR in the Hb may be involved in activation of the descending antinociceptive pathway through glutamatergic neurotransmission via the NMDA receptor.

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.