Proteins In Hippocampal Neurons Research Articles

BDNF-TrkB/proBDNF-p75NTR pathway regulation by lipid emulsion rescues bupivacaine-induced central neurotoxicity in rats

Bupivacaine (BPV) can cause severe central nervous system toxicity when absorbed into the blood circulation system. Rapid intravenous administration of lipid emulsion (LE) could be used to treat local anaesthetic toxicity. This study aimed to investigate the mechanism by which the BDNF-TrkB/proBDNF-p75NTR pathway regulation by LE rescues BPV induced neurotoxicity in hippocampal neurons in rats. Seven- to nine-day-old primary cultured hippocampal neurons were randomly divided into 6 groups: the blank control group (Ctrl), the bupivacaine group (BPV), the lipid emulsion group (LE), the bupivacaine + lipid emulsion group (BPV + LE), the bupivacaine + lipid emulsion + tyrosine kinase receptor B (TrkB) inhibitor group (BPV + LE + K252a), the bupivacaine + lipid emulsion + p75 neurotrophic factor receptor (p75NTR) inhibitor group (BPV + LE + TAT-Pep5). All hippocampal neurons were incubated for 24 h, and their growth state was observed by light microscopy. The relative TrkB and p75NTR mRNA levels were detected by real-time PCR. The protein expression levels of brain-derived neurotrophic factor (BDNF), proBDNF, TrkB, p75NTR and cleaved caspase-3 were detected by western blotting. The results showed that primary hippocampal neuron activity was reduced by BPV. As administration of LE elevated hippocampal neuronal activity, morphology was also somewhat improved. The protein expression and mRNA levels of TrkB and p75NTR were decreased when BPV induced hippocampal neuronal toxicity, while the expression of BDNF was increased. At the same time, BPV increased the original generation of cleaved caspase-3 protein content by hippocampal neurons, while the content of cleaved caspase-3 protein in hippocampal neurons cotreated with LE and BPV was decreased. Thus, this study has revealed LE may reduce apoptosis and promote survival of hippocampal neurons by regulating the BDNF-TrkB pathway and the proBDNF-p75NTR pathway to rescue BPV induced central neurotoxicity in rats.

Deferoxamine Mitigates Ferroptosis and Inflammation in Hippocampal Neurons After Subarachnoid Hemorrhage by Activating the Nrf2/TXNRD1 Axis.

Ferroptosis is a distinct peroxidation-driven form of cell death tightly involved in subarachnoid hemorrhage (SAH). This study delved into the mechanism of deferoxamine (DFO, an iron chelator) in SAH-induced ferroptosis and inflammation. SAH mouse models were established by endovascular perforation method and injected intraperitoneally with DFO, or intraventricularly injected with the Nrf2 pathway inhibitor ML385 before SAH, followed by detection of neurological function, blood-brain barrier (BBB) permeability, and brain water content. Apoptotic level of hippocampal neurons, symbolic changes of ferroptosis, and levels of pro-inflammatory cytokines were assessed using TUNEL staining, Western blotting, colorimetry, and ELISA. The localization and expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) were detected. HT22 cells were exposed to Hemin as in vitro SAH models and treated with FIN56 to induce ferroptosis, followed by evaluation of the effects of DFO on FIN56-treated HT22 cells. The regulation of Nrf2 in thioredoxin reductase 1 (TXNRD1) was analyzed by co-immunoprecipitation and Western blotting. Moreover, HT22 cells were treated with DFO and ML385 to identify the role of DFO in the Nrf2/TXNRD1 axis. DFO extenuated brain injury, and ferroptosis and inflammation in hippocampal neurons of SAH mice. Nrf2 localized at the CA1 region of hippocampal neurons, and DFO stimulated nuclear translocation of Nrf2 protein in hippocampal neurons of SAH mice. Additionally, DFO inhibited ferroptosis and inflammatory responses in FIN56-induced HT22 cells. Nrf2 positively regulated TXNRD1 protein expression. Indeed, DFO alleviated FIN56-induced ferroptosis and inflammation via activation of the Nrf2/TXNRD1 axis. DFO alleviated neurological deficits, BBB disruption, brain edema, and brain injury in mice after SAH by inhibiting hippocampal neuron ferroptosis via the Nrf2/TXNRD1 axis. DFO ameliorates SAH-induced ferroptosis and inflammatory responses in hippocampal neurons by activating the Nrf2/TXNRD1 axis.

Effects of Camk2b Overexpression and Underexpression on the Proteome of Rat Hippocampal Neurons

Recent studies have demonstrated that Camk2b expression is modified in neuropsychiatric illnesses and potentially affects synaptic plasticity. However, the molecular events arising from Camk2b dysregulation are not fully elucidated and need to be comprehensively explored. In the present study, we first induced over-expression and under-expression of Camk2b in cultured rat hippocampal neurons through transfection with lentivirus plasmids. Then isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics followed by bioinformatics analyses were carried out to explore the impacts of Camk2b dysexpression on the proteome of the neurons. Compared with the respective controls, a total of 270 proteins in the Camk2b-overexpression group and 209 proteins in the Camk2b-underexpression group were experienced a divergence in expression. Gene ontology and pathway analyses indicated that Camk2b overexpression and under-expression respectively induced two different change profiles of protein expressions and functions, reflecting the potential differences in cellular processes and biological events. Through cross comparison, several candidate target proteins regulated directly by Camk2b were revealed. Further network and immunoblot analyses demonstrated that Mapk3 could be an important linker and Camk2b-Mapk3 might serve as a new potential pathway affecting the expression of synaptic proteins in hippocampal neurons. Collectively, the present results offer a new comprehension of the regulatory molecular mechanism of Camk2b and thereby increase our understanding of Camk2b-mediated synaptogenesis in synaptic plasticity.

Effect of acupuncture on expression of endoplasmic reticulum Ca2+ and Caspase-12 protein in hippocampal neurons of rats with convulsive brain injury

To observe the effect of acupuncture on endoplasmic reticulum calcium, apoptosis number and Caspase-12 protein expression in hippocampal neurons of convulsive rats, so as to explore its mechanisms underlying improvement of convulsion. SD rats were randomly divided into normal control, model and acupuncture groups, with 36 rats in each group. Rats in the normal control group received intraperitoneal injection (i.p.) of normal saline (2 mL), and those of the other 2 groups received i.p. of pentylenetetrazole (50 mg/kg) for establishing convulsion model. Manual acupuncture stimulation was applied to "Baihui"(GV20) and "Dazhui"(GV14) for 30 min after modeling. The hippocampal tissues were taken at 2, 12 and 48 h after modeling. The endoplasmic reticulum Ca2+ concentration (optical density, OD) was detected by using fluorescence probe technique and laser confocal microscopy, and the number of apoptosis of hippocampal neurons at the 3 time points detected by using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) stain. The expression of Caspase-12 protein in hippocampus at 3 time points was observed by immunohistochemistry. In comparison with the normal group, the number of apoptotic cells of hippocampal neurons and the expression levels of Caspase-12 protein in hippocampus at 2, 12 and 48 h after seizures were obviously increased (P<0.01), and the OD value of Ca2+ at 3 time points significantly decreased (P<0.01) in the model group.Following acupuncture intervention, the increased levels of the number of apoptotic cells of hippocampal neurons and the expression of Caspase-12 protein in hippocampus at 3 time points and the decreased levels of OD value of Ca2+ at 3 time points were reversed in the acupuncture group (P<0.05, P<0.01). Acupuncture intervention is effective in reducing the apoptosis of hippocampal neurons in convulsion rats, which may be related to its functions in down-regulating Caspase-12 expression and promoting influx of Ca2+ in the hippocampal neurons.

TLR3 deletion inhibits programmed necrosis of brain cells in neonatal mice with sevoflurane-induced cognitive dysfunction.

This research aimed to explore the influence of TLR deletion on sevoflurane-induced postoperative cognitive dysfunction in neonatal mice. Herein, WT and TLR3 KO neonatal mice, each with 24, were randomly divided into control group, sevoflurane group, and TLR3−/−+sevoflurane group. The hippocampal neurons of WT, TLR3 KO and RIP3 KO neonatal mice in C group, SEV group, TLR3−/−+SEV group and RIP3−/−+SEV group were extracted for in vitro experiments. The results revealed the degeneration and necrosis of nerve cells in SEV group. Microscopic findings indicated that nerve cells showed shrinkage and nuclear hyperchromatism, along with lessening or even disappearance of nuclei and enlargement of cell spaces, and apoptotic cells in the brain tissues were evidently increased. Compared with SEV group, TLR3−/−+SEV group displayed reductions in these phenomena. Additionally, SEV group showed the reduced SHP2 expression and the increased expressions of proteins associated with TLR signaling pathway and apoptosis. Furthermore, there were no obvious differences in the expressions of such proteins in hippocampal neurons between RIP3−/−+SEV and TLR3−/−+SEV groups. The results confirmed that inhibiting RIP3 phosphorylation and suppressing TLR3 expressions exerted the same influence on the expressions of these proteins in the hippocampus of neonatal mice with sevoflurane-induced cognitive dysfunction. Based on these, it is speculated that TLR3 influences neonatal mice with sevoflurane-induced cognitive dysfunction probably by regulating RIP3 phosphorylation.

Administration of miR-195 Inhibitor Enhances Memory Function Through Improving Synaptic Degradation and Mitochondrial Dysfunction of the Hippocampal Neurons in SAMP8 Mice.

Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and miR-195 is involved in mitochondrial disorder through targeting MFN-2 protein in hippocampal neurons of AD. To clarify if administration of miR-195 inhibitor could enhance the memory deficits through improving hippocampal neuron mitochondrial dysfunction in SAMP8 mice. The expression of miR-195 was detected by RT-qPCR in primary hippocampal neurons and HT-22 cells treated with Aβ1-42. Morris water maze (MWM) was used to assess the learning and memory function in SAMP8 mice administrated with antagomir-195. Transmission electron microscopy was employed to determine the morphological changes of synapses and mitochondria of hippocampus in SAMP8 mice. Mitochondrial respiration was measured using a high-resolution oxygraph. The expression of miR-195 were upregulated in the primary hippocampal neurons and HT-22 cells induced by Aβ1-42. Inhibition of miR-195 ameliorated the mitochondrial dysfunction in HT-22 cells induced by Aβ1-42, including mitochondrial morphologic damages, mitochondrial membrane potential, respiration function, and ATP production. Administration of antagomir-195 by the third ventricle injection markedly ameliorated the cognitive function, postsynaptic density thickness, length of synaptic active area, mitochondrial aspect ratio, and area in hippocampus of SAMP8 mice. Finally, antagomir-195 was able to promote an increase in the activity of respiratory chain complex CI and II in SAMP8 mice. This study demonstrated that miR-195 inhibitor ameliorated the cognitive impairment of AD mice by improving mitochondrial structure damages and dysfunction in the hippocampal neurons, which provide an experimental basis for further exploring the treatment strategy of AD.

Effect of acupuncture serum on expression of microtubule associated protein-2 and nerve growth associated protein-43 in Mg2+-free-cultured hippocampal neurons of neonatal rats

To observe the effect of acupuncture serum on the expression of microtubule associated protein-2 (MAP-2) and nerve growth associated protein-43 (GAP-43) in cultured hippocampal neurons of convulsive rats. The acute convulsion model was induced by intraperitoneal injection of pentylenetetrazol in SD rats who were then randomized into model group and acupuncture group. Rats of the acupuncture group received manual acupuncture stimulation of "Baihui" (GV20) and "Dazhui" (GV14) for 30 min, once daily for 7 days. Then, the blood samples taken from the abdominal aorta of rats in the convulsion model and acupuncture groups were processed into serum samples, i.e. non-acupuncture serum and acupuncture se-rum. The primary-cultured hippocampal neurons of fetal rats were cultured for 10 days and then divided into normal extracellular fluid (normal) group, magnesium (Mg2+) free extracellular fluid group, acupuncture serum group and non-acupuncture serum group. At the 10th day, the neurons in the normal group were cultured continuously in extracellular fluid for 3 h, and then cultured in DMEM/F12(1∶1) medium (planting fluid); neurons in the Mg2+ free group were cultured in magnesium-free fluid medium to induce epileptic-like discharge; neurons in the acupuncture serum group were cultured in the mixed medium of planting fluid and 10% acupuncture serum; and neurons in the non-acupuncture serum were cultured in the mixed culture medium of planting fluid and non-acupuncture serum (10%). At last, these neurons in the above-mentioned groups were cultured in the magnesium-free extracellular fluid continuously for 2, 12 and 48 h, respectively, followed by detecting the expression levels of MAP-2 and GAP-43 proteins at the 3 time points by using immunofluorescence and Western blot, separately. The rate of MAP-2 positive cells and protein expression at 2, 12 and 48 h, and the rate of GAP-43 positive cells and protein expression at 12 and 48 h in the hippocampal neurons were significantly down-regulated in the Mg2+ free group in contrast to the normal group (P<0.05，P<0.01). Compared to the Mg2+ free group, the rates of MAP-2 and GAP-43 positive cells and protein expression at 2, 12 and 48 h were considerably up-regulated in the acupuncture serum group (P<0.05，P<0.01), but not in the non-acupuncture serum group (P>0.05). Acupuncture serum can significantly up-regulate the expression of MAP-2 and GAP-43 proteins in hip-pocampal neurons, which may play a positive role in improving synaptic plasticity and neuronal damage in convulsion rats.

Effect of chlorpyrifos on autophagy-related protein expression in hippocampal neurons of SD rats

Objective: To analyze the effect of chlorpyrifos on the expression of autophagy related proteins in rat hippocampal neurons, and to explore the role of autophagy in central nerve injury caused by acute chlorpyrifos poisoning. Methods: In October 2018, 35 male clean grade SD rats were randomly divided into 7 groups according to the observation time point, namely 0.5 d, 1 d, 2 d, 3 d, 5 d and 7 d groups and the control group, with 5 rats in each group. Each observation group was given 81.5 mg/kg chlorpyrifos by gavage, and the control group was given olive oil by gavage. The general conditions and poisoning symptoms of rats were observed continuously after exposure. The expressions of autophagy related proteins Beclin1, P62/SQSTM1 and LC3 in hippocampus were detected by Western blot. The cell morphology and LC3 expression in brain were observed by immunohistochemical staining. Results: Western blot results showed that compared with the control group, the expression of Beclin1 protein in hippocampal neurons of rats in the 1 d, 2 d, and 3 d groups increased, while the expression of P62/SQSTM1 protein in the 0.5 d, 1 d, and 2 d groups decreased, and the expression of LC3 protein was decreased in the 2 d group, and the differences were statistically significant (P<0.05) . The results of immunohistochemistry showed that the hippocampal neurons of rats in the 5 d group were arranged disorderly, and some nuclei contours disappeared, especially in the 7 d group. The LC3 protein was expressed in the cytoplasm, and the expression level gradually increased, reaching a peak on the second day. Conclusion: The early activation of autophagy in rats with acute chlorpyrifos poisoning may be involved in chlorpyrifos induced hippocampal neuronal injury.

The Search for Citrus Components with Neuroprotective and Anti-dementia Effects in the Mouse Brain

Citrus kawachiensis (Kawachi Bankan), is a citrus species grown in Ehime, Japan, and its peel is abundant in 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF). We have recently revealed that HMF, one of the citrus flavonoids, has anti-inflammatory activity and neuroprotective abilities in the brain against global cerebral ischemia. HMF rescued neuronal cell death in the hippocampus and suppressed the activation of microglia, whose activation have been shown to significantly aggravate neurological deficit scores for ischemic mice. In the Y-maze test, HMF showed protection against ischemia-induced short-term memory dysfunction; in addition, HMF enhanced the expression of brain-derived neurotrophic factor and accelerated neurogenesis in the hippocampus. Similarly, the powder of the peel of C. kawachiensis showed anti-inflammatory activity and neuroprotective abilities in the ischemic brain. To further examine the effect of the peel of C. kawachiensis, we administered it to senescence-accelerated-mouse prone 8 (SAMP8) mice, which show memory impairment and brain inflammation, tau hyperphosphorylation, and neuronal dysfunction. The C. kawachiensis treatment inhibited microglial activation and the hyperphosphorylation of tau protein in hippocampal neurons, and also relieved the suppression of neurogenesis in the dentate gyrus of the hippocampus in SAMP8. These results suggest that HMF and the peel of C. kawachiensis have potential effects as neuroprotective and anti-dementia agents.

Carnitine palmitoyltransferase 1 (CPT1) alleviates oxidative stress and apoptosis of hippocampal neuron in response to beta-Amyloid peptide fragment Aβ25-35

ABSTRACT CPT1C, which is expressed in hippocampus, influences ceramide level, endogenous cannabinoid and oxidation process, as well as plays an important role in various brain functions such as learning. This study aimed to investigate the role of CPT1C in Alzheimer’s disease (AD) and its underlying mechanism. We established a model of Alzheimer’s disease in vitro by exposing primary hippocampal neurons to beta-Amyloid peptide fragment 25–35 (Aβ25-35). The cell viability, lactate dehydrogenase (LDH) level, expressions of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) were detected using Cell Counting Kit-8 (CCK-8), LDH assay, ROS kits, malondialdehyde (MDA) kits and SOD kits, respectively. Moreover, the expression of oxidative stress-related proteins as well as the expressions of amyloid precursor protein (App), p-Tau andβ-site APP-cleaving enzyme1 (Bace-1) were measured using quantitative reverse transcription PCR (RT-qPCR) and western blot. Tunel and western blot were adopted to detect apoptosis as well as its related proteins. After the treatment of peroxisome proliferators-activated receptor alpha (PPARα), CPT1C expression was detected with the application of RT-qPCR and western blot. CPT1C expression was reduced in Aβ25-35-induced HT22 cells. Overexpression of CPT1C relieved cell viability and toxic injury as well as attenuated oxidative stress, apoptosis and expression levels of AD marker proteins. Moreover, higher doses of PPARα agonist activate the expression of CPT1C in Aβ25-35-induced HT22 cells. In conclusion, CPT1C alleviates Aβ25-35-induced oxidative stress, apoptosis and deposition of AD marker proteins in hippocampal neurons, suggesting that CPT1C has favorable effects on alleviating AD and participates in PPARα activation.

Neuroprotective effects of the peel of Citrus kawachiensis (Kawachi Bankan) and auraptene in the hippocampus of hyperglycemia mice and global cerebral ischemia/reperfusion injury mice

The peel of Citrus kawachiensis (Kawachi Bankan), a citrus species grown in Ehime, Japan, is abundant in auraptene. Auraptene, a coumarin compound, have been shown to exert anti-inflammatory effects in peripheral tissues, but it was still unclear of the effect in the brain. Hyperglycemia and brain ischemia induce inflammation and oxidative stress and cause massive damage in the brain; therefore, we examined the anti-inflammatory and other effects of the dried peel powder of C. kawachiensis and auraptene in a hyperglycemia and global cerebral ischemia models. The C. kawachiensis treatment inhibited astroglial activation in the hippocampus and the hyperphosphorylation of tau protein in hippocampal neurons, and also relieved the suppression of neurogenesis in the dentate gyrus of the hippocampus in the type 2 diabetic db/db mice. The C. kawachiensis treatment inhibited microglial and astroglial activation, and neuronal cell death in the hippocampus of transient global cerebral ischemia mice. It was suggested that the dried peel powder of C. kawachiensis exerts anti-inflammatory and neuroprotective effects in the brain. We attempted to demonstrate the effect of auraptene in the brains in streptozotocin-induced hyperglycemic mice and transient global cerebral ischemia mice. Auraptene administration showed the similar effects as the peel of C. kawachiensis in the hippocampus of these mice models. These results suggested that auraptene have potential effects as a neuroprotective agent in the peel of C. kawachiensis.

Auxin-mediated rapid degradation of target proteins in hippocampal neurons.

Genetic manipulation of protein levels is a promising approach to identify the function of a specific protein in living organisms. Previous studies demonstrated that the auxin-inducible degron strategy provides rapid and reversible degradation of various proteins in fungi and mammalian mitotic cells. In this study, we employed this technology to postmitotic neurons to address whether the auxin-inducible degron system could be applied to the nervous system. Using adeno-associated viruses, we simultaneously introduced enhanced green fluorescent protein (EGFP) fused with an auxin-inducible degron tag and an F-box family protein, TIR1 from Oryza sativa (OsTIR1), into hippocampal neurons from mice. In dissociated hippocampal neurons, EGFP enhanced green fluorescent protein fluorescence signals rapidly decreased when adding a plant hormone, auxin. Furthermore, auxin-induced enhanced green fluorescent protein degradation was also observed in hippocampal acute slices. Taken together, these results open the door for neuroscientists to manipulate protein expression levels by the auxin-inducible degron system in a temporally controlled manner.

Simplicity, Flexibility, and Interpretability in a Model of Dendritic Protein Distributions

In this issue of Neuron, Fonkeu etal. (2019) present a mathematical model of mRNA and protein synthesis, degradation, diffusion, and trafficking in neuronal dendrites. The model can predict the spatial distribution and temporal dynamics of proteins along dendrites. The authors use the model to account for in situ imaging data of CaMKII⍺ mRNA and protein in hippocampal neurons.

Neuroprotective effects of the peel of &lt;i&gt;Citrus kawachiensis&lt;/i&gt; (Kawachi Bankan) and auraptene in the hippocampus of hyperglycemia mice and global cerebral ischemia/reperfusion injury mice.

The peel of Citrus kawachiensis (Kawachi Bankan), a citrus species grown in Ehime, Japan, is abundant in auraptene. Auraptene, a coumarin compound, have been shown to exert anti-inflammatory effects in peripheral tissues, but it was still unclear of the effect in the brain. Hyperglycemia and brain ischemia induce inflammation and oxidative stress and cause massive damage in the brain; therefore, we examined the anti-inflammatory and other effects of the dried peel powder of C. kawachiensis and auraptene in a hyperglycemia and global cerebral ischemia models. The C. kawachiensis treatment inhibited astroglial activation in the hippocampus and the hyperphosphorylation of tau protein in hippocampal neurons, and also relieved the suppression of neurogenesis in the dentate gyrus of the hippocampus in the type 2 diabetic db/db mice. The C. kawachiensis treatment inhibited microglial and astroglial activation, and neuronal cell death in the hippocampus of transient global cerebral ischemia mice. It was suggested that the dried peel powder of C. kawachiensis exerts anti-inflammatory and neuroprotective effects in the brain. We attempted to demonstrate the effect of auraptene in the brains in streptozotocin-induced hyperglycemic mice and transient global cerebral ischemia mice. Auraptene administration showed the similar effects as the peel of C. kawachiensis in the hippocampus of these mice models. These results suggested that auraptene have potential effects as a neuroprotective agent in the peel of C. kawachiensis.

Influence of mTOR signaling pathway on ketamine-induced injuries in the hippocampal neurons of rats

ABSTRACTObjective: To explore the influences of mammalian target of rapamycin (mTOR) signaling pathway on ketamine-induced apoptosis, oxidative stress and Ca2+ concentration in the hippocampal neurons of rats.Methods: The primary hippocampal neurons isolated from fetal Sprague Dawley rats were treated with ketamine (0, 50, 100 and 500 μM) for 4 days to observe its effect on mTOR signaling pathway and apoptosis of rat hippocampal neurons. Then, the hippocampal neurons were divided into C (Control), R (Rapamycin, an inhibitor of mTOR signaling pathway), K (Ketamine) and R + K (Rapamycin + Ketamine) groups to detect the apoptosis, reactive oxygen species (ROS) production, and Ca2+ concentration via the terminal transferase uridyl nick end labelling (TUNEL) assay, dichloro-dihydro-fluorescein diacetate (DCFH-DA) method and Fluo-3 acetoxymethyl ester (Fluo-3AM) staining, respectively. The expressions of mTOR signaling pathway and apoptosis-related proteins in hippocampal neurons were examined by qRT-PCR and Western blot.Results: Ketamine could dose-dependently promote the apoptosis of rat hippocampal neurons with upregulation of p-mTOR and its downstream regulators (p-4E-BP-1 and p-p70S6K). However, ketamine-induced apoptosis in hippocampal neurons was reversed significantly by the administration of rapamycin, as evident by the decrease in expressions of pro-apoptotic proteins (Bax and cleaved Caspase-3) and the increase in anti-apoptotic protein (Bcl-2). Meanwhile, the ROS generation and Ca2+ concentration was inhibited accompanied with reduced malonildialdehyde levels but elevated superoxide and glutathione peroxidase activities.Conclusion: Inhibition of mTOR signaling pathway protected rat hippocampal neurons from ketamine-induced injuries via reducing apoptosis, oxidative stress, as well as Ca2+ concentration.Abbreviations: mTOR: mammalian target of rapamycin; SD: Sprague-Dawley; SPF: Specific-pathogen free; ROS: reactive oxygen species; TUNEL: terminal transferase uridyl nick end labelling; DCFH-DA: Dichloro-dihydro-fluorescein diacetate; Fluo-3A: Fluo-3 acetoxymethyl ester; NMDAR: non-competitive N-methyl-D-aspartame glutamate receptor; 4E-BP1: 4E binding protein 1; p70S6K: p70 S6 Kinase; PCR: Polymerase chain reaction; MDA: malonildialdehyde; GSH-PX: glutathione peroxidase; ANOVA: One-way Analysis of Variance.

Brain-derived neurotrophic factor inhibits hyperglycemia-induced apoptosis and downregulation of synaptic plasticity-related proteins in hippocampal neurons via the PI3K/Akt pathway.

It is not known whether brain-derived neurotrophic factor (BDNF) protects hippocampal neurons from high glucose-induced apoptosis and/or synaptic plasticity dysfunction. The present study aimed to assess whether BDNF exerted a neuroprotective effect in rat hippocampal neurons exposed to high glucose and examine the underlying mechanisms. The apoptosis of primary hippocampal neurons was assessed by Annexin V-fluorescein isothiocyanate/propidium iodide staining. The mRNA and protein expression levels were measured by reverse transcription- quantitative polymerase chain reaction and western blot experiments, respectively. Synaptic plasticity was evaluated by the immunolocalization of synaptophysin (Syn). Exposure of the hippocampal neurons to high glucose (75 mM for 72 h) resulted in cell apoptosis, decreased mRNA and protein expression levels of three synaptic plasticity-related proteins (Syn, Arc and cyclic AMP response element-binding protein), and changes in the cellular distribution of Syn, indicating loss of synaptic density. These effects of high glucose were partially or completely reversed by prior administration of BDNF (50 ng/ml for 24 h). Pre-treatment with wortmannin, a phosphatidylinositol-3-kinase (PI3K) inhibitor, suppressed the ability of BDNF to inhibit the effects of high glucose. In addition, BDNF significantly upregulated the tropomyosin-related kinase B, its cognate receptor, Akt and phosphorylated Akt at the protein levels under high glucose conditions. In conclusion, high glucose induced apoptosis and downregulated synaptic plasticity-related proteins in hippocampal neurons. These effects were reversed by BDNF via the PI3K/Akt signaling pathway.

Expression of Bcl-2 and Bad in hippocampus of status epileptic rats and molecular mechanism of intervened recombinant human erythropoietin

Injury of hippocampal neurons in status epilepticus (SE) SD rats kindled by pentylenetetrazol (PTZ) were studied, and the changes of apoptosis neurons, protein expression of Bad and Bcl-2 alone and combined application of phosphatidyl inositol 3-kinase (PI3K) inhibitor LY294002 and recombinant human erythropoietin (rHuEpo) were evaluated for the possible mechanisms of rHuEpo. The SE rats kindled by the PTZ were randomly divided into normal control group [normal saline (NS)], model group (PTZ + NS), rHuEpo treated group (PTZ + rHuEpo), LY294002 treated group (PTZ + LY294002 + rHuEpo) and LY294002 control group (rHuEpo + PTZ + DMSO). Apoptosis of hippocampal neurons was detected by TUNEL method; expression of phosphorylation protein kinase B (p-PKB/p-Akt), Bcl-2 and Bad were detected by immunohistochemistry; the expression of Bcl-2 mRNA, Bad mRNA in hippocampal neurons of rats were detected through reverse transcription polymerase chain reaction (RT-PCR); the expression of Akt, p-Akt and Bcl-2, Bad protein in hippocampal neurons of rats were detected by western blotting. The amount of apoptotic neurons was less in the rHuEpo treated group and the LY294002 control group than in the LY294002 treated group (P<0.05). The expression of p-Akt protein and Bcl-2 protein increased while the Bad protein decreased significantly in the rHuEpo treated group and the LY294002 control group compared with the LY294002 treated group (P<0.05). The expression of Bad protein and Bad mRNA in hippocampus increased while the p-Akt, Bcl-2, Bcl-2 mRNA decreased significantly in the LY294002 treated group compared with the rHuEpo treated group (P<0.05). The PI3K/Akt signaling pathway is one of the pathways of rHuEpo neuroprotective effects and was confirmed from both the of positive and negative aspects. rHuEpo regulates the expression of mitochondrial apoptotic pathway related factors Bad and Bcl-2 to inhibit apoptosis and promotes neuronal survival.

Aging Does Not Affect Axon Initial Segment Structure and Somatic Localization of Tau Protein in Hippocampal Neurons of Fischer 344 Rats.

Little is known about the specific contributions of aging to the neuron dysfunction and death in Alzheimer’s disease (AD). AD is characterized by the pathological accumulation of abnormal tau (a microtubule-associated protein), and the mislocalization of tau from the axon to the somatodendritic compartment is thought to play an important role in disease pathogenesis. The axon initial segment (AIS) is thought to play a role in the selective localization of tau in the axonal compartment. Thus, disruption in the AIS barrier may allow tau to diffuse freely back into the somatodendritic compartment and potentially lead to neurotoxicity. Here, we analyzed AISs using stereological methods and protein immunoblotting, and the localization of tau was assessed with immunofluorescence optical density measurements and protein immunoblotting. None of the outcome measurements assessed, including AIS structure, AIS protein levels, the distribution of tau in neurons of the hippocampus (HP), and total tau or phospho-tau protein levels were different in young, middle-, and old-age Fischer 344 rats. The outcome measurements assessed, including AIS structure, AIS protein levels, the distribution of tau in neurons of the HP, and total tau or phospho-tau protein levels were not different in young, middle-, and old-age Fischer 344 rats, with the exception of a small reduction in AIS volume and diameter in the CA2 region of aged animals. These data suggest that aging largely has no effect on these properties of the AIS or tau distribution, and thus, may not contribute directly to tau mislocalization.

Effect of GAPT extract on expression of tau protein and its phosphorylation related enzymes in hippocampal neurons of APPV717I transgenic mice.

To investigate the effect of GAPT, an extract mixture from Radix Ginseng, Rhizoma Acor tatarinowii, Radix Polygalae and Radix Curcuma (containing ingredient of turmeric), etc. on expression of tau protein and its phosphorylation related enzyme in hippocampal neurons of APPV717I transgenic mice. Sixty three-month-old APPV717I transgenic mice were randomly divided into model group, donepezil group [0.92 mg/(kg•d)], the low, medium and high dosage of GAPT groups [0.075, 0.15, 0.30 g/(kg•d), 12 in each group], and 12 three-month-old C57BL/6J mice were set as a normal control group, treatments were administered orally once a day respectively, and both the normal group and model group were given 0.5% sodium carboxymethyl cellulose solution. Immunohistochemistry (IHC) and Western blot analysis were used to detect the expression of total tau protein (Tau-5), cyclin-dependent kinase 5 (CDK5) and protein phosphatase 2A (PP2A) in hippocampal neurons of experimental mice after 8-month drug administration (11 months old). In the model group, the expression of Tau-5 and CDK5 were increased, whereas the expression of PP2A was decreased in hippocampal neurons, which were signifificantly different compared with that in the normal group (all P<0.01). IHC test indicated the number and area of either Tau-5 or CDK5 positive cells were decreased with a dose-depended way in GAPT groups, and an increase of PP2A. Compared with the model group, the changes were signifificant in GAPT groups (P<0.05 or P<0.01). Similar results were shown by Western blot. GAPT could attenuate abnormal hyperphosphorylation of tau protein in hippocampal neurons of APPV717I transgenic mice via inhibiting the expression of CDK5 and activating the expression of PP2A.

Effect of Heparin on the Expressions of Serum Intercellular Adhesion Molecule-1 and Hippocampus S100β Protein after Cardiopulmonary Resuscitation in Rabbits

Objective Use heparin during cardiac arrest (CA) in rabbits and observe the serum intercellular adhesion molecule-1 (ICAM-1) and hippocampal S100β protein expressions after cardiopulmonary resuscitation (CPR). Methods Thirty-two New Zealand rabbits were randomly divided into, Group I, control group; Group II, saline group; Group III, heparin group. Each animal underwent continuous hemodynamic monitoring including mean arterial pressure (MBP), heart rate (HR), and the end-tidal carbon dioxide partial pressure (PetCO2). Twenty-four hours after resuscitation, serum and hippocampal neurons were collected from all animals. Enzyme linked immunosorbent assay was used to detect serum ICAM-1 and immunohistochemistry to detect the S100β protein in hippocampal neurons. According to the rate of positive cells, each hippocampal specimen was categorized into four expression levels. Results The differences in the serum ICAM-1 concentration in the three groups were statistically significant. The expression of S100β protein in the hippocampus showed eight cases in group I at level 1 and none in groups II and III. There was 1 case in group II and 7 cases in group III at level 2; five cases in group II and 2 cases in group III at level 3; 2 cases in group II and 1 case in group III at level 4. The expression strength of S100β protein in the three groups differed significantly (P &lt; 0.05). Conclusions Heparin therapy can reduce the expression of serum ICAM-1 and S100β protein in hippocampal neurons during CPR. It is possible that heparin can have a positive effect on brain protection during CPR.