急性缺氧和急性低糖对脑片tau蛋白磷酸化的影响

为探讨急性缺氧对tau蛋白磷酸化的影响,将Wistar大鼠脑片进行不同时间的缺氧培养后,对tau蛋白的磷酸化状态及相关磷酸酯酶的活性和表达进行检测.结果显示,急性缺氧使tau蛋白多个丝氨酸位点磷酸化水平下降,蛋白磷酸酯酶～2A(PP-2A)的活性升高,其催化亚单位表达上调,而蛋白磷酸酯酶-1(PP-1)的活性及催化亚单位表达均无明显改变.该研究结果表明:急性缺氧可能通过蛋白磷酸酯酶-2A的上调而使tau蛋白多个丝氨酸位点发生去磷酸化作用.     

糖尿病大鼠脑GSK-3与PP-2A失调诱导tau蛋白过度磷酸化

探讨胰岛素缺乏的糖尿病大鼠皮层糖原合酶激酶-3(GSK-3)及蛋白磷酯酶-2A(PP-2A)变化及其对tau蛋白磷酸化的作用.用链脲佐菌素(streptozotocin,STZ)建立胰岛素缺乏的糖尿病大鼠模型,用放射性配体结合实验检测了GSK-3和PP-2A的活性,蛋白质印迹检测了tau蛋白的磷酸化水平及PP-2A的表达.结果提示:在糖尿病大鼠皮层,GSK-3活性升高,PP-2A活性及表达降低,tau蛋白在Ser198/Ser199/Ser202和Ser396/Ser404位点磷酸化.应用GSK-3的选择性抑制剂Li2CO3后,GSK-3活性降低,PP-2A活性及表达恢复,tau蛋白在Ser198/Ser199/Ser202和Ser396/Ser404位点磷酸化水平降低.研究提示:糖尿病大鼠皮层GSK-3升高可能抑制PP-2A的活性,升高的GSK-3和降低的PP-2A协同促进tau蛋白的磷酸化.     

Val97Leu mutant presenilin-1 induces tau hyperphosphorylation and spatial memory deficit in mice and the underlying mechanisms

Although the pathological role of presenilin-1 mutation in early onset familial Alzheimer's disease has been widely studied, few focused on how the presenilin-1 mutations result in memory impairment and tau hyperphosphorylation. In the present study, we expressed human Val97Leu mutant presenilin-1, which is reported in Chinese pedigrees by our group, in transgenic mice and found that the mutant presenilin-1 induced spatial memory deficit and tau hyperphosphorylation at PHF-1, pS199/202, pT231 and pS396 epitopes, but not at pS214 and pS422 epitopes. Pearson analysis showed that the memory deficit was only significantly correlated with tau phosphorylation level at PHF-1, pS199/202, pT231 and pS396 epitopes. Additionally, the hyperphosphorylated tau and tangle-like argentophilic structures were detected at CA3 and CA4, but not CA1, region of hippocampus, and we also found tangle-like structure and wizened degenerative neurons in frontal cortex. We demonstrated the tau hyperphosphorylation at the same epitopes in N2a cells expressing the mutant presenilin-1, which is caused by inhibition of phosphoinositol-3 kinase/Akt and activation of glycogen synthase kinase-3 specifically. Our data demonstrated that human Val97Leu mutant presenilin-1 causes spatial memory deficit in mice and increases tau phosphorylation level in glycogen synthase kinase-3-dependent manner.     

Inhibition by veratridine of carbachol-stimulated inositol tetrakisphosphate accumulation in rat brain cortical slices

The present studies examined the inhibitory effect of veratridine (a Na⁺ channel activator) on carbachol (a cholinergic agonist) stimulated inositol 1,3,4,5-tetrakisphosphate accumulation in rat brain cortical slices. Veratridine inhibited carbachol stimulation of inositol 1,3,4,5-tetrakisphosphate formation (after a delay of about 30 seconds) at 60 or 120 seconds when there was little inhibition of inositol 1,4,5 trisphophate accumulation. The inhibitory effect of veratridine on carbachol stimulated inositol 1,3,4,5-tetrakisphosphate accumulation was abolished in the presence of ouabain or tetrodotoxin but was unaffected in low calcium conditions. Veratridine reduced the total ATP content and this effect was abolished by tetrodotoxin. The inhibitory effect of 10 but not 30 M veratridine on inositol 1,3,4,5-tetrakisphosphate accumulation in the presence of carbachol was reversed by the presence of exogenous 8-bromo cyclic AMP or forskolin which activates adenylylcyclase. However, the decrease in brain slice ATP seen in the presence of veratridine was unaffected by forskolin. Our results are compatible with the hypothesis that veratridine inhibition of carbachol-stimulated inositol 1,3,4,5-tetrakisphosphate formation is due to depletion of ATP at the site of Ins 1,3,4,5-P₄ formation from Ins 1,4,5-P₃.Abbreviations used Ins 1,4,5-P₃ inositol 1,4,5 trisphosphate - Ins 1,3,4,5-P₄ inositol 1,3,4,5-tetrakisphosphate - PMA phorbol 12-myristate 13-acetate     

Compromised mitochondrial function results in dephosphorylation of tau through a calcium-dependent process in rat brain cerebral cortical slices

Mitochondria play an important role in modulating intracellular levels of calcium, and therefore compromised mitochondrial function often leads to disruptions in calcium homeostasis. In this study, the effects of two uncouplers of oxidative phosphorylation, carbonyl cyanide-3-chlorophenylhydrazone (CCCP) and p-trifluoromethoxyphenylhydrazone (FCCP), on calcium-mediated modifications of the microtubule-associated protein, tau, in rat brain slices were examined. Incubation of slices with CCCP or FCCP resulted in an increase in electrophoretic mobility of several of the tau isoforms, with no apparent loss of intact tau or the appearance of degradation products. These data indicated that disrupting mitochondrial function by dissipating the transmembrane potential resulted in the dephosphorylation of tau. This finding was confirmed by using a front phosphorylation assay to demonstrate a CCCP-induced decrease in the phosphorylation state of tau. The dephosphorylation of tau induced by the proton-ionophores appeared to be calcium-dependent since the effect was blocked by EGTA. In addition, the CCCP-induced dephosphorylation of tau was blocked by cyclosporin A, a selective inhibitor of the calcium-dependent phosphatase, calcineurin. These data strongly indicate that tau is a substrate for calcineurin in vivo. Finally, the levels of ATP were depleted to a similar extent in brain slices incubated in the presence of CCCP or CCCP and EGTA. These results demonstrated depletion of ATP alone was not sufficient to stimulate the dephosphorylation of tau in this experimental paradigm.     

Calcium rises induced by AMPA and nicotine receptors in the ventral tegmental area show differences in mouse brain slices prenatally exposed to nicotine

Nicotine exposure during gestation is associated with a higher risk of adverse behavioral outcomes including a heightened liability for dependency to drugs of abuse, which can exhibit drug‐specificity influenced by gender. This enhanced liability suggests that nicotine use during pregnancy alters neural development in circuits involved in motivation and reward‐based learning. The ventral tegmental area (VTA) is critical in motivated behaviors and we hypothesized that gestational exposure to nicotine alters the development of excitatory circuits in this nucleus. Accordingly, in VTA brain slices from male and female mice exposed to nicotine during the prenatal period (PNE) and controls, we compared cellular rises in calcium induced by AMPA receptor and nicotinic acetylcholine receptor (nAChR) stimulation by use of the ratiometric calcium binding dye, Fura‐2AM. We found that AMPA induced smaller amplitude calcium rises in the PNE VTA, which was an effect only detected in males. Further, while the amplitude did not vary between treatment and control in females, a greater number of cells responded with rises in calcium in the PNE. Conversely, the proportions of cells responding with calcium rises induced by nAChR stimulation did not change in either gender according to treatment. However, larger rises in calcium in PNE females were detected. When taken together our data show that excitatory signaling in the VTA is altered in a gender‐specific manner by PNE and suggest that alterations in signaling could play a role in drug‐specific differences in maladaptive, motivated behaviors exhibited by males and females born to mothers exposed to nicotine during pregnancy. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 2018     

低氧对小鼠学习记忆力及脑中tau蛋白磷酸化的影响

目的：研究不同低氧暴露对小鼠学习记忆及脑中tau蛋白磷酸化的影响。方法：雄性昆明小鼠40只,随机分为4组（n=10）：对照组（control）、8h低氧暴露组（8h）、7d低氧暴露组（7d）和28d低氧暴露组（28d）。将低氧暴露模型组置于模拟高原海拔5500m的低压氧舱,每天低氧暴露8h,避暗和旷场实验检测其活动能力及学习记忆能力：免疫印迹技术检测小鼠海马和皮层中tau蛋白磷酸化水平。结果：随着低氧时间的增加,小鼠短期学习记忆力及活动能力下降程度增大,28d低氧暴露后其下降程度最大;海马中tau蛋白多个位点的磷酸化水平呈现升高趋势,28d时tau蛋白磷酸化程度最高（P〈0．05）;皮层中的磷酸化水平在低氧暴露7d时达到最高,低氧暴露28d时略有降低,但与control组相比仍有明显差异（P〈0．05）。结论：慢性间歇性低氧可导致小鼠学习记忆能力下降,其机制可能与tau蛋白过度磷酸化相关。     

Effect of anoxia and reoxygenation on antioxidant enzyme activities in immortalized brain endothelial cells

Summary The effects of anoxia and reoxygenation on major antioxidant enzyme activities were investigatedin vitro in immortalized rat brain endothelial cells (RBE₄ cells). A sublethal anoxic period of 12 h was assessed for RBE₄ cells using the neutral red uptake test. Anoxia markedly influenced the specific activity of catalase and superoxide dismutase, with no major effect on glutathione peroxidase or glutathione reductase. After 24 h postanoxia, the superoxide dismutase activity modulated by the presence or absence of oxygen returned to control value. Damage and recovery of RBE₄ immortalized rat brain endothelial cells in culture after exposure to free radicals and other oxygen-derived species provides a usefulin vitro model to study anoxia-reoxygenation trauma at the cellular level.     

Oxidative stress induces transient O‐GlcNAc elevation and tau dephosphorylation in SH‐SY5Y cells

O‐linked β‐N‐acetlyglucosamine or O‐GlcNAc modification is a dynamic post‐translational modification occurring on the Ser/Thr residues of many intracellular proteins. The chronic imbalance between phosphorylation and O‐GlcNAc on tau protein is considered as one of the main hallmarks of Alzheimer's disease. In recent years, many studies also showed that O‐GlcNAc levels can elevate upon acute stress and suggested that this might facilitate cell survival. However, many consider chronic stress, including oxidative damage as a major risk factor in the development of the disease. In this study, using the neuronal cell line SH‐SY5Y we investigated the dynamic nature of O‐GlcNAc after treatment with 0.5 mM H₂O₂ for 30 min. to induce oxidative stress. We found that overall O‐GlcNAc quickly increased and reached peak level at around 2 hrs post‐stress, then returned to baseline levels after about 24 hrs. Interestingly, we also found that tau protein phosphorylation at site S262 showed parallel, whereas at S199 and PHF1 sites showed inverse dynamic to O‐Glycosylation. In conclusion, our results show that temporary elevation in O‐GlcNAc modification after H₂O₂‐induced oxidative stress is detectable in cells of neuronal origin. Furthermore, oxidative stress changes the dynamic balance between O‐GlcNAc and phosphorylation on tau proteins.     

Protein kinase involvement in land snail aestivation and anoxia: Protein kinase A kinetic properties and changes in second messenger compounds during depressed metabolism

In response to environmental stress (low water, low oxygen) snails sharply suppress their metabolic rate, a process that is coordinated at the molecular level by reversible protein phosphorylation of key enzymes and functional proteins. Factors affecting protein kinase activity are, therefore, critical to metabolic suppression. Changes in the concentration of protein kinase second messenger compounds were followed over the first 24 h of aestivation and anoxia exposure in the terrestrial snail Otala lactea (Muller) (Pulmonata, Helicidae). The results showed declining concentrations of cyclic AMP over the first 24 h of anoxia exposure and aestivation in foot. Cyclic AMP concentrations in hepatopancreas transiently decreased with the lowest concentration observed at 4 h in both anoxic and aestivating animals. A transient increase in foot muscle cyclic GMP concentrations was apparent 4 h after the start of aestivation whereas a slow, steady increase was seen in anoxic foot muscle. Foot muscle 1,4,5-inositol triphosphate (IP3) concentrations decreased transiently during anoxia exposure and aestivation. Hepatopancreas IP₃ concentrations were significantly lower in 24 h anoxic snails and foot IP3 concentrations were significantly lower in 24 h aestivating snails. Kinetic characterization of purified PKA catalytic subunit was also performed. Snail PKA catalytic subunit had an absolute requirement for Mg²⁺ ion but was inhibited at Mg²⁺ concentrations above 0.5 mM. Increasing concentrations of neutral salts and phosphate also inhibited activity although the inhibition by phosphate appeared to be specific since the inhibition constant (I₅₀ = 39 mM) was much lower than that of the neutral salts (I₅₀ 240 mM). The enzyme exhibited a broad pH optimum between pH 6.5–8.5. Arrhenius plots gave an activation energy of 13.3 kcal/mol corresponding to a Q₁₀ value of 2.3. The relationship between these results and temporal control of enzyme phosphorylation is discussed.Abbreviations CAMP adenosine 3:5-cyclic monophosphate - cGMP-guanosine 3:5-cyclic monophosphate - H-89N [2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCI - IP₃ d-myo-inositol 1,4,5-triphosphate - I₅₀ the concentration of inhibitor required to reduce the velocity to one half its original value - PKA cAMP dependent protein kinase - PKAc PKA catalytic subunit - PKA-I PKA inhibitor protein - PKC calcium and phospholipid-dependent protein kinase - PKC-I PKC inhibitor protein - PKG cGMP dependent protein kinase - mU nmol of phosphate transferred per minute     

Alzheimer neurofibrillary degeneration: significance, etiopathogenesis, therapeutics and prevention

Alzheimer disease (AD) is multi-factorial and heterogeneous. Independent of the aetiology, this disease is characterized clinically by chronic and progressive dementia and histopathologically by neurofibrillary degeneration of abnormally hyperphosphorylated tau seen as intraneuronal neurofibrillary tangles, neuropil threads and dystrophic neurites, and by neuritic (senile) plaques of beta-amyloid. The neurofibrillary degeneration is apparently required for the clinical expression of AD, and in related tauopathies it leads to dementia in the absence of amyloid plaques. While normal tau promotes assembly and stabilizes microtubules, the abnormally hyperphosphorylated tau sequesters normal tau, MAP1 and MAP2, and disrupts microtubules. The abnormal hyperphosphorylation of tau also promotes its self-assembly into tangles of paired helical and or straight filaments. Tau is phosphorylated by a number of protein kinases. Glycogen synthase kinase-3 (GSK-3) and cyclin dependent protein kinase 5 (cdk5) are among the kinases most implicated in the abnormal hyperphosphorylation of tau. Among the phosphatases which regulate the phosphorylation of tau, protein phosphatase-2A (PP-2A), the activity of which is down-regulated in AD brain, is by far the major enzyme. The inhibition of abnormal hyperphosphorylation of tau is one of the most promising therapeutic targets for the development of disease modifying drugs. A great advantage of inhibiting neurofibrillary degeneration is that it can be monitored by evaluating the levels of total tau and tau phosphorylated at various known abnormally hyperphosphorylated sites in the cerebrospinal fluid of patients, obtained by lumbar puncture. There are at least five subgroups of AD, each is probably caused by a different etiopathogenic mechanism. The AD subgroup identification of patients can help increase the success of clinical trials and the development of specific and potent disease modifying drugs.     

Role of calcineurin and protein phosphatase-2A in the regulation of DARPP-32 dephosphorylation in neostriatal neurons

DARPP-32, a dopamine- and cyclic AMP-regulated phosphoprotein of Mr 32 kDa, is phosphorylated on Thr34 by cyclic AMP-dependent protein kinase, resulting in its conversion to a potent inhibitor of protein phosphatase-1 (PP-1). Conversely, Thr34-phosphorylated DARPP-32 is dephosphorylated and inactivated in vitro by calcineurin and protein phosphatase-2A (PP-2A). We have investigated the relative contributions of these protein phosphatases to the regulation of DARPP-32 dephosphorylation in mouse neostriatal slices. Cyclosporin A (5 microM), a calcineurin inhibitor, maximally increased the level of phosphorylated DARPP-32 by 17+/-2-fold. Okadaic acid (1 microM), an inhibitor of PP-1 and PP-2A, had a smaller effect, increasing phospho-DARPP-32 by 5.1+/-1.3-fold. The effect of okadaic acid on DARPP-32 phosphorylation was shown to be due to inhibition of PP-2A activity. Incubation of slices in the presence of cyclosporin A plus either okadaic acid or calyculin A, another PP-1/PP-2A inhibitor, caused a synergistic increase in the level of phosphorylated DARPP-32. The use of Ca2(+)-free/EGTA medium mimicked the effects of cyclosporin A on DARPP-32 phosphorylation, supporting the conclusion that the action of cyclosporin on DARPP-32 phosphorylation was attributable to blockade of the Ca2(+)-dependent activation of calcineurin. The results indicate that calcineurin and PP-2A, but not PP-1, act synergistically to maintain a low level of phosphorylated DARPP-32 in neostriatal slices.     

Effects of fasting and diabetes on some enzymes and transport of glutamate in cortex slices or synaptosomes from rat brain

Phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase (GAD) were assayed in homogenates and synaptosomes obtained from starved (48 hr or 120 hr) and diabetic (streptozotocin) rat brain cortex. Glutamine synthetase (GS) was assayed in homogenates, microsomal and soluble fractions, from brain cortex of similarly treated rats.l-Glutamate uptake and exit rates were determined in cortex slices and synaptosomes under the same conditions. The specific activity (s.a.) of PAG, a glutamate producing enzyme, decreased (50%) in the homogenate after 120-hr starvation. In synaptosomes it decreased (25%) only after 48-hr starvation. The s.a of GAD and GS, which are glutamate-consuming enzymes, were progressively increased with time of starvation, reaching 39% and 55% respectively after 120 hr. GS in the microsomes or the soluble fraction and GAD in the synaptosomes showed no change in s.a. under these conditions. Diabetes increased (40%) microsomal GS s.a. and decreased GAD s.a. (18%) in the homogenate. Thel-glutamate uptake rate was decreased (48%) by diabetes in slices but not in synaptosomes. It is suggested that a) enzymes of the glutamate system respond differently in different subcellular fractions towards diabetes or deprivation of food and b) diabetes may affect the uptake system in glial cells but not in neurons.Abbreviations used AET 2-aminoethylisourethonium bromide - GAD glutamic acid decarboxylase - GS glutamine synthetase - GSH glutathione - PAG phosphate-activated glutaminase - PLP pyridoxal phosphate - r.c.f. relative centrifugal force - s.a. specific activity     

N-glycosylation induced changes in tau protein dynamics reveal its role in tau misfolding and aggregation: A microsecond long molecular dynamics study

Various posttranslational modifications like hyperphosphorylation, O-GlcNAcylation, and acetylation have been attributed to induce the abnormal folding in tau protein. Recent in vitro studies revealed the possible involvement of N-glycosylation of tau protein in the abnormal folding and tau aggregation. Hence, in this study, we performed a microsecond long all atom molecular dynamics simulation to gain insights into the effects of N-glycosylation on Asn-359 residue which forms part of the microtubule binding region. Trajectory analysis of the stimulations coupled with essential dynamics and free energy landscape analysis suggested that tau, in its N-glycosylated form tends to exist in a largely folded conformation having high beta sheet propensity as compared to unmodified tau which exists in a large extended form with very less beta sheet propensity. Residue interaction network analysis of the lowest energy conformations further revealed that Phe378 and Lys353 are the functionally important residues in the peptide which helped in initiating the folding process and Phe378, Lys347, and Lys370 helped to maintain the stability of the protein in the folded state.     

Sub-neurotoxic neonatal anoxia induces subtle behavioural changes and specific abnormalities in brain group-I metabotropic glutamate receptors in rats

Anoxia in the first week of life can induce neuronal death in vulnerable brain regions usually associated with an impairment of cognitive function that can be detected later in life. We set-up a model of subneurotoxic anoxia based on repeated exposures to 100% nitrogen during the first 7 days of post-natal life. This mild post-natal exposure to anoxia specifically modified the behaviour of the male adult rats, which showed an attention deficit and an increase in anxiety, without any impairment in spatial learning and any detectable brain damage (magnetic resonance imaging and histological analysis). Post-anoxic rats showed a reduction in the expression of group-I metabotropic glutamate receptors (i.e. mGlu1 and mGlu5 receptors) in the hippocampus and cerebral cortex, whereas expression of the mGlu 2/3 receptors, the NR1 subunit of NMDA receptors, and the GluR1 subunit of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors was unchanged. mGlu1 and mGlu5 receptor signalling was also impaired in postanoxic rats, as revealed by a reduced efficacy of the agonist (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) to stimulate polyphosphoinositide hydrolysis in hippocampal slices. We conclude that rats subjected to subneurotoxic doses of anoxia during the early post-natal life develop behavioural symptoms that are frequently encountered in the inattentive subtype of the attention deficit hyperactivity disorder, and that group-I mGlu receptors may be involved in the pathophysiology of these symptoms.     

高压氧治疗创伤性脑损伤的效用及机制研究

目的:研究高压氧(HBO)对大鼠创伤性脑损伤(TBI)治疗效用并观察脑组织星形胶质细胞活化及胶质细胞源性神经营养因子(GDNF)和神经生长因子(NGF)表达的变化以探讨作用机制。方法:SD雄性大鼠54只,随机分为3组(n=18):假手术组、TBI组和HBO治疗组。采用Feeney法建立大鼠TBI模型,假手术组只开放骨窗,不予打击。HBO治疗组大鼠于脑损伤后6 h采用动物高压舱,以3ATA压力纯氧治疗60 min。TBI后48 h测量神经功能,然后分离脑组织,其中18只用干湿法测定脑含水量;18只脑组织用于切片,部分进行尼氏染色后作形态学观察,部分进行免疫组织化学染色,检测星形胶质细胞标记物胶质纤维酸性蛋白(GFAP)、波形蛋白(vimentin)与S100蛋白的表达;另18只大鼠取伤侧脑半球,进行Western blot分析,观察GDNF和NGF的表达。结果:HBO治疗能减轻神经功能障碍,降低脑含水量,减少海马部位神经细胞丢失,进一步激活损伤侧皮质与海马部位GFAP、vimentin与S-100阳性表达星形胶质细胞,促进损伤侧脑组织GDNF与NGF的表达。结论:HBO对创伤性脑损伤有较好治疗效果,其机制与上调GDNF和NGF的表达有关。     

Morphological and physiological studies of rat kidney cortex slices undergoing isosmotic swelling and its reversal: A possible mechanism for ouabain-resistant control of cell volume

Summary Slices of rat kidney cortex were induced to swell by preincubation at 1°C in an isotonic Ringer's solution, and their capacity to reverse swelling, by net extrusion of cellular water, was studied during subsequent incubation at 25°C. The recovery from swelling was prevented by the respiratory inhibitor, antimycin A. On the other hand, extrusion of water was little affected by ouabain. The extrusion of water continuing in the presence of ouabain (but not that in its absence) was significantly reduced when furosemide was added or when medium Cl^– was replaced by NO ₃ ^– , or I^–. There was substantial variability in the morphological appearance of cells within the cortical slices. Different segments of the nephron showed different structural changes during swelling and its reversal, the proximal tubules being most markedly affected. Proximal tubular cells of swollen slices showed disorganization of brush borders and expansion of their apical surfaces, and contained vesicles in their apical cytoplasm. Upon recovery at 25°C, the apical portions of these cells showed reversal of the expansion, but some apical vesicles remained. These vesicles were much more numerous after recovery in the presence of ouabain, but they were much reduced in numbers, or totally absent, when recovery took place in the presence of furosemide or absence of Cl^–, with or without ouabain. The vesicles seen in the presence of ouabain alone appeared to fuse with each other and with infoldings of the basolateral plasma membrane. Rather similar results were obtained with distal tubular cells in the slices. We suggest that volume regulation in the proximal and distal tubular cells proceeds by way of two mechanisms. The first consists of extrusion of water coupled to the ouabain-sensitive transport of Na⁺ and K⁺. The other proceeds by way of an ouabain-resistant entry of water into apical cytoplasmic vesicles, following furosemide-sensitive movements of Cl^– and Na⁺; the vesicles then expel their contents by exocytosis at the basolateral cell borders.     

The involvement of glycogen synthase kinase-3 and protein phosphatase-2A in lactacystin-induced tau accumulation

Here, we demonstrated that lactacystin inhibited proteasome dose-dependently in HEK293 cells stably expressing tau. Simultaneously, it induces accumulation of both non-phosphorylated and hyperphosphorylated tau and decreases the binding of tau to the taxol-stabilized microtubules. Lactacystin activates glycogen synthase kinsase-3 (GSK-3) and decreases the phosphorylation of GSK-3 at serine-9. LiCl inhibits GSK-3 and thus reverses the lactacystin-induced accumulation of the phosphorylated tau. Lactacystin also inhibits protein phosphase-2A (PP-2A) and it significantly increases the level of inhibitor 1 of PP-2A. These results suggest that inhibition of proteasome by lactacystin induces tau accumulation and activation of GSK-3 and inhibition of PP-2A are involved.     

Effects of oxidative stress on phospholipid signaling in rat cultured astrocytes and brain slices

Although reactive oxygen species (ROS) are conventionally viewed as toxic by-products of cellular metabolism, a growing body of evidence suggests that they may act as signaling molecules. We have studied the effects of hydrogen peroxide (H(2)O(2))-induced oxidative stress on phospholipid signaling in cultured rat cortical astrocytes. H(2)O(2) stimulated the formation of phosphatidic acid and the accumulation of phosphatidylbutanol, a product of the phospholipase D (PLD)-catalyzed transphosphatidylation reaction. The effect of exogenous H(2)O(2) on the PLD response was mimicked by menadione-induced production of endogenous H(2)O(2). Oxidative stress also elicited inositol phosphate accumulation resulting from phosphoinositide phospholipase C (PLC) activation. The PLD response to H(2)O(2) was totally suppressed by chelation of both extracellular and cytosolic Ca(2+) with EGTA and BAPTA/AM, respectively. Furthermore, H(2)O(2)-induced PLD stimulation was completely abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide and chelerythrine and by PKC down-regulation. Activation of PLD by H(2)O(2) was also inhibited by the protein-tyrosine kinase inhibitor genistein. Finally, H(2)O(2) also stimulated both PLC and PLD in rat brain cortical slices. These results show for the first time that oxidative stress elicits phospholipid breakdown by both PLC and PLD in rat cultured astrocytes and brain slices.