Cytoplasm Of Hippocampal Neurons Research Articles

Attenuation of propofol-induced hippocampal neuron damage in developing rats by dexmedetomidine

This study explored the mechanism of dexmedetomidine attenuating propofol damage to hippocampal neurons in rats. By constructing rat hippocampal neuron model and carrying out targeted intervention; enzyme linked immunosorbent assay (ELISA) detected IL-1β and IL-18 levels; while Nissl staining observed hippocampus Histopathology; flow cytometry quantitatively analyzed the number of hippocampal neuron cells and apoptosis rate. Moreover, levels of PKA, Caspase-1 and NOD-like receptor thermal protein domain associated protein 3(NLRP3) and corresponding proteins were measured. Results showed that, there were more Nissl bodies in cytoplasm of hippocampal neurons in control group, and distribution in the cytoplasm was relatively uniform. Moreover, IL-1β and IL-18 in model group continued to increase; while dexmedetomidine effectively inhibited apoptosis of hippocampal neurons, which may be related to decreased expression of protein kinase A (PKA). After using PKA inhibitors, apoptosis was significantly inhibited, and when the expression of PKA was reduced, Caspase-1 was down-regulated along with reduced NLRP3 level, which improved the injury of hippocampal neurons. Dexmedetomidine can therefore down-regulate the level of Caspase-1 in hippocampal neurons by inhibiting the PKA signaling pathway, improving apoptosis, reducing the genetic and protein expressions of NLRP3, and slowing down the damage of hippocampal neurons.

Effects on Autophagy of Moxibustion at Governor Vessel Acupoints in APP/PS1double-Transgenic Alzheimer's Disease Mice through the lncRNA Six3os1/miR-511-3p/AKT3 Molecular Axis.

Objective To explore the effect and mechanism of moxibustion at acupoints of the governor vessel on lncRNA Six3os1 in amyloid precursor protein/presenilin1 (APP/PS1) double-transgenic Alzheimer's disease (AD) mice. Methods Twenty-four specific pathogen-free and APP/PS1 double-transgenic male mice were randomly allocated into the AD model and moxibustion groups, with 12 cases in each group. Twelve syngeneic C57BL/6J mice were selected as the control group. Mice in the moxibustion group received aconite cake-separated moxibustion at the Baihui acupoint. Suspension moxibustion was applied at Fengfu and Dazhui for 15 minutes each day. All treatments were conducted over two weeks. Control and AD model mice were routinely fed without any intervention. Behavioral observation tests were conducted before and after the intervention. The autophagosome in the hippocampus was observed using transmission electron microscopy. Immunohistochemistry was performed to detect Aβ1-42 expression. LC3B and P62 expressions were evaluated by immunofluorescence. The expression levels of the lncRNAs Six3os1, miR-511-3p, and AKT3 were detected by qRT-PCR. The differential expression of PI-3K, AKT3, mTOR, LC3B-II/I, and P62 proteins in the hippocampus was detected by western blot. The dual-luciferase assay was undertaken to examine the targeting relationships of the lncRNAs Six3os1, miR-511-3p, and AKT3. Results Compared with the control group, the AD model showed higher escape latency in the Morris Water Maze and reduced autophagic vacuoles in the cytoplasm of hippocampal neurons (both p < 0.01). Compared with the control group, the AD model showed higher expression of Aβ1-42, the lncRNAs Six3os1, PI-3K, mTOR, P62, and AKT3 protein (all p < 0.01); but lower mir-511-3p and LC3B (both p < 0.01). Compared with the AD model group, the moxibustion group had a shorter escape latency, more autophagic bubbles in the hippocampus, and lower expression of positive Aβ1-42, the lncRNAs Six3os1, PI-3K, mTOR, P62, and AKT3 protein (all p < 0.01). In contrast, the levels of miR-511-3p and LC3B proteins were considerably increased in the moxibustion group compared to the AD model group (both p < 0.01). Based on the dual-luciferase assay, there was a targeting link among the lncRNAs Six3os1, miR-511-3p, and AKT3. Conclusion Moxibustion at acupoints of the governor vessel can suppress the lncRNA Six3os1 expression, promote cell autophagy, accelerate Aβ1-42 clearance and alleviate cognitive dysfunction of AD mediated by the PI3K/AKT/mTOR signaling pathway through the lncRNA Six3os1/miR-511-3p/AKT3 axis.

Translocation of High Mobility Group Box 1 From the Nucleus to the Cytoplasm in Depressed Patients With Epilepsy.

Depression is a common psychiatric comorbidity in patients with epilepsy, especially those with temporal lobe epilepsy (TLE). The aim of this study was to assess changes in high mobility group box protein 1 (HMGB1) expression in epileptic patients with and without comorbid depression. Sixty patients with drug-resistant TLE who underwent anterior temporal lobectomy were enrolled. Anterior hippocampal samples were collected after surgery and analyzed by immunofluorescence (n = 7/group). We also evaluated the expression of HMGB1 in TLE patients with hippocampal sclerosis and measured the level of plasma HMGB1 by enzyme-linked immunosorbent assay. The results showed that 28.3% of the patients (17/60) had comorbid depression. HMGB1 was ubiquitously expressed in all subregions of the anterior hippocampus. The ratio of HMGB1-immunoreactive neurons and astrocytes was significantly increased in both TLE patients with hippocampal sclerosis and TLE patients with comorbid depression compared to patients with TLE only. The ratio of cytoplasmic to nuclear HMGB1-positive neurons in the hippocampus was higher in depressed patients with TLE than in nondepressed patients, which suggested that more HMGB1 translocated from the nucleus to the cytoplasm in the depressed group. There was no significant difference in the plasma level of HMGB1 among patients with TLE alone, TLE with hippocampal sclerosis, and TLE with comorbid depression. The results of the study revealed that the translocation of HMGB1 from the nucleus to the cytoplasm in hippocampal neurons may play a previously unrecognized role in the initiation and amplification of epilepsy and comorbid depression. The direct targeting of neural HMGB1 is a promising approach for anti-inflammatory therapy.

Effects of environmental enrichment on cognitive behavior and the expression of adenosine triphosphate binding cassette transporter A7 in hippocampus of adolescent mice with high fat diet.

To investigate the effects of environmental enrichment on cognitive behavior and the expression of adenosine triphosphate binding cassette transporter A7 (ABCA7) in hippocampus of the adolescent mice with high fat diet. A total of healthy 3-week-old male C57BL/6J mice were randomly divided into 3 groups: a control (Con) group, a high fat diet (HFD) group, and a high fat diet+environmental enrichment (HFD+EE) group, with 10 mice in each group. The Con group was given normal diet. The HFD group was given high fat diet. The HFD+EE group was given high fat diet; at the same time, they treated by environmental enrichment. After 10 weeks, open field test was used to detect activity. Novel object recognition test and Y maze test were used to detect cognitive behavior. After the test, the brain was collected and used to detect the protein expression of ABCA7 in the hippocampus by immunohistochemistry and Western blotting. And quantitative RT-PCR (RT-qPCR) was used to detect the ABCA7 mRNA expression level in the hippocampus. There was no significant difference in the total movement distance in the mice among the 3 groups (P>0.05). In the novel object recognition test, the discrimination index of the HFD group was much lower than that of the Con group, and the difference was significant (P<0.01). The discrimination index of the HFD+EE group was higher than that of the HFD group, and the difference was significant (P<0.01). In the Y maze test, there was no significant difference in the percentage of time spent on the new arm among the mice in the 3 groups (P=0.1279). The percentage of entries in new arm in the HFD group was much lower than that in the Con group, and the difference was significant (P<0.01). The percentage of the entries in new arm in the HFD+EE group was significantly higher than that in the HFD group (P<0.05). The results of immunohistochemistry showed that ABCA7 was positively expressed in the cytoplasm of hippocampal neurons in the mice from these 3 groups, and the expression of ABCA7 in the hippocampus of the HFD group was lower than that of the Con group (CA1: P<0.01, CA3: P=0.06), while the expression of ABCA7 in hippocampus of HFD+EE group was higher than that of HFD group (CA1: P=0.23, CA3: P<0.05). Western blotting results showed that compared with the Con group, the protein level of ABCA7 in the hippocampus of the HFD group was significantly reduced (P<0.05), while compared with the Con group, the protein level of ABCA7 in the hippocampus of the HFD+EE group showed an upward trend (P=0.06). The results of RT-qPCR showed that the mRNA level of ABCA7 in the hippocampus of HFD group was significantly lower than that of the Con group (P<0.01), while the mRNA level of ABCA7 in the hippocampus of HFD+EE group was significantly higher than that of the HFD group (P<0.01). High fat diet in adolescent can impair cognitive function with a decrease in the expression of ABCA7 in hippocampus, which can be ameliorate by environmental enrichment.

Effect of electroacupuncture on the expression of insulin signal pathway related proteins in hippocampus in mice with Alzheimer's disease

To observe the effect of electroacupuncture (EA) on the expression of insulin phosphatidylinositol-3 kinase/glycogen synthetase kinase-3α (PI3K/GSK3α) signal pathway related proteins in the hippocampus in mice with Alzheimer's disease (AD), and to explore the regulatory mechanism of EA on improving the pathological characteristics of AD. Twelve male APP/PS1 double transgenic mice were randomly divided a model group and a treatment group, 6 mice in each group; another 6 wild-type male mice were taken as the control group. The mice in the treatment group were treated with EA (continuous wave, 2 Hz of frequency) at "Baihui" (GV 20) and bilateral "Shenshu" (BL 23), once a day; 7-day treatment was taken as a course of treatment, and 2 courses of treatment were given. The immunohistochemistry method and Western blot method were used to detect the distribution and expression level of hippocampal PI3K/GSK3α signal pathway related proteins P85α, P110α, GSK3α and pS21GSK3α, and the number of hippocampal senile plaques (SP) was observed. The proteins of P85α, P110α, GSK3α and pS21GSK3α were mainly distributed in the cytoplasm of hippocampal neurons, and the GSK3α was also distributed in the axons of neurons in the model group and the treatment group. The immunohistochemistry results showed that the distribution level of GSK3α in the hippocampus in the model group was significantly higher than that in the control group (P<0.001), and the distribution level of pS21GSK3α, P85α and P110α was significantly decreased (P<0.01, P<0.001); compared with the model group, the distribution level of GSK3α in the hippocampus in the treatment group was significantly decreased (P<0.001), and the distribution level of pS21GSK3α, P85α and P110α in hippocampus was significantly increased (P<0.05, P<0.001). The Western blot results showed compared with the control group, the expression of pS21GSK3α, P85α and P110α as well as the ratio of pS21GSK3α/GSK3α in the hippocampus in the model group were significantly decreased (P<0.001), and the expression of GSK3α was increased (P<0.05); compared with the model group, the expression of pS21GSK3α, P85α, P110α and the ratio of pS21GSK3α/GSK3α in the hippocampus in the treatment group were significantly increased (P<0.01, P<0.001), and the expression of GSK3α was decreased (P<0.05). Compared with the control group, the number of hippocampal SP in the model group was significantly increased (P<0.001); compared with the model group, the number of hippocampal SP in the treatment group was significantly decreased (P<0.01). EA could effectively regulate the expression of PI3K/GSK3α signal pathway related proteins in the hippocampus in mice with AD, so as to reduce the formation and deposition of SP.

Hippocampal tau oligomerization early in tau pathology coincides with a transient alteration of mitochondrial homeostasis and DNA repair in a mouse model of tauopathy

Insoluble intracellular aggregation of tau proteins into filaments and neurodegeneration are histopathological hallmarks of Alzheimer disease (AD) and other tauopathies. Recently, prefibrillar, soluble, oligomeric tau intermediates have emerged as relevant pathological tau species; however, the molecular mechanisms of neuronal responses to tau oligomers are not fully understood. Here, we show that hippocampal neurons in six-month-old transgenic mouse model of tauopathy, THY-Tau22, are enriched with oligomeric tau, contain elongated mitochondria, and display cellular stress, but no overt cytotoxicity compared to the control mice. The levels of several key mitochondrial proteins were markedly different between the THY-Tau22 and control mice hippocampi including the mitochondrial SIRT3, PINK1, ANT1 and the fission protein DRP1. DNA base excision repair (BER) is the primary defense system against oxidative DNA damage and it was elevated in six-month-old transgenic mice. DNA polymerase β, the key BER DNA polymerase, was enriched in the cytoplasm of hippocampal neurons in six-month-old transgenic mice and localized with and within mitochondria. Polβ also co-localized with mitochondria in human AD brains in neurons containing oligomeric tau. Most of these altered mitochondrial and DNA repair events were specific to the transgenic mice at 6 months of age and were not different from control mice at 12 months of age when tau pathology reaches its maximum and oligomeric forms of tau are no longer detectable. In summary, our data suggests that we have identified key cellular stress responses at early stages of tau pathology to preserve neuronal integrity and to promote survival. To our knowledge, this work provides the first description of multiple stress responses involving mitochondrial homeostasis and BER early during the progression of tau pathology, and represents an important advance in the etiopathogenesis of tauopathies.

Granulovacuolar degeneration: a neurodegenerative change that accompanies tau pathology.

Granule-containing vacuoles in the cytoplasm of hippocampal neurons are a neuropathological feature of Alzheimer's disease. Granulovacuolar degeneration (GVD) is not disease-specific and can be observed in other neurodegenerative disorders and even in the brains of non-demented elderly people. However, several studies have reported much higher numbers of neurons undergoing GVD in the hippocampus of Alzheimer's disease cases. Recently, a neuropathological staging system for GVD has facilitated neuropathological assessment. Data obtained by electron microscopy and immunolabeling suggest that GVD inclusions are a special form of autophagic vacuole. GVD frequently occurs together with pathological changes of the microtubule-associated protein tau, but to date, the relationship between the two lesions remains elusive. Originally identified in hematoxylin- and silver-stained sections, immunolabeling has shown that the granules are composed of a variety of proteins, including those related to tau pathology, autophagy, diverse signal transduction pathways, cell stress and apoptosis. Several of these proteins serve as markers of GVD. Most researchers and authors have interpreted the sequestration of proteins into GVD inclusions as either a cellular defense mechanism or one that leads to the impairment of important cellular functions. This review provides a detailed overview of the various aspects of GVD and focuses on the relationship between tau pathology and GVD.

Prefibrillar Tau oligomers alter the nucleic acid protective function of Tau in hippocampal neurons in vivo

The accumulation of DNA and RNA oxidative damage is observed in cortical and hippocampal neurons from Alzheimer's disease (AD) brains at early stages of pathology. We recently reported that Tau is a key nuclear player in the protection of neuronal nucleic acid integrity in vivo under physiological conditions and hyperthermia, a strong inducer of oxidative stress. In a mouse model of tauopathy (THY-Tau22), we demonstrate that hyperthermia selectively induces nucleic acid oxidative damage and nucleic acid strand breaks in the nucleus and cytoplasm of hippocampal neurons that display early Tau phosphorylation but no Tau fibrils. Nucleic acid-damaged neurons were exclusively immunoreactive for prefibrillar Tau oligomers. A similar association between prefibrillar Tau oligomers and nucleic acid oxidative damage was observed in AD brains. Pretreatment with Methylene Blue (MB), a Tau aggregation inhibitor and a redox cycler, reduced hyperthermia-induced Tau oligomerization as well as nucleic acid damage.This study clearly highlights the existence of an early and critical time frame for hyperthermia-induced Tau oligomerization, which most likely occurs through increased oxidative stress, and nucleic acid vulnerability during the progression of Tau pathology. These results suggest that at early stages of AD, Tau oligomerization triggers the loss of the nucleic acid protective function of monomeric Tau. This study highlights the existence of a short therapeutic window in which to prevent the formation of pathological forms of Tau and their harmful consequences on nucleic acid integrity during the progression of Tau pathology.

ATBF1 is a novel amyloid-β protein precursor (AβPP) binding protein that affects AβPP expression.

The cytoplasmic C-terminal domain of amyloid-β protein precursor (AβPP) binds to several proteins that regulate the trafficking and processing of AβPP and affects amyloid-β (Aβ) production. We previously reported that levels of AT-motif binding factor 1 (ATBF1) are increased in the brains of 17-month-old Tg2576 mice compared with wild-type controls, and that Aβ42 increases ATBF1 expression, inducing death in primary rat cortical neurons. Here, we show that ATBF1 levels are increased in the cytoplasm of hippocampal neurons in Alzheimer's disease (AD) brains compared with non-AD brains. Furthermore, cotransfection of human embryonic kidney (HEK293T) and human neuroblastoma (SH-SY5Y) cells with ATBF1 and AβPP695 increased steady-state levels of AβPP via the binding of ATBF1 to the AβPP cytoplasmic domain (amino acids 666-690), resulting in increased Aβ production and cellular and soluble AβPP (sAβPP) levels without affecting the activity or levels of AβPP processing enzymes (α-, β-, or γ-secretase). Conversely, knockdown of endogenous ATBF1 reduced levels of cellular AβPP, sAβPP, and Aβ in HEK293 cells overexpressing human AβPP695. Our findings provide insight into the dynamics of AβPP processing and Aβ production, and suggest that ATBF1 is a novel AβPP binding protein that may be a suitable therapeutic target for AD.

Cellular expression and localization of DGKζ-interacting NAP1-like proteins in the brain and functional implications under hypoxic stress.

Diacylglycerol kinase (DGK) catalyzes conversion of a lipid second messenger diacylglycerol to another messenger molecule phosphatidic acid. Consequently, DGK plays a pivotal role in cellular pathophysiology by regulating the levels of these two messengers. We reported previously that DGKζ translocates from the nucleus to cytoplasm in hippocampal neurons under ischemic/hypoxic stress. In addition, we also identified nucleosome assembly protein 1 (NAP1)-like proteins NAP1L1 and NAP1L4 as novel DGKζ-interacting partners using a proteomic approach and revealed that these NAP1-like proteins induce cytoplasmic translocation of DGKζ in overexpressed cells because NAP1-like proteins associate with the nuclear localization signal of DGKζ and block its nuclear import via importin α. In the present study, we examined whether NAP1-like proteins are expressed in the brain and whether the molecular interaction of DGKζ and NAP1-like proteins would be changed in the brain after hypoxic stress. Immunohistochemistry revealed that NAP1L1 and NAP1L4 are widely expressed in neurons and glial cells in the brain with some differences. After 3 days of transient whole-body hypoxic stress, DGKζ translocated from the nucleus to cytoplasm in hippocampal pyramidal neurons, whereas NAP1-like proteins remained in the cytoplasm. Contrary to our expectations, NAP1-like proteins showed no change in their expression levels. The molecular interaction between DGKζ and NAP1-like proteins was attenuated after hypoxic stress. These results suggest that DGKζ cytoplasmic translocation in neurons under hypoxic stress is regulated by some mechanism which differs from that mediated by NAP1-like proteins.

Deficiency of G3BP1, the stress granules assembly factor, results in abnormal synaptic plasticity and calcium homeostasis in neurons

Ras-GAP SH3-domain-binding protein, G3BP, is an important component in the assembly of stress granules (SGs), which are cytoplasmic aggregates assembled following translational stress. To assess the physiological function of G3BP, we generated viable G3bp1-knockout (KO) mice, which demonstrated behavioral defects linked to the CNS-associated with ataxia phenotype. Immunohistochemistry pinpointed high expression of G3BP in the cytoplasm of hippocampal neurons and Purkinje cells of the cerebellum of wild-type mice. Also, electrophysiological measurements revealed that the absence of G3BP1 leads to an enhancement of short-term potentiation (STP) and long-term depression in the CA1 area of G3bp1 KO mice compared with wild-type mice. Consistently, G3BP1 deficiency in neurons leads to an increase in intracellular calcium and calcium release in response to (S)-3,5-Dihydroxyphenylglycine, a selective agonist of group I metabotropic glutamate receptors. These results show, for the first time, a requirement for G3BP1 in the control of neuronal plasticity and calcium homeostasis and further establish a direct link between SG formation and neurodegenerative diseases.

DGKζ is degraded through the cytoplasmic ubiquitin–proteasome system under excitotoxic conditions, which causes neuronal apoptosis because of aberrant cell cycle reentry

Recent reports have described the involvement of the diacylglycerol kinase (DGK) family in various pathological conditions. In an animal model of transient ischemia, DGKζ containing a nuclear localization signal (NLS) is shown to translocate quickly from the nucleus to the cytoplasm in hippocampal neurons and to disappear gradually after reperfusion. Those neurons die a delayed neuronal death because of glutamate excitotoxicity. This study investigated the molecular mechanism and functional relation linking DGKζ and neuronal death. In primary cultured neurons, transient exposure to excitotoxic concentration of glutamate led to cytoplasmic accumulation of DGKζ followed by its down-regulation. Results showed that DGKζ down-regulation was caused by proteolytic degradation through the ubiquitin–proteasome system (UPS) rather than transcriptional inhibition. DGKζ polyubiquitination was inhibited in the presence of nuclear export inhibitor leptomycin B. Furthermore, NLS-deleted mutant DGKζΔNLS, which mainly localizes to the cytoplasm, was ubiquitinated more heavily than wild-type DGKζ. From a functional perspective, in vitro gene silencing of DGKζ via specific siRNA enhanced DNA fragmentation in cultured neurons after glutamate exposure. At the organismal level, hippocampal neurons of DGKζ-deficient mice showed vulnerability to kainate-induced seizures. In addition, DGKζ-deficient hippocampus exhibited a significant increase in Ser807/811 phosphorylated retinoblastoma protein levels together with up-regulation of the expression of type D and E cyclins, indicative of cell cycle reentry. Collectively, these results suggest that 1) glutamate excitotoxicity induces nucleocytoplasmic translocation of DGKζ followed by its degradation through the cytoplasmic UPS in hippocampal neurons and that 2) DGKζ-deficient neurons do not succumb directly to apoptosis, although they are more vulnerable to excitotoxicity because of aberrant cell cycle reentry.

Nuclear relocation of DGKζ in cardiomyocytes under conditions of ischemia/reperfusion.

Diacylglycerol (DG) and phosphatidic acid (PA) are generated under various conditions, such as ligand stimulation and several stresses. They serve as second messengers to respond to pathophysiological conditions. DG kinase (DGK) catalyzes DG to produce PA. It is regarded as a regulator of these lipid messengers. Previous studies show that DGKζ, a nuclear isozyme, translocates from the nucleus to the cytoplasm in hippocampal neurons under transient ischemia and never relocates to the nucleus after reperfusion. This study examined whether a similar phenomenon is observed in cardiomyocytes, which represent another type of postmitotic, terminally differentiated cell. We performed immunostaining on ischemic hearts induced by occlusion of the left anterior descending coronary artery and on primary cultured cardiomyocytes under oxygen-glucose deprivation (OGD). In the animal model, 10 min ischemia is sufficient to cause DGKζ to disappear from the nucleus in cardiomyocytes. However, DGKζ is observed again in the nucleus at 10 min following reperfusion after 10 min ischemia, which contrasts sharply with ischemic hippocampal neurons. Similar results were obtained from experiments using primary cultured cardiomyocytes under OGD conditions, except that DGKζ relocates autonomously, if at all, to the nucleus, even under continuous OGD conditions. Results suggest that DGKζ is involved in the acute phase of cellular response to ischemic stress in cardiomyocytes in a similar, but not identical, manner to that of neurons.

A possible pivotal role of mitochondrial free calcium in neurotoxicity mediated by N-methyl- d-aspartate receptors in cultured rat hippocampal neurons

We have previously shown that mitochondrial membrane potential disruption is involved in mechanisms underlying differential vulnerabilities to the excitotoxicity mediated by N-methyl- d-aspartate (NMDA) receptors between primary cultured neurons prepared from rat cortex and hippocampus. To further elucidate the role of mitochondria in the excitotoxicity after activation of NMDA receptors, neurons were loaded with the fluorescent dye calcein diffusible in the cytoplasm and organelles for determination of the activity of mitochondrial permeability transition pore (mPTP) responsible for the leakage of different mitochondrial molecules. The addition of CoCl 2 similarly quenched the intracellular fluorescence except mitochondria in both cultured neurons, while further addition of NMDA led to a leakage of the dye into the cytoplasm in hippocampal neurons only. An mPTP inhibitor prevented the NMDA-induced loss of viability in hippocampal neurons, while an activator of mPTP induced a similarly potent loss of viability in cortical and hippocampal neurons. Although NMDA was more effective in increasing rhodamine-2 fluorescence as a mitochondrial calcium indicator in hippocampal than cortical neurons, a mitochondrial calcium uniporter inhibitor significantly prevented the NMDA-induced loss of viability in hippocampal neurons. Expression of mRNA was significantly higher for the putative uniporter uncoupling protein-2 in hippocampal than cortical neurons. These results suggest that mitochondrial calcium uniporter would be at least in part responsible for the NMDA neurotoxicity through a mechanism relevant to promotion of mPTP orchestration in hippocampal neurons.

Aging-related alterations in the expression and distribution of GluR2 and PICK1 in the rat hippocampus

Deficit in synaptic plasticity in the hippocampus frequently occurs during normal aging. Although the protein level and calcium permeability of AMPARs alter with aging, the alteration of AMPARs and their regulatory proteins during aging are far from understanding. Dynamics of GluR2 subunit are dependent on the function of protein interacting with Cα kinase 1 (PICK1), PKCα and calcineurin (CaN). Here, we firstly show that the expression of PICK1 and CaN B decreased significantly in the hippocampus of old rats compared to that of young and adult rats. The decrease was accompanied by a reduction of GluR2 and PKCα and an increase in CaN A. Next, we found that in young and adult rats, the distribution of PICK1 and GluR2 diffused in the cytoplasm of hippocampal neurons, but closely around perinuclear in the hippocampal neurons of old rats. These results suggest that the expression of GluR2, PICK1, PKCα and CaN B significant decreased in the hippocampus and these alterations may lead to altered distribution of GluR2 and PICK1 during aging.

Excitotoxicity by kainate-induced seizure causes diacylglycerol kinase ζ to shuttle from the nucleus to the cytoplasm in hippocampal neurons

Diacylglycerol kinase (DGK), which consists of several isozymes, plays a pivotal role in lipid second-messenger diacylglycerol metabolism. A nuclear isozyme, DGKζ, which is translocated from the nucleus to the cytoplasm in hippocampal neurons under transient ischemic stress, is implicated in nuclear events of delayed neuronal death. Kainate (KA)-induced seizure is another model used to study excitotoxic stress. Therefore, we examined whether DGKζ is implicated in a different type of degenerative excitotoxicity in hippocampal neurons. We conducted immunohistochemical analysis of rat hippocampi after KA-induced seizures. DGKζ in hippocampal neurons shuttles from the nucleus to the cytoplasm. It never relocates to the nucleus during KA-induced seizures. Marked change in the immunoreactivity is first observed in CA1 pyramidal neurons 2 h after injection during stage 3 seizures. Immunoreactivity for DGKι remains unchanged in the cytoplasm. That for NeuN remains mostly unchanged in the nucleus. Results show that nucleocytoplasmic translocation of DGKζ also occurs in a different model of excitotoxicity that results in apoptotic neuronal death. Cytoplasmic translocation of DGKζ might be involved in early events of the apoptotic cell death pathway in hippocampal neurons under stressed conditions.

Cytoplasmic BK Ca channel intron-containing mRNAs contribute to the intrinsic excitability of hippocampal neurons

High single-channel conductance K + channels, which respond jointly to membrane depolarization and micromolar concentrations of intracellular Ca 2+ ions, arise from extensive cell-specific alternative splicing of pore-forming α-subunit mRNAs. Here, we report the discovery of an endogenous BK Ca channel α-subunit intron-containing mRNA in the cytoplasm of hippocampal neurons. This partially processed mRNA, which comprises ≈10% of the total BK Ca channel α-subunit mRNAs, is distributed in a gradient throughout the somatodendritic space. We selectively reduced endogenous cytoplasmic levels of this intron-containing transcript by RNA interference without altering levels of the mature splice forms of the BK Ca channel mRNAs. In doing so, we could demonstrate that changes in a unique BK Ca channel α-subunit intron-containing splice variant mRNA can greatly impact the distribution of the BK Ca channel protein to dendritic spines and intrinsic firing properties of hippocampal neurons. These data suggest a new regulatory mechanism for modulating the membrane properties and ion channel gradients of hippocampal neurons.

Quinone reductase (NQO1), a sensitive redox indicator, is increased in Alzheimer's disease

NAD(P)H:quinone oxidoreductase 1 (NQO1), a redox-regulated flavoenzyme, plays a central rolein monitoring cellular redox state. NQO1 acts to protect against oxidative stress induced by a variety of metabolic situations, including metabolism of quinones and other xenobiotics, by: (i)functioning as a two electron donor to provide a shunt that competes with the formation of reactive oxygen species; (ii) maintaining reduced coenzyme Q; and (iii) regulating the stress activated kinase pathway. In Alzheimer's disease, while there is abundant evidence for the involvement of oxidative stress, the cause or the consequences are largely unresolved. We suspected that increased NQO1 could signal a major shift in redox balance in Alzheimer's disease and, in this study, found that NQO1 is localized not only to neurofibrillary tangles but also the cytoplasm of hippocampal neurons. By marked contrast, there is very little NQO1 in the same neuronal populations in young and age-matched controls. This novel association of NQO1 further buttresses the nexus of oxidative stress, via free radicals, with selective neuronal vulnerability and also supports a fundamental abnormality in redox balance in Alzheimer's disease.

Region-specific targets of p42/p44MAPK signaling in rat brain.

In vitro studies indicate that p42/p44MAPK phosphorylate both nuclear and cytoplasmic proteins. However, the functional targets of p42/p44MAPK activation in vivo remain unclear. To address this question, we localized activated p42/p44MAPK in hippocampus and cortex and determined their signaling effects after electroconvulsive shock treatment (ECT) in rats. Phosphorylated p42/p44MAPK content increased in the cytoplasm of hippocampal neurons in response to ECT. Consistent with this cytoplasmic localization, inhibition of ECT-induced p42/p44MAPK activation by the extracellular signal-regulated kinase kinase inhibitor PD098059 blocked phosphorylation of the cytoplasmic protein microtubule-associated protein 2c (MAP2c), but failed to inhibit the induction of the nuclear protein c-Fos in response to ECT. In contrast to hippocampal neurons, cortical neurons exhibited an increase in amount of phosphorylated p42/p44MAPK in both the nucleus and cytoplasm after ECT. Accordingly, PD098059 blocked the induction of Fos-like immunoreactivity in the nuclei of cortical neurons as well as MAP2c phosphorylation in the cytoplasm. Our data indicate that both nuclear and cytoplasmic substrates can be activated by p42/p44MAPK in vivo. However, the functional targets of p42/p44MAPK signaling depend on the precise location of p42/p44MAPK within different subcellular compartments of brain regions. These results indicate unique functional pathways of p42/p44MAPK-mediated signal transduction within different brain regions in vivo.

Zinc supplementation in ethanol-treated pregnant rats increases the metabolic activity in the fetal hippocampus

As a possible preventative measure for brain dysfunction in the fetal alcohol syndrome, the effect of zinc supplementation together with ethanol on the fetal cerebrum, especially the hippocampus, was investigated. Female rats of an ethanol group and a control group received 30% ethanol and water, respectively. During pregnancy half of both groups received 0.01% zinc together with ethanol or water. Fetuses on gestational day 21 were examined biochemically and ultrastructurally. Administration of zinc with ethanol resulted in an increased cerebral weight and cerebral RNA content compared to administration of ethanol alone. However, these better results did not reach those of controls without ethanol. In the cytoplasm of hippocampal neurons from dams only given zinc with ethanol confronting cisternae were frequently observed between the rough endoplasmic reticulum and the rough endoplasmic reticulum or the nuclear envelope. Although the functional roles of the confronting cisternae were not definite, it is reasonable to conclude that the confronting cisternae were a repair mechanism of zinc against the inhibitory effect on RNA and protein syntheses of ethanol. This study shows evidence of prevention of the brain dysfunction on ethanol treatment.