Pathological analysis of Prader-Willi syndrome using adipocytes.

Prader-Willi syndrome (PWS) is a complex epigenetic disorder caused by the deficiency of paternally expressed genes in chromosome 15q11-q13. This syndrome also includes endocrine dysfunction, leading to short stature, hypogonadism, and obscure hyperphagia. Although recent progress has been made toward understanding the genetic basis for PWS, the molecular mechanisms underlying its pathology in obesity remain unclear. In this study, we examined the adipocytic characteristics of two PWS-induced pluripotent stem cell (iPSC) lines: those with the 15q11-q13 gene deletion (iPWS cells) and those with 15q11-q13 abnormal methylation (M-iPWS cells). The transcript levels of the lipid-binding protein aP2 were decreased in iPWS and M-iPWS adipocytes. Flow-cytometry analysis showed that PWS adipocytes accumulated more lipid droplets than did normal individual adipocytes. Furthermore, glucose uptake upon insulin stimulation was attenuated compared to that in normal adipocytes. Overall, our results suggest a significantly increased lipid content and defective in glucose metabolism in PWS adipocytes.

Defects in early synaptic formation and neuronal function in Prader-Willi syndrome.

Prader-Willi syndrome (PWS), which is a complex epigenetic disorder caused by the deficiency of paternally expressed genes in chromosome 15q11-q13, is associated with several psychiatric dimensions, including autism spectrum disorder. We have previously reported that iPS cells derived from PWS patients exhibited aberrant differentiation and transcriptomic dysregulation in differentiated neural stem cells (NSCs) and neurons. Here, we identified SLITRK1 as a downregulated gene in NSCs differentiated from PWS patient iPS cells by RNA sequencing analysis. Because SLITRK1 is involved in synaptogenesis, we focused on the synaptic formation and function of neurons differentiated from PWS patient iPS cells and NDN or MAGEL2 single gene defect mutant iPS cells. Although ßIII tubulin expression levels in all the neurons were comparable to the level of differentiation in the control, pre- and postsynaptic markers were significantly lower in PWS and mutant neurons than in control neurons. PSD-95 puncta along ßIII tubulin neurites were also decreased. Membrane potential responses were measured while exposed to high K+ stimulation. The neuronal excitabilities in PWS and mutant neurons showed significantly lower intensity than that of control neurons. These functional defects in PWS neurons may reflect phenotypes of neurodevelopmental disorders in PWS.

Carbon nanotube recognition by human Siglec-14 provokes inflammation.

For the design and development of innovative carbon nanotube (CNT)-based tools and applications, an understanding of the molecular interactions between CNTs and biological systems is essential. In this study, a three-dimensional protein-structure-based in silico screen identified the paired immune receptors, sialic acid immunoglobulin-like binding lectin-5 (Siglec-5) and Siglec-14, as CNT-recognizing receptors. Molecular dynamics simulations showed the spatiotemporally stable association of aromatic residues on the extracellular loop of Siglec-5 with CNTs. Siglec-14 mediated spleen tyrosine kinase (Syk)-dependent phagocytosis of multiwalled CNTs and the subsequent secretion of interleukin-1ß from human monocytes. Ectopic in vivo expression of human Siglec-14 on mouse alveolar macrophages resulted in enhanced recognition of multiwalled CNTs and exacerbated pulmonary inflammation. Furthermore, fostamatinib, a Syk inhibitor, blocked Siglec-14-mediated proinflammatory responses. These results indicate that Siglec-14 is a human activating receptor recognizing CNTs and that blockade of Siglec-14 and the Syk pathway may overcome CNT-induced inflammation.

Abnormal DNA methylation in pluripotent stem cells from a patient with Prader-Willi syndrome results in neuronal differentiation defects.

DNA methylation is a common method of gene expression regulation, and this form of regulation occurs in the neurodevelopmental disorder Prader-Willi syndrome (PWS). Gene expression regulation via methylation is important for humans, although there is little understanding of the role of methylation in neuronal differentiation. We characterized the cellular differentiation potential of iPS cells derived from a patient with PWS with abnormal methylation (M-iPWS cells). A comparative genomic hybridization (CGH) array revealed that, unlike iPWS cells (deletion genes type), the abnormally methylated M-iPWS cells had no deletion in the15q11.2-q13 chromosome region. In addition, methylation-specific PCR showed that M-iPWS cells had strong methylation in CpG island of the small nuclear ribonucleoprotein polypeptide N (SNRPN) on both alleles. To assess the effect of abnormal methylation on cell differentiation, the M-iPWS and iPWS cells were induced to differentiate into embryoid bodies (EBs). The results suggest that iPWS and M-iPWS cells are defective at differentiation into ectoderm. Neural stem cells (NSCs) and neurons derived from M-iPWS cells had fewer NSCs and mature neurons with low expression of NSCs and neuronal markers. We conclude that expression of the downstream of genes in the PWS region regulated by methylation is involved in neuronal differentiation.

Sir2D, a Sirtuin family protein, regulates adenylate cyclase A expression through interaction with the MybB transcription factor early in Dictyostelium development upon starvation.

Sirtuin interacts with many regulatory proteins involved in energy homeostasis, DNA repair, cell survival, and lifespan extension. We investigated the functional roles of Sir2D during early Dictyostelium development upon starvation. We found that ectopic expression of Sir2D accelerated development among three Sirtuins containing highly homologous catalytic domain sequences to mouse Sirt1. Sir2D expression upregulated adenylate cyclase A (aca) mRNA expression 2, 4 and 6 h after starvation. We have previously reported that nicotinamide, a Sirt1 inhibitor, treatment delayed the development and decreased the expression of aca at 4 h after starvation. Sir2D expressing cells showed resistance against the nicotinamide effect. RNAi-mediated Sir2D knockdown cells were generated, and their development was also delayed. Aca expression was decreased 4 h after starvation. Sir2D expression restored the developmental impairment of Sir2D knockdown cells. The induction of aca upon starvation starts with transcriptional activation of MybB. The ectopic expression of MybB accelerated the development and increased the expression of aca 2 and 4 h after starvation but did not restore the phenotype of Sir2D knockdown cells. Sir2D expression had no effects on MybB-null mutant cells during early development. Thus, MybB is necessary for the upregulation of aca by Sir2D, and Sir2D is necessary for the full induction of aca after 4 h by MybB. MybB was coimmunoprecipitated with Sir2D, suggesting an interaction between MybB and Sir2D. These results suggest that Sir2D regulates aca expression through interaction with the MybB transcription factor early in Dictyostelium development upon starvation.

Neuronal differentiation defects in induced pluripotent stem cells derived from a Prader-Willi syndrome patient.

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder caused by a lack of expression of paternally inherited genes located in the15q11.2-q13 chromosome region. An obstacle in the study of human neurological diseases is the inaccessibility of brain material. Generation of induced pluripotent stem cells (iPSC cells) from patients can partially overcome this problem. We characterized the cellular differentiation potential of iPS cells derived from a PWS patient with a paternal 15q11-q13 deletion. A gene tip transcriptome array revealed very low expression of genes in the 15q11.2-q13 chromosome region, including SNRPN, SNORD64, SNORD108, SNORD109, and SNORD116, in iPS cells of this patient compared to that in control iPS cells. Methylation-specific PCR analysis of the SNRPN gene locus indicated that the PWS region of the paternal chromosome was deleted or methylated in iPS cells from the patient. Both the control and patient-derived iPS cells were positive for Oct3/4, a key marker of pluripotent cells. After 11 days of differentiation into neural stem cells (NSCs), Oct3/4 expression in both types of iPS cells was decreased. The NSC markers Pax6, Sox1, and Nestin were induced in NSCs derived from control iPS cells, whereas induction of these NSC markers was not apparent in NSCs derived from iPS cells from the patient. After 7 days of differentiation into neurons, neuronal cells derived from control iPS cells were positive for ßIII-tubulin and MAP2. However, neuronal cells derived from patient iPS cells only included a few immunopositive neurons. The mRNA expression levels of the neuronal marker ßIII-tubulin were increased in neuronal cells derived from control iPS cells, while the expression levels of ßIII-tubulin in neuronal cells derived from patient iPS cells were similar to those of NSCs. These results indicate that iPS cells derived from a PWS patient exhibited neuronal differentiation defects.

Nse1 and Nse4, subunits of the Smc5-Smc6 complex, are involved in Dictyostelium development upon starvation.

The Smc5-Smc6 complex contains a heterodimeric core of two SMC proteins and non-Smc elements (Nse1-6), and plays an important role in DNA repair. We investigated the functional roles of Nse4 and Nse1 in Dictyostelium discoideum. Nse4 and Nse3 expressed as Flag-tagged fusion proteins were highly enriched in nuclei, while Nse1 was localized in whole cells. Using yeast two-hybrid assays, only the interaction between Nse3 and Nse1 was detected among the combinations. However, all of the interactions among these three proteins were recognized by co-immunoprecipitation assay using cell lysates prepared from the cells expressing green fluorescent protein (GFP)- or Flag-tagged fusion proteins. GFP-tagged Nse1, which localized in whole cells, was translocated to nuclei when co-expressed with Flag-tagged Nse3 or Nse4. RNAi-mediated Nse1 and Nse4 knockdown cells (Nse1 KD and Nse4 KD cells) were generated and found to be more sensitive to UV-induced cell death than control cells. Upon starvation, Nse1 and Nse4 KD cells had increases in the number of smaller fruiting bodies that formed on non-nutrient agar plates or aggregates that formed under submerged culture. We found a reduction in the mRNA level of pdsA, in vegetative and 8 h-starved Nse4 KD cells, and pdsA knockdown cells displayed effects similar to Nse4 KD cells. Our results suggest that Nse4 and Nse1 are involved in not only the cellular DNA damage response but also cellular development in D. discoideum.

GPR87 mediates lysophosphatidic acid-induced colony dispersal in A431 cells.

We have previously reported that an orphan G protein-coupled receptor GPR87 was activated by lysophosphatidic acid (LPA) and that it induced an increase in the intracellular Ca(2+) levels in the CHO cells genetically engineered to express GPR87-Gα16 fusion protein. Because the Ca(2+) response was blocked by the LPA receptor antagonist Ki16425, GPR87 was suggested to be a putative LPA receptor. However, further studies are required to confirm whether GPR87 is an LPA receptor. A previous study showed that colonies of A431 cells treated with LPA showed rapid and synchronized dissociation. Because A431 cells have been shown to express GPR87, we used these cells to examine whether GPR87 acted as an LPA receptor. When A431 cells were treated with gpr87-specific siRNA, the expression of GPR87 was decreased and LPA-induced colony dispersal was significantly reduced. Treatment of the cells with lpa1 siRNA had an additive effect in decrease in the colony dispersal. Studies on the LPA-mediated signaling pathway in A431 cells indicated that transactivation of the epidermal growth factor receptor (EGFR) by LPA led to cell scattering. PD153035, an inhibitor of tyrosine-kinase of EGFR, and BB94, an inhibitor of metalloprotease which produces a ligand for EGFR, significantly prevented the LPA-induced scattering of A431 cells pretreated with lpa1 or gpr87-siRNA. These results strongly suggested that GPR87 acts as an LPA receptor and induces colony dispersal via the transactivation of EGFR in A431 cells.

Ketamine reduces amyloid ß-protein degradation by suppressing neprilysin expression in primary cultured astrocytes.

Pathological accumulation of cortical amyloid ß-protein (Aß) is an early and consistent feature of Alzheimer's disease (AD). Aß levels in the brain are determined by production and catabolism. Previous studies have suggested that deficits in the brain expression of neprilysin (NEP) and the insulin-degrading enzyme (IDE), which are both proteases involved in amyloid degradation, may promote Aß deposition in patients with sporadic late-onset AD. Because the incidence of AD increases after surgical intervention, we examined whether ketamine, which is a general anaesthetic with neuroprotective properties for excitotoxic ischaemic damage, is associated with Aß degradation by inducing NEP and IDE expression. The non-competitive N-methyl-d-aspartate receptor antagonist ketamine and MK801 significantly decreased the expression of NEP, but not IDE, in a concentration- and time-dependent manner through the dephosphorylation of p38 mitogen-activated protein kinase (MAPK) in cultured rat astrocytes. Furthermore, NEP-reduced reagents significantly suppressed the degradation of exogenous Aß in cultured astrocytes. These results suggested that ketamine suppresses the Aß degradation of NEP by reducing p38 MAPK-mediated pathway activity.

Propofol and thiopental suppress amyloid fibril formation and GM1 ganglioside expression through the γ-aminobutyric acid A receptor.

BACKGROUND: The incidence of Alzheimer disease may increase after surgical interventions. Amyloid ß-protein (Aß) fibrillogenesis, which is closely related to Alzheimer disease, is reportedly accelerated by exposure to anesthetics. However, the effects of GM1 ganglioside (GM1) on Αß fibrillogenesis have not yet been reported. The current study was designed to examine whether the anesthetics propofol and thiopental are associated with Αß assembly and GM1 expression on the neuronal cell surface. METHODS: PC12N cells and cultured neuronal cells were treated with propofol or thiopental, and GM1 expression in treated and untreated cells was determined by the specific binding of horseradish peroxidase-conjugated cholera toxin subunit B (n = 5). The effects of an inhibitor of the γ-aminobutyric acid A receptor was also examined (n= 5). In addition, the effects of the anesthetics on GM1 liposome-induced Αß assembly were investigated (n = 5). Finally, the neurotoxicity of the assembled Αß fibrils was studied by the lactate dehydrogenase release assay (n = 6). RESULTS: Propofol (31.2 ± 4.7%) and thiopental (34.6 ± 10.5%) decreased GM1 expression on the cell surface through the γ-aminobutyric acid A receptor. The anesthetics inhibited Αß fibril formation from soluble Αß in cultured neurons. Moreover, propofol and thiopental suppressed GM1-induced fibril formation in a cell-free system (propofol, 75.8 ± 1.9%; thiopental, 83.6 ± 1.9%) and reduced the neurotoxicity of a mixture containing Aß and GM1 liposomes (propofol, 35.3 ± 16.4%; thiopental, 21.3 ± 11.6%). CONCLUSIONS: Propofol and thiopental have direct and indirect inhibitory effects on Αß fibrillogenesis.

Brain insulin resistance accelerates Aß fibrillogenesis by inducing GM1 ganglioside clustering in the presynaptic membranes.

Type 2 diabetes mellitus is thought to be a significant risk factor for Alzheimer's disease. Insulin resistance also affects the central nervous system by regulating key processes, such as neuronal survival and longevity, learning and memory. However, the mechanisms underlying these effects remain uncertain. To investigate whether insulin resistance is associated with the assembly of amyloid ß-protein (Aß) at the cell surface of neurons, we inhibited insulin-signalling pathways of primary neurons. The treatments of insulin receptor (IR)-knockdown and a phosphatidylinositol 3-kinase inhibitor (LY294002), but not an extracellular signal-regulated kinase inhibitor, induced an increase in GM1 ganglioside (GM1) levels in detergent-resistant membrane microdomains of the neurons. The aged db/db mouse brain exhibited reduction in IR expression and phosphorylation of Akt, which later induced an increase in the high-density GM1-clusters on synaptosomes. Neurons treated with IR knockdown or LY294002, and synaptosomes of the aged db/db mouse brains markedly accelerated an assembly of Aßs. These results suggest that ageing and peripheral insulin resistance induce brain insulin resistance, which accelerates the assembly of Aßs by increasing and clustering of GM1 in detergent-resistant membrane microdomains of neuronal membranes.

Insulin inhibits Abeta fibrillogenesis through a decrease of the GM1 ganglioside-rich microdomain in neuronal membranes.

Type 2 diabetes is a risk factor for late-onset Alzheimer's disease. However, the underlying mechanisms remain unknown. To investigate whether insulin is associated with the assembly of amyloid beta-protein from the cell surface, we treated nerve growth factor (NGF)-treated rat pheochromocytoma 12 (PC12) cells with insulin, which is related to the development of diabetes. Insulin treatment induced a decrease in GM1 ganglioside (GM1) levels in detergent-resistant membrane microdomains of NGF-treated PC12 cells. The insulin-induced effects on GM1 levels were regulated by a phosphatidylinositol 3-kinase inhibitor, but not by an extracellular signal-regulated kinase inhibitor. Pre-treatment with a protein synthesis inhibitor did not inhibit the decrease in GM1 levels induced by insulin. In addition, insulin failed to induce formation of fibrils from soluble amyloid beta-protein or to accelerate GM1-induced fibril formation. Furthermore, assembly of amyloid beta-protein in cultures of NGF-treated PC12 cells was significantly decreased by insulin. These results suggest that insulin inhibits amyloid beta-protein assembly by decreasing GM1 expression in detergent-resistant membrane microdomains of neuronal membranes.

Possible protection by notoginsenoside R1 against glutamate neurotoxicity mediated by N-methyl-D-aspartate receptors composed of an NR1/NR2B subunit assembly.

Notoginsenoside R1 (NTR1) is the main active ingredient in Panax notoginseng, a herbal medicine widely used in Asia for years. The purpose of this study was to investigate pharmacological properties of NTR1 on neurotoxicity of glutamate (Glu) in primary cultured mouse cortical neurons along with its possible mechanism of action. We found that NTR1 significantly protected neurons from the loss of cellular viability caused by brief exposure to 10 microM Glu for 1 hr in a dose-dependent manner at concentrations from 0.1 to 10 microM, without affecting the viability alone. NTR1 significantly inhibited the increased number of cells positive to propidium iodide (PI) staining, increase of intracellular free Ca(2+) ions, overproduction of intracellular reactive oxygen species, and depolarization of mitochondrial membrane potential in cultured neurons exposed to Glu, in addition to blocking decreased Bcl-2 and increased Bax expression levels. We further evaluated the target site at which NTR1 protects neurons from Glu toxicity by using the acquired expression strategy of N-methyl-D-aspartate (NMDA) receptor subunits in human embryonic kidney 293 cells. We found that 10 microM NTR1 protected NR1/NR2B subunit expressing cells from cell death by 100 microM NMDA, but not cells expressing NR1/NR2A subunits, when determined by PI staining. These results suggest that NTR1 may preferentially protect neurons from Glu excitotoxicity mediated by NMDA receptor composed of an NR1/NR2B subunit assembly in the brain.

A critical importance of polyamine site in NMDA receptors for neurite outgrowth and fasciculation at early stages of P19 neuronal differentiation.

We have investigated the role of N-methyl-d-aspartate receptors (NMDARs) and gamma-aminobutyric acid receptors type A (GABA(A)Rs) at an early stage of P19 neuronal differentiation. The subunit expression was profiled in 24-hour intervals with RT-PCR and functionality of the receptors was verified via fluo-3 imaging of Ca(2+) dynamics in the immature P19 neurons showing that both NMDA and GABA excite neuronal bodies, but only polyamine-site sensitive NMDAR stimulation leads to enhanced Ca(2+) signaling in the growth cones. Inhibition of NR1/NR2B NMDARs by 1 muM ifenprodil severely impaired P19 neurite extension and fasciculation, and this negative effect was fully reversible by polyamine addition. In contrast, GABA(A)R antagonism by a high dose of 200 microM bicuculline had no observable effect on P19 neuronal differentiation and fasciculation. Except for the differential NMDAR and GABA(A)R profiles of Ca(2+) signaling within the immature P19 neurons, we have also shown that inhibition of NR1/NR2B NMDARs strongly decreased mRNA level of NCAM-180, which has been previously implicated as a regulator of neuronal growth cone protrusion and neurite extension. Our data thus suggest a critical role of NR1/NR2B NMDARs during the process of neuritogenesis and fasciculation of P19 neurons via differential control of local growth cone Ca(2+) surges and NCAM-180 signaling.

Up-regulation of ciliary neurotrophic factor receptor expression by GABAA receptors in undifferentiated neural progenitors of fetal mouse brain.

In this study, we evaluated the role of the GABA(A) receptor (GABA(A)R), expressed by undifferentiated neural progenitors isolated from fetal mouse neocortex, in the mechanisms relevant to self-replication and differentiation toward neuronal and astroglial lineages. Round spheres were formed with clusters of proliferating cells within 2-4 days during culture with epidermal growth factor (EGF), whereas the size of these clusters was drastically increased in proportion to increasing durations up to 10 days. Sustained exposure to the GABA(A)R agonist muscimol at 100 microM led to significant increases in the size of neurospheres cultured for 6-10 days, with increased proliferative activity and unchanged lactate dehydrogenase release in a manner sensitive to the GABA(A)R antagonist bicuculline. Muscimol also significantly increased the incorporation of 5-bromo-2'-deoxyuridine in neurospheres in a bicuculline-sensitive manner, whereas both high potassium and nifedipine significantly decreased the neurosphere area with increased numbers of apoptotic cells. Prior activation of GABA(A)R significantly promoted the subsequent expression of an astroglial marker protein in cells differentiated by ciliary neurotrophic factor (CNTF) toward an astroglial lineage after the removal of EGF, with a concomitant decrease in neuronal marker protein expression. In neurospheres with GABA(A)R activation, a significant and predominant increase was seen in mRNA expression of CNTF receptors. These results suggest that prior tonic activation of GABA(A)R may preferentially promote the differentiation by CNTF of neural progenitor cells toward an astroglial lineage through selective up-regulation of CNTF receptor expression in the developing mouse brain.

Promotion of neuronal differentiation through activation of N-methyl-D-aspartate receptors transiently expressed by undifferentiated neural progenitor cells in fetal rat neocortex.

Neural progenitor cell is a generic term for undifferentiated cell populations composed of neural stem, neuronal progenitor, and glial progenitor cells with abilities for self-renewal and multipotentiality. In this study, we have attempted to evaluate the possible functional expression of N-methyl-D-aspartate (NMDA) receptors by neural progenitor cells prepared from neocortex of 18-day-old embryonic rats. Cells were cultured in the presence of basic fibroblast growth factor (bFGF) for different periods up to 12 days under floating conditions. Reverse transcription-polymerase chain reaction and fluorescence imaging analyses revealed transient expression of functional NMDA receptors in neurospheres formed by clustered progenitors during the culture with bFGF. A similarly potent increase was seen in the fluorescence intensity after brief exposure to NMDA in cells differentiated after the removal of bFGF under adherent conditions, and an NMDA receptor antagonist invariably prevented these increases by NMDA. Moreover, sustained exposure to NMDA not only inhibited the formation of neurospheres when exposed for 10 days from day 2 to day 12 but also promoted spontaneous and induced differentiation of neurospheres to cells immunoreactive for a neuronal marker protein on immunocytochemistry and Western blotting analyses. These results suggest that functional NMDA receptors may be transiently expressed to play a role in mechanisms underlying the modulation of proliferation along with the determination of subsequent differentiation fate toward a neuronal lineage in neural progenitor cells of developing rat neocortex.

Modulation of cellular proliferation and differentiation through GABA(B) receptors expressed by undifferentiated neural progenitor cells isolated from fetal mouse brain.

In this study, we have attempted to evaluate the possible role of metabotropic GABA(B) receptors (GABA(B)R) expressed by neural progenitor cells prepared from neocortex of embryonic Std-ddY mice. Immunocytochemical analysis confirmed the validity of isolation procedures of neural progenitors, while round spheres were formed with clustered cells during culture with epidermal growth factor (EGF) for 10 days. A reverse transcription polymerase chain reaction analysis revealed constitutive expression of GABA(A)R, GABA(B)R, and GABA(C)R subtypes in undifferentiated progenitors and neurospheres formed within 10 days. Exposure to GABA led to concentration-dependent increases in the total area and proliferation activity of neurospheres at 10-300 microM, while the GABA(B)R agonist baclofen at 100 microM significantly increased the size of neurospheres expressing both GABA(B)R1 and GABA(B)R2 subunits in a manner sensitive to a GABA(B)R antagonist. By contrast, a significant decrease was seen in the total areas of neurospheres prepared from mice deficient of the GABA(B)R1 subunit. In neurospheres of GABA(B)R1-null mice, a significant increase was induced in the number of cells immunoreactive for a glial marker protein, with a concomitant decrease in that of a neuronal marker protein, upon spontaneous differentiation after the removal of EGF. These results suggest that GABA(B)R may be functionally expressed by neural progenitor cells to preferentially promote the commitment toward a neuronal lineage after the activation of cellular proliferation toward self-replication in the developing mouse brain.

Upregulation of Myo6 expression after traumatic stress in mouse hippocampus.

Traumatic stress has been believed to result in a variety of unusual alterations of the integrity and the functionality in the hippocampus. In this study, we searched for genes responsive to traumatic stress in the mouse hippocampus to elucidate the underlying mechanisms. Adult male mice were subjected to water-immersion restraint stress (WIRS) for 3h as an extremely stressful experience, followed by dissection of the hippocampus and subsequent extraction of RNA for differential display polymerase chain reaction (PCR) analysis. The actin-based molecular motor protein myosin VI (Myo6) was identified as a gene markedly upregulated by traumatic stress in the mouse hippocampus 24h after WIRS. Real-time PCR and Western blotting analyses clearly revealed a significant increase in the expression of both mRNA and corresponding protein for Myo6 in the hippocampus within 24h after WIRS, while WIRS failed to significantly affect the expression of Myo6 protein in the cerebral cortex, cerebellum and olfactory bulb. Immunohistochemistry analysis revealed that Myo6 protein was ubiquitously expressed throughout the mouse brain, with an extremely high level in the olfactory bulb. These results suggest that Myo6 may be selectively and rapidly upregulated to play a hitherto unidentified role in the maintenance of the integrity and functionality in the hippocampus after traumatic stress.

Insensitivity to glutamate neurotoxicity mediated by NMDA receptors in association with delayed mitochondrial membrane potential disruption in cultured rat cortical neurons.

We have attempted to elucidate mechanisms underlying differential vulnerability to glutamate (Glu) using cultured neurons prepared from discrete structures of embryonic rat brains. Brief exposure to Glu led to a significant decrease in the mitochondrial activity in hippocampal neurons cultured for 9 or 12 days at 10 muM to 1 mM with an apoptosis-like profile, without markedly affecting that in cortical neurons. Brief exposure to Glu also increased lactate dehydrogenase release along with a marked decrease in the number of cells immunoreactive for a neuronal marker protein in hippocampal, but not cortical, neurons. Similar insensitivity was seen to the cytotoxicity by NMDA, but not to that by tunicamycin, 2,4-dinitrophenol, hydrogen peroxide or A23187, in cortical neurons. However, NMDA was more efficient in increasing intracellular free Ca2+ levels in cortical neurons than in hippocampal neurons. Antagonists for neuroprotective metabotropic Glu receptors failed to significantly affect the insensitivity to Glu, while NMDA was more effective in disrupting mitochondrial membrane potentials in hippocampal than cortical neurons. These results suggest that cortical neurons would be insensitive to the apoptotic neurotoxicity mediated by NMDA receptors through a mechanism related to mitochondrial membrane potentials, rather than intracellular free Ca2+ levels, in the rat brain.

Group III metabotropic glutamate receptor activation suppresses self-replication of undifferentiated neocortical progenitor cells.

We evaluated the possible functional expression of metabotropic glutamate receptors (mGluRs) by neural progenitors from embryonic mouse neocortex. Constitutive expression was seen with group I, II, and III mGluRs in undifferentiated cells and neurospheres formed by clustered cells during culture with epidermal growth factor. The group III mGluR agonist, L-2-amino-4-phosphonobutyrate, drastically reduced proliferation activity at 1-100 microM without inducing cell death, with group I and group II mGluR agonists being ineffective, in these neurospheres. Both forskolin and a group III mGluR antagonist significantly increased the proliferation alone, but significantly prevented the suppression by L-2-amino-4-phosphonobutyrate. Activation of group III mGluR significantly decreased mRNA expression of the cell cycle regulator cyclinD1, in addition to inhibiting the transactivation mediated by cAMP of cyclinD1 gene in the pluripotent P19 progenitor cells. Prior activation of group III mGluR led to a significant decrease in the number of cells immunoreactive for a neuronal marker, with an increase in that for an astroglial marker irrespective of differentiation inducers. These results suggest that group III mGluR may be functionally expressed to suppress self-renewal capacity through a mechanism related to cAMP formation with promotion of subsequent differentiation into astroglial lineage in neural progenitors.