Biomedical subjects

Mami Noda

Publications and source records attributed to Mami Noda.

29 records · Page 2Linked to original sources

PubMed2004

Role of ubiquitin carboxy terminal hydrolase-L1 in neural cell apoptosis induced by ischemic retinal injury in vivo.

Ubiquitin is thought to be a stress protein that plays an important role in protecting cells under stress conditions; however, its precise role is unclear. Ubiquitin expression level is controlled by the balance of ubiquitinating and deubiquitinating enzymes. To investigate the function of deubiquitinating enzymes on ischemia-induced neural cell apoptosis in vivo, we analyzed gracile axonal dystrophy (gad) mice with an exon deletion for ubiquitin carboxy terminal hydrolase-L1 (UCH-L1), a neuron-specific deubiquitinating enzyme. In wild-type mouse retina, light stimuli and ischemic retinal injury induced strong ubiquitin expression in the inner retina, and its expression pattern was similar to that of UCH-L1. On the other hand, gad mice showed reduced ubiquitin induction after light stimuli and ischemia, whereas expression levels of antiapoptotic (Bcl-2 and XIAP) and prosurvival (brain-derived neurotrophic factor) proteins that are normally degraded by an ubiquitin-proteasome pathway were significantly higher. Consistently, ischemia-induced caspase activity and neural cell apoptosis were suppressed approximately 70% in gad mice. These results demonstrate that UCH-L1 is involved in ubiquitin expression after stress stimuli, but excessive ubiquitin induction following ischemic injury may rather lead to neural cell apoptosis in vivo.

Animals↗

PubMed2004

Two closely related ubiquitin C-terminal hydrolase isozymes function as reciprocal modulators of germ cell apoptosis in cryptorchid testis.

The experimentally induced cryptorchid mouse model is useful for elucidating the in vivo molecular mechanism of germ cell apoptosis. Apoptosis, in general, is thought to be partly regulated by the ubiquitin-proteasome system. Here, we analyzed the function of two closely related members of the ubiquitin C-terminal hydrolase (UCH) family in testicular germ cell apoptosis experimentally induced by cryptorchidism. The two enzymes, UCH-L1 and UCH-L3, deubiquitinate ubiquitin-protein conjugates and control the cellular balance of ubiquitin. The testes of gracile axonal dystrophy (gad) mice, which lack UCH-L1, were resistant to cryptorchid stress-related injury and had reduced ubiquitin levels. The level of both anti-apoptotic (Bcl-2 family and XIAP) and prosurvival (pCREB and BDNF) proteins was significantly higher in gad mice after cryptorchid stress. In contrast, Uchl3 knockout mice showed profound testicular atrophy and apoptotic germ cell loss after cryptorchid injury. Ubiquitin level was not significantly different between wild-type and Uchl3 knockout mice, whereas the levels of Nedd8 and the apoptotic proteins p53, Bax, and caspase3 were elevated in Uchl3 knockout mice. These results demonstrate that UCH-L1 and UCH-L3 function differentially to regulate the cellular levels of anti-apoptotic, prosurvival, and apoptotic proteins during testicular germ cell apoptosis.

Animals↗

PubMed2004

Multiple signal transduction pathways mediated by 5-HT receptors.

Among human serotonin (5-HT) receptor subtypes, each G protein-coupled receptor subtype is reported to have one G protein-signaling cascade. However, the signaling may not be as simple as previously thought to be. 5-HT5A receptors are probably the least well understood among the 5-HT receptors, but the authors found that 5-HT5A receptors couple to multiple signaling cascades. When the 5-HT5A receptors were expressed in undifferentiated C6 glioma cells, they modulated the level of second messengers. For example, activation of 5-HT5A receptors inhibited the adenylyl cyclase activity and subsequently reduced the cAMP level, as previously reported. In addition to this known signaling via Gi/Go, 5-HT5A receptors are coupled to the inhibition of ADP-ribosyl cyclase and cyclic ADP ribose formation. On the other hand, activation of 5-HT5A receptors transiently opened the K+ channels, presumably due to the increase in intracellular Ca2+ after formation of inositol (1,4,5) trisphosphate. The K+ currents were inhibited by both heparin and pretreatment with pertussis toxin, suggesting the cross-talk between Gi/Go protein and phopholipase C cascade. Thus, the authors results indicate that 5-HT5A receptors couple to multiple second messenger systems and may contribute to the complicated physiological and pathophysiological states. Although this multiple signaling has been reported only for 5-HT5A/5-HT1 receptors so far, it is possible that other 5-HT receptor subtypes bear similar complexity. As a result, in addition to the wide variety of expression patterns of each 5-HT receptor subtype, it is possible that multiple signal transduction systems may add complexity to the serotonergic system in brain function. The investigation of these serotonergic signaling and its impairment at cellular level may help to understand the symptoms of brain diseases.

ADP-ribosyl Cyclase↗

PubMed2003

Ubiquitin carboxy-terminal hydrolase L1 binds to and stabilizes monoubiquitin in neuron.

Mammalian neuronal cells abundantly express a deubiquitylating enzyme, ubiquitin carboxy-terminal hydrolase 1 (UCH L1). Mutations in UCH L1 are linked to Parkinson's disease as well as gracile axonal dystrophy (gad) in mice. In contrast to the UCH L3 isozyme that is universally expressed in all tissues, UCH L1 is expressed exclusively in neurons and testis/ovary. We found that UCH L1 associates and colocalizes with monoubiquitin and elongates ubiquitin half-life. The gad mouse, in which the function of UCH L1 is lost, exhibited a reduced level of monoubiquitin in neurons. In contrast, overexpression of UCH L1 caused an increase in the level of ubiquitin in both cultured cells and mice. These data suggest that UCH L1, with avidity and affinity for ubiquitin, insures ubiquitin stability within neurons. This study is the first to show the function of UCH L1 in vivo.

Animals↗

PubMed2003

Alterations of structure and hydrolase activity of parkinsonism-associated human ubiquitin carboxyl-terminal hydrolase L1 variants.

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a neuron-specific ubiquitin recycling enzyme. A mutation at residue 93 and polymorphism at residue 18 within human UCH-L1 are linked to familial Parkinson's disease and a decreased Parkinson's disease risk, respectively. Thus, we constructed recombinant human UCH-L1 variants and examined their structure (using circular dichroism) and hydrolase activities. We confirmed that an I93M substitution results in a decrease in kcat (45.6%) coincident with an alteration in alpha-helical content. These changes may contribute to the pathogenesis of Parkinson's disease. In contrast, an S18Y substitution results in an increase in kcat (112.6%) without altering the circular dichroistic spectrum. These data suggest that UCH-L1 hydrolase activity may be inversely correlated with Parkinson's disease risk and that the hydrolase activity is protective against the disease. Furthermore, we found that oxidation of UCH-L1 by 4-hydroxynonenal, a candidate for endogenous mediator of oxidative stress-induced neuronal cell death, results in a loss of hydrolase activity. Taken together, these results suggest that further studies of altered UCH-L1 hydrolase function may provide new insights into a possible common pathogenic mechanism between familial and sporadic Parkinson's disease.

Aldehydes↗

PubMed2003

Expression and function of bradykinin receptors in microglia.

Expression of bradykinin (BK) receptors and their cellular function were investigated in microglia. Microglial cells were isolated from mixed cultures of cerebrocortical cells from postnatal day 3 Wistar rats. Reverse transcription-PCR (RT-PCR) showed that rat primary microglia express mRNAs for the type 2 bradykinin (B(2)) receptor subtype but not the type 1 (B(1)) receptor subtype under our experimental condition. However, the expression of B(1) receptor was greatly up-regulated after the treatment of microglia with BK for 24 hours. The expression of B(2) receptor in microglia was further confirmed by immunocytochemistry. Membrane currents were measured using whole-cell recording under voltage-clamp conditions. In 14% of patched cells (12/85 cells), BK (100-200 nM) induced an outward current at the holding potential of -20 mV, with oscillations in 2 cases. The BK-induced outward current was transient and desensitized rapidly. TEA inhibited the BK-induced outward current in a dose-dependent manner. These results suggest that microglia express B(2) receptors and presumably increase the intracellular Ca(2+) concentration via inositol trisphosphate with the subsequent activation of Ca(2+)-dependent K(+) channels. Our data provide the first evidence that microglia express functional BK receptors and support the idea that microglia play an important role in CNS inflammatory responses.

Animals↗

PubMed2003

Translocation of glutamate transporter subtype excitatory amino acid carrier 1 protein in kainic acid-induced rat epilepsy.

Glutamate excitotoxicity has been implicated in the pathophysiology of epilepsy. Systemic injection of kainic acid (KA) in the rat produces an animal model of human temporal lobe epilepsy. We examined the temporal expression of the sodium-dependent neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), in KA-induced rat epilepsy. As an early alteration, perinuclear deposits of EAAC1 protein were found mainly in the large pyramidal neurons at the hippocampus, neocortex, piriform cortex, and amygdala with the reduction of neuropil staining 6 hours after KA injection. Immunoelectron microscopic study revealed that the perinuclear EAAC1 immunoreactivity corresponded to the translocation to the Golgi complex. At this time point, EAAC1 mRNA was down-regulated. The intracellular aggregation of EAAC1 primarily disappeared by 24 hours. In vitro studies indicated that internalization of EAAC1 from the plasma membrane to the intracellular compartment by KA treatment was associated with the reduction of electrogenic transporter currents. Our results suggest that the transient EAAC1 internalization participates in the modulation of the transporter function preventing excessive glutamate uptake to pyramidal neurons during the early stage of epilepsy.

Amino Acid Transport System X-AG↗

PubMed2003

Recombinant human serotonin 5A receptors stably expressed in C6 glioma cells couple to multiple signal transduction pathways.

Human serotonin 5A (5-HT5A) receptors were stably expressed in undifferentiated C6 glioma. In 5-HT5A receptors-expressing cells, accumulation of cAMP by forskolin was inhibited by 5-HT as reported previously. Pertussis toxin-sensitive inhibition of ADP-ribosyl cyclase was also observed, indicating a decrease of cyclic ADP ribose, a potential intracellular second messenger mediating ryanodine-sensitive Ca2+ mobilization. On the other hand, 5-HT-induced outward currents were observed using the patch-clamp technique in whole-cell configuration. The 5-HT-induced outward current was observed in 84% of the patched 5-HT5A receptor-expressing cells and was concentration-dependent. The 5-HT-induced current was inhibited when intracellular K+ was replaced with Cs+ but was not significantly inhibited by typical K+ channel blockers. The 5-HT-induced current was significantly attenuated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the patch pipette. Depleting intracellular Ca2+ stores by application of caffeine or thapsigargin also blocked the 5-HT-induced current. Blocking G protein, the inositol triphosphate (IP3) receptor, or pretreatment with pertussis toxin, all inhibited the 5-HT-induced current. IP3 showed a transient increase after application of 5-HT in 5-HT5A receptor-expressing cells. It was concluded that in addition to the inhibition of cAMP accumulation and ADP-ribosyl cyclase activity, 5-HT5A receptors regulate intracellular Ca2+ mobilization which is probably a result of the IP3-sensitive Ca2+ store. These multiple signal transduction systems may induce complex changes in the serotonergic system in brain function.

ADP-ribosyl Cyclase↗

PubMed2003

Subtype-specific coupling with ADP-ribosyl cyclase of metabotropic glutamate receptors in retina, cervical superior ganglion and NG108-15 cells.

Cyclic ADP-ribose (cADP-ribose) is a putative second messenger or modulator. However, the role of cADP-ribose in the downstream signals of the metabotropic glutamate receptors (mGluRs) is unclear. Here, we show that glutamate stimulates ADP-ribosyl cyclase activity in rat or mouse crude membranes of retina via group III mGluRs or in superior cervical ganglion via group I mGluRs. The retina of mGluR6-deficient mice showed no increase in the ADP-ribosyl cyclase level in response to glutamate. GTP enhanced the initial rate of basal and glutamate-stimulated cyclase activity. GTP-gamma-S also stimulated basal activity. To determine whether the coupling mode of mGluRs to ADP-ribosyl cyclase is a feature common to individual cloned mGluRs, we expressed each mGluR subtype in NG108-15 neuroblastoma x glioma hybrid cells. The glutamate-induced stimulation of the cyclase occurs preferentially in NG108-15 cells over-expressing mGluRs1, 3, 5, and 6. Cells expressing mGluR2 or mGluRs4 and 7 exhibit inhibition or no coupling, respectively. Glutamate-induced activation or inhibition of the cyclase activity was eliminated after pre-treatment with cholera or pertussis toxin, respectively. Thus, the subtype-specific coupling of mGluRs to ADP-ribosyl cyclase via G proteins suggests that some glutamate-evoked neuronal functions are mediated by cADP-ribose.

ADP-ribosyl Cyclase↗

PubMed2002

Measurement of adenylyl cyclase by separating cyclic AMP on silica gel thin-layer chromatography.

A procedure for isolation of cyclic AMP (cAMP) by thin-layer chromatography on silica gel is described. One-dimensional ascending chromatograms were developed using [H(2)O/C(2)H(5)OH/NH(4)HCO(3) (30%:70%:0.2M)] as the mobile phase. This procedure separated [32P]cAMP from other radioactive metabolites of [32P]ATP in up to 19 samples on one sheet (20 x 10 cm) over 40-60 min at room temperature (21 degrees C). This simple and rapid isolation method provides a novel and convenient technique for the assay of adenylyl cyclase.

Adenylyl Cyclases↗

PubMedDate not supplied

Kinin receptors in cultured rat microglia.

Kinins are produced and act at the site of injury and inflammation in various tissues. They are likely to initiate a particular cascade of inflammatory events, which evokes physiological and pathophysiological responses including an increase in blood flow and plasma leakage. In the central nervous system (CNS), kinins are potent stimulators of the production and release of pro-inflammatory mediators represented by prostanoids and cytotoxins. They are known to induce neural tissue damage. Many of the cytotoxins such as cytokines and free radicals and prostanoids are released from glial cells. Among glial cells, astrocytes and oligodendrocytes are known to possess bradykinin (BK) B(2) receptors that phosphoinositide (PI) turnover and raise intracellular Ca(2+) concentration. The presence of bradykinin receptors in microglia has been of great significance. We recently showed that rat primary microglia express kinin receptors. In resting microglia, B(2) receptors but not B(1) receptors are expressed. When the microglia are activated by bradykinin, B(1) receptors are up-regulated, while B(2) receptors are down-regulated. As observed in other glial cells, electrophysiological measurements suggest that B(2) receptors in phosphoinositide turnover and intracellular Ca(2+) concentration in microglia. Release of cytotoxins is likely consequent upon the activation of BK receptors. Our study provides the first evidence that microglia express functional kinin receptors and suggests that microglia play an important role in CNS inflammatory responses.

Animals↗