N-acetyl-D-glucosamine kinase interacts with dynein light-chain roadblock type 1 at Golgi outposts in neuronal dendritic branch points

N-acetylglucosamine kinase (GlcNAc kinase or NAGK) is a ubiquitously expressed enzyme in mammalian cells. Recent studies have shown that NAGK has an essential structural, non-enzymatic role in the upregulation of dendritogenesis. In this study, we conducted yeast two-hybrid screening to search for NAGK-binding proteins and found a specific interaction between NAGK and dynein light-chain roadblock type 1 (DYNLRB1). Immunocytochemistry (ICC) on hippocampal neurons using antibodies against NAGK and DYNLRB1 or dynein heavy chain showed some colocalization, which was increased by treating the live cells with a crosslinker. A proximity ligation assay (PLA) of NAGK-dynein followed by tubulin ICC showed the localization of PLA signals on microtubule fibers at dendritic branch points. NAGK-dynein PLA combined with Golgi ICC showed the colocalization of PLA signals with somal Golgi facing the apical dendrite and with Golgi outposts in dendritic branch points and distensions. NAGK-Golgi PLA followed by tubulin or DYNLRB1 ICC showed that PLA signals colocalize with DYNLRB1 at dendritic branch points and at somal Golgi, indicating a tripartite interaction between NAGK, dynein and Golgi. Finally, the ectopic introduction of a small peptide derived from the C-terminal amino acids 74-96 of DYNLRB1 resulted in the stunting of hippocampal neuron dendrites in culture. Our data indicate that the NAGK-dynein-Golgi tripartite interaction at dendritic branch points functions to regulate dendritic growth and/or branching.

Glucose-Insulin-Potassium Solution Protects Ventricular Myocytes of Neonatal Rat in an In Vitro Coverslip Ischemia/Reperfusion Model

BACKGROUND AND OBJECTIVES: The benefit of high glucose-insulin-potassium (GIK) solution in clinical applications is controversial. We established a neonatal rat ventricular myocyte (NRVM) in vitro coverslip ischemia/reperfusion (I/R) model and investigated the effects of GIK solution on suppressing reactive oxygen species (ROS) and upregulating O-GlcNacylation, which protects cells from ischemic injury. MATERIALS AND METHODS: NRVMs were isolated from postnatal day 3-4 Sprague-Dawley rat pups and grown in Dulbecco's modified Eagle's medium containing high glucose (4.5 g/L), fetal bovine serum, and penicillin/streptomycin. The effects of the GIK solution on ROS production, apoptosis, and expression of O-GlcNAc and O-GlcNAc transferase (OGT) were investigated in the coverslip I/R model. RESULTS: Covering the 24-well culture plates for 3 hr with 12 mm diameter coverslips resulted in the appropriate ischemic shock. Glucose and insulin synergistically reduced ROS production, protected NRVM dose-dependently from apoptosis, and altered O-GlcNAc and OGT expression. CONCLUSION: The high GIK solution protected NRVM from I/R injury in vitro by reducing ROS and altering O-GlcNacylation.

Unique Hippocampal Changes and Allodynia in a Model of Chronic Stress

Sustained stress can have numerous pathologic effects. There have been several animal models for chronic stress. We tried to identify the changes of pain threshold and hippocampus in a model of chronic stress. Male Sprague-Dawley rats were kept in a cage filled with 23degrees C water to a height of 2.2 cm for 7 days. Nociceptive thresholds, expressed in grams, were measured with a Dynamic Plantar Aesthesiometer. Golgi staining was used to identify hippocampal changes. To demonstrate how long allodynia was lasting, behavioral test was repeated daily on another experiment. Compared to control group, chronic stress group showed bilateral mechanical hyper-responsiveness on days 5 (P = 0.047) and 7 (P = 0.032). In general, dendrite atrophic changes within hippocampus of chronic stress model were much more prominent in comparison with control. Compared to control, decreased spine number (P < 0.001) and spine length (P < 0.001) on Golgi staining were seen in the hippocampus of animals with chronic stress. Bilateral mechanical hyperresponsiveness was recovered on day 19 in animals with chronic stress. Chronic stress may bring about central sensitization and hippocampal changes in rats.

Dendritic eIF4E-binding Protein 1 (eIF4E-BP1) mRNA Is Upregulated by Neuronal Activation

Long-term synaptic plasticity requires addition of new proteins at the synaptic site. The local protein synthesis at subsynaptic sites confers advantageous mechanisms that would regulate the protein composition in local domains on a moment-by-moment basis. However, our information on the identities of 'dendritic' mRNAs is very limited. In this study we investigated the expression of the protein and mRNA for eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) in cultured rat hippocampal neurons. Immunocytochemistry (ICC) showed that 4EBP1 protein is highly localized to the nucleus. In dendrites most 4EBP1 punctae were not colocalized with those of eIF4E. In situ hybridization (ISH) and Fluorescence ISH (FISH) revealed that 4EBP1 mRNA was present in dendrites. The FISH signals formed clusters along dendrites that colocalized with ICC signals for Staufen, a marker for RNA granules. The neuronal activation by KCl (60 mM, 10 min) significantly increased the density of 4EBP1 FISH signals in the nucleus after 2 hr, and both in the nucleus and dendrites after 6 hr. Our results indicate that 4EBP1 and its mRNA are present in dendrites, and the mRNA is upregulated and transported to dendritic domains in RNA granules upon neuronal activation.

Neuronal activation increases the density of eukaryotic translation initiation factor 4E mRNA clusters in dendrites of cultured hippocampal neurons

Activity-dependent dendritic translation in CNS neurons is important for the synapse-specific provision of proteins that may be necessary for strengthening of synaptic connections. A major rate-limiting factor during protein synthesis is the availability of eukaryotic translation initiation factor 4E (eIF4E), an mRNA 5'-cap-binding protein. In this study we show by fluorescence in situ hybridization (FISH) that the mRNA for eIF4E is present in the dendrites of cultured rat hippocampal neurons. Under basal culture conditions, 58.7 +/- 11.6% of the eIF4E mRNA clusters localize with or immediately adjacent to PSD-95 clusters. Neuronal activation with KCl (60 mM, 10 min) very significantly increases the number of eIF4E mRNA clusters in dendrites by 50.1 and 74.5% at 2 and 6 h after treatment, respectively. In addition, the proportion of eIF4E mRNA clusters that localize with PSD-95 increases to 74.4 +/- 7.7% and 77.8 +/- 7.6% of the eIF4E clusters at 2 and 6 h after KCl treatment, respectively. Our results demonstrate the presence of eIF4E mRNA in dendrites and an activity-dependent increase of these clusters at synaptic sites. This provides a potential mechanism by which protein translation at synapses may be enhanced in response to synaptic stimulation.

Hypoxia-A Possibility in Fibromyalgia Syndrome Pathogenesis / 대한류마티스학회지

OBJECTIVE: We studied the expression of pain-related molecules such as substance P involved in chronic pain of fibromyalgia syndrome (FMS) patients using rat cortical cells in hypoxia. METHODS: We sacrificed pregnant Sprague-Dawley rat and got embryo. We cultured the cortical cells and compared the expression of pain-related molecules in 1st, 3rd, 5th day cortical cells exposed to hypoxia (37 degrees C, 5% CO2, 98% N2) to control by immunohistochemistry. We measured the density at soma using softwear 'Scion image'. RESULTS: The expression of substance P was increased in hypoxic cortical cell group than control (control mean: 49.9 vs. hypoxia 1st day: 75.4 (p<0.001), 3rd day: 65.6 (p<0.001), 5th day: 79.9 (p<0.001)). The expression of kainate receptor was increased in hypoxic cortical cell group than control (control mean: 58.4 vs. hypoxia 1st day: 64.9 (p<0.001), 3rd day: 63.3 (p<0.001), 5th day: 62.9 (p<0.001)). The expression of N-methyl-D-aspartate receptor 2B was increased in hypoxic cortical cell group than control (control mean : 59.4 vs. hypoxia 1st day: 60.8 (p<0.001), 3rd day: 62.6 (p<0.001), 5th day: 67.1 (p<0.001)). But, the expression of calcitonin gene related peptide was decreased in hypoxic cortical cell group than control (control mean: 76.8 vs. hypoxia 1st day: 76.4 (p<0.001), 3rd day: 71.5 (p<0.001), 5th day: 61.3 (p<0.001)). CONCLUSION: Hypoxia during night could increase the expression of some pain-related molecules, which might be the cause of chronic pain in FMS patients.

Up-regulation of PSD-95 in the Rat Hippocampal Formation after Chronic Renal Failure / 대한해부학회지

The mechanism of central nervous system (CNS) dysfunction in uremia are multifactorial and only partially characterized. Studies using hippocampal formation (HF) evaluate the relationship between the uremia and memory impairment. Immunoblots with calcium permeable NMDA (N-methyl-D-aspartate) and AMPA (2-amino-3-hydroxy-5-methylisoxazole-4-propinoic acid) receptors and their associated PSD-95 proteins after chronic renal failure (CRF) provided significant new informations. CRF rats induced by 5/6 nephrectomized had significant effects on up-regulation of PSD-95 protein rather than those of calcium permeable NMDA and AMPA receptor subunits. Up-regulation of PSD-95 after CRF might be associated with the enhanced activity of NMDA and/or AMPA receptors, thereby leads to the intracellular Ca2+ accumulation and functional neuronal cell damage subsequently. Degradation of intermediate filament 200 (NF200) in the axon after CRF may induce an impairment of intracellular transport and eventual cellular dysfunction through destruction of the neuronal cytoarchitecture. These data suggest that up-regulation of PSD-95 in CRF may increase the functional derangement between the nerve cells and ultimately lead to memory impairment.

Nonspecific association of 2',3'-cyclic nucleotide 3'-phosphodiesterase with the rat forebrain postsynaptic density fraction

The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a protein of unknown function in vivo, is abundantly expressed in myelinating glia in two isoforms, CNP1 and CNP2. In this study, immunoblot analysis showed that CNP1 is the major isoform in adult forebrain, and that both isoforms are included in the postsynaptic density (PSD) fraction and tyrosine-phosphorylated at the basal level. However, subcellular distribution and detergent extraction data showed that CNP is nonspecifically associated with the PSD fraction. Immunocytochemistry revealed that CNP is detected, in a weak but punctate pattern, in dissociated rat hippocampal neurons of 3 days to 2 weeks in vitro. The CNP-positive punctae were distributed throughout soma and dendrites, and distinct from PSD95-positive ones. Immunoblot analysis indicated that CNP is also expressed in neuronal stem cell lines, HiB5 and F11. Interestingly, in addition to the known two isoforms, a new CNP isoform of MW 45 kDa was expressed in these cell lines and was the major type of isoform in F11 cells. Taken together, our data suggest that CNP is expressed in the early stage of in vitro development and nonspecifically included in the adult rat PSD fraction.

Expression of the NR2A and 2B Subunits of N-Methyl-D-aspartate Receptors in Hypoxic Neonatal Rat Hippocampus

PURPOSE: The developing brain has been reported to be extremely susceptible to toxicity of ischemia and/or hypoxia during a restricted developmental period. Hippocampal neuronal cell death is a typical type of perinatal hypoxic brain lesion and often coexists with other forms of cerebral hypoxic injuries. In the present study, we examined whether transcriptional changes of NR2A and NR2B subunits of the N-Methyl-D-aspartate(NMDA) receptors related to the neuronal cell death to hypoxic toxicity are involved in developing neurons in the hippocampus. METHODS: We examined the lesion produced by in vivo direct exposure of 92% N2 and 8% O2 for 2 hours at postnatal day 7. Hippocampal sections from the 7th and 14th days after hypoxia were obtained, and the amount of the NR2A and NR2B mRNA subunits were measured by in situ hybridization using the antisense probe to the NR2A and NR2B subunits. To determine the effects of molecular changes of NMDA receptor subunits, morphological changes of neurons and/or accompanying astrocytosis were evaluated by H&E and immunohistochemical stain. RESULTS: Fourteen days after hypoxia, the expression of NR2B significantly increased whereas NR2A showed distinct reduction compared with that of control rat pups. At this time, unexpectedly, neurons in CA3 region showed prominant reduction of the actual numbers and accompanied reactive astrocytosis. CONCLUSION: Alteration of NR2A and NR2B expression to hypoxic insults, suggest the possibility that changes of the NR2 subunits which can alter the function of the NMDA receptor play a crucial role in the occurrence of developmentally specific hippocampal neuronal injury.

Reactive Astrocytosis in Post-ischemic Rat Hippocampal Formation / 대한해부학회지

In the present studies, changes of the glial fibrillary acidic protein (GFAP) expression in the astrocytes of the rat hippocampal formation were examined in response to the bilateral carotid artery occlusion for 10 minutes along with a decrease of mean arterial blood pressure (MABP) to 50 mmHg. Their relations to neuronal viability were also studied by H&E staining. In early postischemic period, mild increase of the GFAP expression was observed and this was not only confined to the mild-necrotic (CA3 and dentate gyrus) regions but also in the non-necrotic regions (CA1 and subiculum) at postischemic 8 h. This suggest that astrocytosis during early postischemic period may be resulted from nonspecific reaction associated with changes in brain environment. In contrast, in late phase of the postischemia, a marked increase of the GFAP expression was observed at day 4. Moreover, cell bodies were significantly larger and many prominent and numerous processes were observed, suggesting that this may also contribute to the significant increase in the GFAP expression. Importantly, these cellular changes were only confined to the regions of massive necrosis such as subiculum and inner granular cell layer of dentate gyrus and were not observed in the non-necrotic regions (except CA1). In contrast, the GFAP expression in astrocytes were returned to control levels in mildly damaged CA3 region by 4 days. Thus reactive astrocytosis with upregulation of the GFAP in the late postischemic period with structural transformation in the regions of massive necrosis may contribute to the damages in the neighboring neurons.

Effects of Transient Cerebral Ischemia on the Expression of N-methyl-D-aspartate receptor subunit 2A, 2B and Neurofilament 200 in the Rat Cerebral Cortex / 대한해부학회지

Transient cerebral ischemia was induced by bilateral common carotid artery ligation with reperfusion to understand its effect on the expression of NMDA receptor subunits 2A (NR2A), 2B (NR2B), and NF200 The changes of the expressions of NR2A, NR2B, and NF200 in cerebral postsynaptic density (PSD) were evaluated through immunoblot analyses. The expressions of NR2A and NF200 were markedly decreased until 18 hours after reperfusion, while that of NR2B was increased. The immunohistochemistry with NFIGO antibody showed that NF200 protein, which is a marker for neuronal damage, was also significantly decreased at this time point indicating neuronal damages, and the morphological damages of neuronal cells were evident by hyperchromatic condensation of nucleus, irregular cell membrane, displacement of nucleus, and chromatolysis of Nissl substances in toluidine blue stain. However, from 18 hours to 3 day after reperfusion, immunoblot analyses showed that NF200 was increased significantly, while the expression of NR2A were recovered to the control level and that of NR2B was returned to somewhat higher level than control. The NR1/NR2B-type receptor is known to have a longer offset decay time than NR1/NR2A-type ones, and to be more potent in Ca2 influxing. Therefore, our results suggest that, until 18 hours, neurons are damaged by overinflux of Ca2 through NR1/NR2B receptors which helps to degrade NF200 by Ca2 sensitive professes resulting in damages to intracellular transport. The fact that the expression of NF200 was increased even though the NR2A and NR2B are control level during 18 hours to 3 days after damage suggests that NMDA receptor subunits expressed at this time may not form functional receptors. The worsening of some neuronal damages after 3 days may indicate that an abnormal reorganization of elevated NF200 between 18 hours to 3 days further disturb intracellular transport and functions of cell membrane which cause cell death.

NR1/NR2B receptor Activation induces Cytoskeletal Proteolysis in Partial Ischemic Rat Hippocampal Formation / 대한해부학회지

In the rat brain, partial ischemia causes a delayed neuronal degeneration that occurs hours to days after reoxygenation. It is generally thought that the ischemic damage is initiated by neurotoxicity mediated through glutamate receptors, particulaly NMDA subtypes. Calcium entry through the NMDA receptor is responsible for the synaptic plasiticity and neuronal pathology. Degradation of MAP-2 and NF200, a major components of neuronal cytoskeleton, by Ca2+-dependent protease after NMDA receptor activation has been postulated in delayed neuronal damage. Calcium-activated protease calpain, excessive degradation of MAP-2, together with the calpain-sensitive microtubule and neurofilaments, would be expected to disrupt intracellular transport- and membrane-related functions that is vital to neurons. Changed of NR subunit 2A, 2B, MAP2 and NF200 in rat hippncampal postsynaptic density[PSD] after partial ischemic injury were investigated though immunoblot analyses. To understand the effect of Ca2+, influx through NMDA receptors on neuronal damage which is manifested by cytoskeletal disruption, morphological change was examined through immunohistochemistry and routine staining method. We found that immunoreactivity to NR2B receptor subuit in the hippocampal formation PSD was upregulated while MAP2 and NF200 was down-regulted at 18 hours after initial partial ischemic insult. On the other hand, morphological changes of neuronal cell in partial ischemic conditions were manifested as eosinophilic inclusion bodies in the cytoplasm which is progression of neuronal damage after 6 days. Calcium influx through NR1/NR2B receptor channel may activate intracellular proteases which would degrade cytoskeleton. Proteolysis of cytoskeleton leads to its reorganization and eventually damages normal function of cell membrane which cause neuronal cell death.

Enhancement of N-methyl-D-aspartateNMDA Receptor 2B-mediated Neurotoxi-city after Hypoxia in the Rat Hippocampal Formation / 대한해부학회지

Ischemic brain hippocampal formation has been developed to understand the relationship between delayed neuronal damage and the expression of NMDA receptor subunits[NR2A, NR2B], MAP2, and NF200 in ttle conditions of hypoxia. Changes of NR subunits[NR2A, 2B], MAP2 6nd NF200 in rat brain postsynaptic density[PSD] after hypoxic injury were investigated through immunoblot analyses. To understand the effect of Ca2+ influx through NMDA receptors on neuronal damage which is manifested by morphological change, cytoskeletal disruption was examined through H & E, toluidine blue and immunohistochemical studies. The expression of NR2B was increased than normal at 30 hours after hypoxia. At this time, the expression of MAP2 and NF200 was markedly decreased and their morphology was more eosinophilic than normal and then became darker with expanded perineuronal space. Irreversible neuronal cell damage in hypoxic hippocampal formation is most prominent in CA3 region of hippocampus and the process is triggered by Ca2+ influx through NR1/MR2B receptor channel at 30 hour after initial hypoxic insult. Ca2+ influx through NR1/MR2B receptor channel may activate intracellular proteases which would degrade cytoskeleton. Proteolysis of cytoskeleton leads to its reorganization and eventually damages normal function of cell membrane which causes neuronal cell death. And, morphological changes of neuronal cells in hypoxic conditions were manifested as red neurons in the stage of reactive change, and as dark neuron in the stage of late hypoxic cell damage.

Up-regulation of NMDA Receptor Subunit 2B Induces Degradation of Cytoskeletons in Hypoxic Rat Cerebral Cortex / 대한해부학회지

In the rat brain, global hypoxia cause a delayed neuronal degeneration that occurs hours to days after reoxygenation. It is generally thought that the ischemic damage is initiated by neurotoxicity mediated through glutamate receptors, particulary NMDA subtypes. Calcium entry through the NMDA receptor is responsible for the synaptic plasiticity and neuronal pathology. Degradation of MAP-2 and NF200, a major components of neuronal cytoskeleton, by Ca2+-dependent protease after NMDA receptor activation has been postulated in delayed neuronal damage. Changes of NR subunit 2B, MAP2 and NF200 in rat brain postsynaptic density[PSD] after hypoxic injury were investigated through immunoblot analyses. To understand the effect of Ca2+ influx through NMDA receptors on neuronal damage which is manifested by cytoskeletal disruption, morphological change was examined through immunohistochemistry and H & E staining. We found that immunoreactivity to NR2B antibody in the cerebral cortex PSD was up-regulated while MAP2 and NF200 was down-regulated at 30 hours after initial hypoxic insult. At this time, morphological changes of neuronal cells in hypoxic conditions were manifested as down-regulation of MAP2 and NF200 immunoreactivities, hyperchromatic condensation of cytoplasm and nucleus, homogenizing cell change, expansion of perineuronal space and dispersion of chromatin. From 3 days, NR2B, MAP2, NF200 were up-regulated simultaneously. On the other hand, morphological alterations in hypoxic neurons were progress further. Our present results suggests that Calcium influx through NR1/NR2B receptor channel is effective whithin 30 hours but ineffective from 30 hours. Delayed neuronal cell death triggered by Ca2+ influx through NR1/NR2B receptor channel within 30 hours, which may activate intracellular profeases. Proteolysis of cytoskeleton by activated protease leads to its abnormal reorganization and eventually damages normal function of cell membrane which causes neuronal cell death.