Kinases and phosphatases and tau sites involved in Alzheimer neurofibrillary degeneration

Microtubule associated protein (MAP) tau is abnormally hyperphosphorylated in Alzheimer's disease (AD) and related tauopathies; in this form it is the major protein subunit of paired helical filaments (PHF)/neurofibrillary tangles. However, the nature of protein kinases and phosphatases and tau sites involved in this lesion has been elusive. We investigated self-assembly and microtubule assembly promoting activities of hyperphosphorylated tau isolated from Alzheimer disease brain cytosol, the AD abnormally hyperphosphorylated tau (AD P-tau) before and after dephosphorylation by phosphoseryl/phosphothreonyl protein phosphatase-2A (PP-2A), and then rephosphorylation by cyclic AMP-dependent protein kinase (PKA), calcium, calmodulin-dependent protein kinase II (CaMKII), glycogen synthase kinase-3beta (GSK-3beta) and cyclin-dependent protein kinase 5 (cdk5) in different kinase combinations. We found that (i) dephosphorylation of AD P-tau by PP-2A inhibits its polymerization into PHF/straight filaments (SF) and restores its binding and ability to promote assembly of tubulin into microtubules; (ii) rephosphorylation of PP-2A-dephosphorylated AD P-tau by sequential phosphorylation by PKA, CaMKII and GSK-3beta or cdk5, and as well as by cdk5 and GSK-3beta, promotes its self-assembly into tangles of PHF similar to those seen in Alzheimer brain, and (iii) phosphorylation of tau sites required for this pathology are Thr231 and Ser262, along with several sites flanking the microtubule binding repeat region. Phosphorylation of recombinant human brain tau(441) yielded similar results as the PP-2A dephosphorylated AD P-tau, except that mostly SF were formed. The conditions for the abnormal hyperphosphorylation of tau that promoted its self-assembly also induced the microtubule assembly inhibitory activity. These findings suggest that activation of PP-2A or inhibition of either both GSK-3beta and cdk5 or one of these two kinases plus PKA or CaMKII might be required to inhibit Alzheimer neurofibrillary degeneration.     

Phosphorylation of tau protein over time in rats subjected to transient brain ischemia

Transient brain ischemia has been shown to induce hyperphosphorylation of the micro- tubule-associated protein tau. To further determine the mechanisms underlying these processes, we investigated the interaction between tau, glycogen synthase kinase （GSK）-313 and protein phos- phatase 2A. The results confirmed that tau protein was dephosphorylated during brain ischemia; in addition, the activity of GSK-3β was increased and the activity of protein phosphatase 2A was de- creased. After reperfusion, tau protein was hyperphosphorylated, the activity of GSK-3β was de- creased and the activity of protein phosphatase 2A remained low. Importantly, the interaction of tau with GSK-3β and protein phosphatase 2A was altered during ischemia and reperfusion. Lithium chloride could affect tau phosphorylation by regulating the interaction of tau with GSK-3β and pro- tein phosphatase 2A, and improve learning and memory ability of rats after transient brain ischemia. The present study demonstrated that it was the interaction of tau with GSK-3β and protein phos- phatase 2A, rather than their individual activities, that dominates the phosphorylation of tau in tran- sient brain ischemia. Hyperphosphorylated tau protein may play an important role in the evolution of brain injury in ischemic stroke. The neuroprotective effects of lithium chloride partly depend on the inhibition of tau phosphorylation during transient brain ischemia.     

脑缺血时NMDA受体通过Src激酶和Ca2+/钙调蛋白依赖性蛋白激酶Ⅱ调控ERKs激活

目的ERKs是钙依赖性激活蛋白,本研究旨在探讨钙依赖性蛋白激酶是否参与了脑缺血后ERK级联的调控。方法采用四动脉结扎诱导大鼠前脑缺血,用免疫印迹的方法观察几个钙依赖性蛋白激酶含量及活性的变化。结果致死性脑缺血以NMDA受体依赖的方式激活ERKs,并差异性上调Src和Ca^2＋／钙调蛋白依赖性蛋白激酶Ⅱ（CaMKⅡ）的活性。Src激酶和CaMKⅡ的抑制剂PP2和KN62能显著的阻止缺血诱导的ERKs激活。然而,缺血诱导的Src过度激活也伴随着ERKs的活性抑制。结论致死性脑缺血刺激NMDA受体通过Src激酶和CaMKⅡ介导ERKs活性上调,但是脑缺血诱导的Src过度激活可能也参与了ERKs信号通路的负性调控。     

Activation of PP2A-like phosphatase and modulation of tau phosphorylation accompany stress-induced apoptosis in cultured oligodendrocytes

In a number of neurodegenerative diseases, tau-positive glial cytoplasmic inclusions (GCIs), immunochemically labeled with antibodies to the small heat shock protein (HSP) alphaB-crystallin, occur in oligodendrocytes. The microtubule-associated protein tau is functionally modulated by phosphorylation. We have shown previously that oxidative stress (OS) and heat shock (HS) induce apoptotic cell death in oligodendrocytes. The present study was undertaken to test whether stress responses in oligodendrocytes cause abnormalities in the expression and posttranslational modification of tau proteins, and whether the dynamic phosphorylation and dephosphorylation of tau are involved in the pathogenesis of glial cells. Cultured rat brain oligodendrocytes were subjected to OS, exerted by hydrogen peroxide, or HS (44 degrees C, 30 min). Immunoblot analysis with a panel of phosphorylation-dependent antibodies shows that OS and HS caused the rapid dephosphorylation of tau proteins at multiple sites, before characteristic features of apoptosis were observed. Concomitantly, ERK1,2 (extracellular signal-regulated kinase) was activated. Tau phosphorylation and rephosphorylation after stress was mediated by glycogen synthase kinase 3beta (GSK-3beta), and not by ERK1,2 and could be suppressed by lithium chloride, a specific inhibitor of GSK-3beta. Stress-induced dephosphorylation could be mimicked by alkaline phosphatase and suppressed by the protein phosphatase inhibitor okadaic acid (OA), indicating that PP2A in oligodendrocytes is activated by stress. OA at low concentrations could prevent stress-induced DNA fragmentation, but eventually exerted cytotoxic effects. Hence, stress-induced activation of PP2A in oligodendrocytes and tau dephosphorylation constitute a major feature of the response to injury in these cells, which eventually undergo apoptotic cell death.     

脑缺血时NMDA受体通过Src激酶和Ca~(2+)/钙调蛋白依赖性蛋白激酶II调控ERKs激活(英文)

目的ERKs是钙依赖性激活蛋白,本研究旨在探讨钙依赖性蛋白激酶是否参与了脑缺血后ERK级联的调控。方法采用四动脉结扎诱导大鼠前脑缺血,用免疫印迹的方法观察几个钙依赖性蛋白激酶含量及活性的变化。结果致死性脑缺血以NMDA受体依赖的方式激活ERKs,并差异性上调Src和Ca2+/钙调蛋白依赖性蛋白激酶II(CaMKII)的活性。Src激酶和CaMKII的抑制剂PP2和KN62能显著的阻止缺血诱导的ERKs激活。然而,缺血诱导的Src过度激活也伴随着ERKs的活性抑制。结论致死性脑缺血刺激NMDA受体通过Src激酶和CaMKII介导ERKs活性上调,但是脑缺血诱导的Src过度激活可能也参与了ERKs信号通路的负性调控。     

In vivo activation and nuclear translocation of phosphorylated glycogen synthase kinase-3beta in neuronal apoptosis: links to tau phosphorylation

The roles of glycogen synthase kinase-3beta (GSK-3beta) and tau phosphorylation were examined in seven-day-old rats injected with the NMDA receptor antagonist (MK801) that is known to induce neuronal apoptosis. Immunoblot and immunohistochemical analysis of brain samples demonstrated a site-specific increase in tau phosphorylation associated with the relocalization of the protein to the nuclear/perinuclear region of apoptotic neurons. In addition, a tau 32-kDa fragment was detected, suggesting that tau was a target of intracellular proteolysis in MK801-treated brains. The proteolytically modified form of tau has reduced ability to bind to microtubules. GSK-3beta kinase assay and immunoblottings of active (tyrosine-216) and inactive (serine-9) forms of GSK-3beta revealed a rapid and transient increase in the kinase activity. Lithium chloride, a GSK-3beta inhibitor, prevented tau phosphorylation suggesting that tau phosphorylation is mediated by the activation of GSK-3beta. Confocal microscopy using double labelling of tau and GSK-3beta revealed that the activation of GSK-3beta in neurons was associated with early (2 h) nuclear translocation of tyrosine-216 GSK-3beta. The execution phase of neuronal apoptosis was accompanied by a selective phosphorylation of serine-9 and dephosphorylation of tyrosine-216 GSK-3beta. These findings demonstrate that in vivo, GSK-3beta kinase activation and nuclear translocation are early stress signals of neuronal apoptosis.     

PSD-95 promotes CaMKII-catalyzed serine phosphorylation of the synaptic RAS-GTPase activating protein SynGAP after transient brain ischemia in rat hippocampus

Recent studies have indicated that cerebral ischemia induces rapid serine phosphorylation of synaptic RAS-GTPase activating protein (SynGAP) by calcium/Camodulin-dependent protein kinase II (CaMKII) in rat hippocampus. To further illustrate the mechanisms underlying these processes, we examined the effects of transient (15 min) brain ischemia followed by reperfusion (0, 30 min, 6 h, 1, 3 days) on serine phosphorylation of SynGAP and interactions involving SynGAP, postsynaptic density protein 95 (PSD95) and CaMKII in rat hippocampus. Transient brain ischemia was induced by the method of four-vessel occlusion in Sprague-Dawley rats. Serine phosphorylation of SynGAP increased immediately after brain ischemia and peaked at 30-min reperfusion, and the increase was maintained for 3 days. The association among SynGAP, PSD95 and CaMKII had a similar trend as serine phosphorylation of SynGAP. Intracrebroventricular infusion of PSD95 antisense oligodeoxynucleotide not only markedly decreased the protein levels of PSD95 but also attenuated the elevated serine phosphorylation of SynGAP and the associations among SynGAP, PSD95 and CaMKII induced by 30-min reperfusion following 15-min brain ischemia. The results suggest that the serine phosphorylation of SynGAP catalyzed by CaMKII is immediately increased and that PSD95 is critical for promoting SynGAP serine phosphorylation after transient brain ischemia.     

Pharmacological approaches of neurofibrillary degeneration

Alzheimer disease (AD) and related tauopathies are all characterized histopathologically by neurofibrillary degeneration. The neurofibrillary changes, whether of paired helical filaments (PHF), twisted ribbons or straight filaments (SF) are made up of abnormally hyperphosphorylated tau. Unlike normal tau which promotes assembly and maintains structure of microtubules, the abnormal tau not only lacks these functions but also sequesters normal tau, MAP1 and MAP2, and causes disassembly of microtubules. This toxic behavior of the abnormal tau is solely due to its hyperphosphorylation because dephosphorylation restores it into a normal-like protein. The abnormal hyperphosphorylation also promotes the self-assembly of tau into PHF/SF. The state of phosphorylation of a phosphoprotein is the function of the activities of protein kinases and as well as of protein phosphatases that regulate the level of phosphorylation. A cause of the abnormal hyperphosphorylation in AD brain is a decrease in the activity of protein phosphatase (PP)-2A, a major regulator of the phosphorylation of tau. A decrease in PP-2A activity results in the abnormal hyperphosphorylation of tau not only by decreased dephosphorylation of tau but also by stimulating the activities of tau kinases like CaMKII, PKA and MAP kinases which are regulated by PP-2A. Thus, the abnormal hyperphosphorylation can be inhibited both by inhibition of the activity/s of a tau protein kinase and as well as by restoration of the activity/s of a tau protein phosphatase. The development of drugs that inhibit neurofibrillary degeneration is a very promising and feasible therapeutic approach to inhibit the progression of AD and related tauopathies.     

Tau hyperphosphorylation correlates with reduced methylation of protein phosphatase 2A

The down-regulation of protein phosphatase 2A (PP2A) activity is thought to play an important role in the formation of tau hyperphosphorylation in the Alzheimer's disease (AD) brain. Methylation of the PP2A catalytic subunit at the L309 site can potently activate PP2A for some substrates via the increasing recruitment of its regulatory subunits into the holoenzyme. Abeta is overproduced yet estrogen is deficient in the brains of the menopausal AD patients. Both Abeta and estrogen deficiency can interact with tau kinases such as protein kinase B and glycogen synthase kinase 3. In the current study, levels of demethylated (-m) PP2A (L309) were significantly increased, and methylated (+m) PP2A (L309) were significantly decreased, which corresponded with the increased tau phosphorylation at the Tau-1 and PHF-1 sites in both mouse N2a cells carrying the human APP with Swedish mutation (APPswe) and transgenic APPswe/presenilin (PS) 1 (A246E) mice. These findings were replicated in wild-type N2a cells treated with Abeta25-35, and to a relatively larger extent, in both wild-type N2a cells and APPswe treated by okadaic acid, as well as in the brains of estrogen receptor (ER) alpha-/- and ERbeta-/- mice that mimic the status of estrogen deficiency in menopausal AD patients. Together, these findings suggested that the increased demethylation of PP2A (L309) mediated by Abeta overproduction or estrogen deficiency (ERalpha-/- and ERbeta-/-) may contribute to the reduced PP2A activity observed in the AD brain, resulting in the compromised dephosphorylation of abnormally hyperphosphorylated tau.     

Enriched environment prevents cognitive impairment and tau hyperphosphorylation after chronic cerebral hypoperfusion

Chronic cerebral hypoperfusion (CCH) has been gradually prevalent in the patients over middle age, especially the old over 60 years. It has been proved that CCH is highly related with cognitive impairment. CCH emerges not only in vascular dementia (VaD), but also in Alzheimer's disease (AD), which regarded as a critical causative for cognitive impairment in these diseases. Nevertheless, the mechanisms underlying cognitive deficit remain elusive. Moreover, there are no dramatically effective preventions. In the present study, by employing a recognized CCH rat model, we found that CCH induced spatial learning/memory deficits with simultaneously increasing tau hyperphosphorylation at multiple Alzheimer-related phosphorylation sites with activation of glycogen synthase kinase-3β (GSK-3β), Cyclin-dependent kinase (Cdk5), Calcium/calmodulin-dependent protein kinase II (CaMKII), and protein kinase B (Akt), and inhibition of protein phosphatase (PP) 2A (PP-2A). Interestingly, enriched environment (EE) treatment, an effect environment stimuli filled with various novel objects, could prevent rats from the EE-induced memory deficits and alterations of tau hyperphosphorylation. Our data suggested that EE might be potentially used for attenuating the detrimental cognition induced by CCH through regulating tau hyperphosphorylation.     

Over activation of hippocampal serine/threonine protein phosphatases PP1 and PP2A is involved in lead-induced deficits in learning and memory in young rats

Serine/threonine protein phosphatases regulate several key cellular events in the brain, including learning and memory. These enzymes, when over-activated, are known to function as a constraint on learning and memory. We investigated whether these phosphatases are implicated in lead (Pb)-induced deficits in learning and memory. Wistar rat pups were exposed to 0.2% Pb-acetate via their dams' drinking water from postnatal day (PND) 1-21 and directly in drinking water until PND 30. Pb levels in blood, brain and hippocampus were measured and expression of PP1, PP2A, PP2B and PP5 in hippocampus was analyzed. Total phosphatase activity, and PP1 and PP2A activities were determined. Tau phosphorylation at various epitopes was determined by Western blot. Spatial learning and memory was determined by Morris water maze test. Pb exposure significantly increased levels of Pb in blood, brain and hippocampus, reduced the number of synapses in hippocampus and impaired learning and long-term memory (LTM). Short-term memory (STM) was only affected in rats at PND21. Pb exposure increased the expression and activity of PP1 and decreased phosphorylation of tau at threonine-231 in hippocampus at both PND21 and PND30. Pb-induced phosphorylation of tau at serine-199/202 (AT8) paralleled with PP2A activity; at PND21 PP2A activity increased and AT8 phosphorylation decreased; at PND30 PP2A activity decreased and AT8 phosphorylation increased. Increased PP1 activity in hippocampus by Pb is associated with learning and LTM impairment, whereas, increased PP2A activity is associated with STM impairment. These findings suggest the overactivation of PP1 and PP2A, together with changes in tau phosphorylation, as a potential mechanism of lead-induced deficits in learning and memory.     

Contributions of protein phosphatases PP1, PP2A, PP2B and PP5 to the regulation of tau phosphorylation

Abnormal hyperphosphorylation of tau is believed to lead to neurofibrillary degeneration in Alzheimer's disease (AD) and other tauopathies. Recent studies have shown that protein phosphatases (PPs) PP1, PP2A, PP2B and PP5 dephosphorylate tau in vitro, but the exact role of each of these phosphatases in the regulation of site-specific phosphorylation of tau in the human brain was unknown. Hence, we investigated the contributions of these PPs to the regulation of tau phosphorylation quantitatively. We found that these four phosphatases all dephosphorylated tau at Ser199, Ser202, Thr205, Thr212, Ser214, Ser235, Ser262, Ser396, Ser404 and Ser409, but with different efficiencies toward different sites. The K(m) values of tau dephosphorylation catalysed by PP1, PP2A and PP5 were 8-12 microm, similar to the intraneuronal tau concentration of human brain, whereas the K(m) of PP2B was fivefold higher. PP2A, PP1, PP5 and PP2B accounted for approximately 71%, approximately 11%, approximately 10% and approximately 7%, respectively, of the total tau phosphatase activity of human brain. The total phosphatase activity and the activities of PP2A and PP5 toward tau were significantly decreased, whereas that of PP2B was increased in AD brain. PP2A activity negatively correlated to the level of tau phosphorylation at the most phosphorylation sites in human brains. Our findings indicate that PP2A is the major tau phosphatase that regulates its phosphorylation at multiple sites in human brain. The abnormal hyperphosphorylation of tau is partially due to a downregulation of PP2A activity in AD brain.     

17β-estradiol attenuates glycogen synthase kinase-3β activation and tau hyperphosphorylation in Akt-independent manner

Decline of estrogen is associated with high incidence of Alzheimer's disease (AD) characterized pathologically with tau hyperphosphorylation, and glycogen synthase kinase-3beta (GSK-3beta) is a major tau kinase. However, the role of estrogen on GSK3beta-induced tau hyperphosphorylation is elusive. Here, we treated N2a cells with wortmannin (Wort) and GF-109203X (GFX) or gene transfection to activate GSK-3beta and to induce tau hyperphosphorylation and then the effects of 17beta-estradiol (betaE2) on tau phosphorylation and GSK-3beta activity were studied. We found that betaE2 could attenuate tau hyperphosphorylation at multiple AD-related sites, including Ser396/404, Thr231, Thr205, and Ser199/202, induced by Wort/GFX or transient overexpression of GSK-3beta. Simultaneously, it increased the level of Ser9-phosphorylated (inactive) GSK-3beta. To study whether the protective effect of betaE2 on GSK-3beta and tau phosphorylation involves protein kinase B (Akt), an upstream effector of GSK-3, we transiently expressed the dominant negative Akt (dnAkt) in the cells. We found that betaE2 could attenuate Wort/GFX-induced GSK-3beta activation and tau hyperphosphorylation with Akt-independent manner. It suggests that betaE2 may arrest AD-like tau hyperphosphorylation by directly targeting GSK-3beta.     

丝裂原活化蛋白激酶ERKs和JNKs在脑缺血损伤中的差异激活及其调控机制

目的　探讨丝裂原活化蛋白激酶家族 (MAPK)两成员ERK1/2和JNK1/2在全脑缺血损伤中的激活及其可能的分子机制。 方法　 采用四动脉结扎模型诱导SD大鼠前脑缺血 ,免疫印迹的方法观察ERK1/2和JNK1/2蛋白激酶特异性Thr和Tyr双位点磷酸化的变化及NMDA受体选择性拮抗剂对其双磷酸化的影响。结果　 缺血诱导海马脑区MAPK家族蛋白激酶两成员显著去磷酸化 ,严重缺血 (30min)ERK1/2而不是JNK1/2活性反弹 ;缺血再灌注ERK1/2活性在 15min首先升至最高而JNK1/2 1h后才逐渐升至峰值 (P <0 .0 5 ) ,2 4h再灌注能诱导两者的再次激活 ,且氯胺酮能显著抑制缺血诱导的ERK1/2而不是JNK1/2的激活。 结论　 前脑缺血明显诱导ERK1/2和JNK1/2的差异激活 ,提示两者可能分享不同的分子机制 ,其中ERK1/2的激活明显与NMDA受体功能上调有关。     

Effects of FK506 on the translocation of protein kinase C and CaM kinase II in the gerbil hippocampal CA1 neurons

To explore biochemical basis for cerebroprotective effect of immunosuppressant FK506, we studied changes in subcellular distribution of protein kinase C gamma (PKC gamma) as well as calcium/calmodulin-dependent protein kinase II (CaMKII) after ischemia. Male Mongolian gerbils were subjected to 5 min forebrain ischemia. FK506 (1 or 3 mg kg-1) was administered at 1 min after recirculation, which was confirmed to be cerebroprotective by histological examination at seven days after ischemia. At the designated time points (before ischemia, 5 min ischemia, 1 and 24 h recovery), heads were frozen and samples were taken from CA1 subfield of hippocampus. Western blot analysis was carried out. Persistent translocations of PKC gamma and CaMKII to synaptosomal P2 fraction were observed in vehicle-treated group. FK506 significantly decreased levels of PKC gamma and CaMKII in P2 fraction at 24 h of recovery. The present results suggest FK506 downregulates translocated PKC gamma and CaMKII, which may contribute to its survival promoting effect after cerebral ischemia.     

Impairment of phosphatase 2A contributes to the prolonged MAP kinase phosphorylation in Alzheimer's disease fibroblasts

The serine/threonine phosphatase 2A (PP2A) has been implicated in the pathogenesis of Alzheimer's disease (AD) due to its important role in regulating dephosphorylation of the microtubule-associated protein tau and mitogen-activated protein (MAP) kinase. In the present study, we show that PP2A was responsible for dephosphorylation of the extracellular signal-regulated kinase 1/2 (Erk1/2) following its activation by BK stimulation. Abnormal gene and protein expressions of PP2A, as well as its activity, were found to contribute to the abnormally prolonged Erk1/2 phosphorylation in the AD fibroblasts. Inhibition of PP2A with okadiac acid produced enhanced and more lasting Erk1/2 phosphorylation after BK stimulation, whereas FK506, an inhibitor of PP2B and FK-binding protein, inhibited the BK-stimulated Erk1/2 phosphorylation. Furthermore, while the phosphorylated Erk1/2 was concentrated in the nucleus of AC cells, it was mainly distributed in the extranuclear compartments of AD cells. These results suggest that the delayed dephosphorylation of Erk1/2 in AD cells following its BK-stimulated activation may be due to deficits of PP2A activity and impaired nuclear translocation of phosphorylated Erk1/2.     

Beneficial Effects of a CaMKIIα Inhibitor TatCN21 Peptide in Global Cerebral Ischemia

Aberrant calcium influx is a common feature following ischemic reperfusion (I/R) in transient global cerebral ischemia (GCI) and causes delayed neuronal cell death in the CA1 region of the hippocampus. Activation of calcium-calmodulin (CaM)-dependent protein kinase IIα (CaMKIIα) is a key event in calcium signaling in ischemic injury. The present study examined the effects of intracerebroventricular (icv) injection of tatCN21 in ischemic rats 3 h after GCI reperfusion. Cresyl violet and NeuN staining revealed that tatCN21 exerted neuroprotective effects against delayed neuronal cell death of hippocampal CA1 pyramidal neurons 10 days post-GCI. In addition, TatCN21 administration ameliorated GCI-induced spatial memory deficits in the Barnes maze task as well as anxiety-like behaviors and spontaneous motor activity in the elevated plus maze and open field test, respectively. Mechanistic studies showed that the administration of tatCN21 decreased GCI-induced phosphorylation, translocation, and membrane targeting of CaMKIIα. Treatment with tatCN21 also inhibited the level of CaMKIIα-NR2B interaction and NR2B phosphorylation. Our results revealed an important role of tatCN21 in inhibiting CaMKIIα activation and its beneficial effects in neuroprotection and memory preservation in an ischemic brain injury model.     

Activation of protein phosphatase 2B and hyperphosphorylation of Tau in Alzheimer's disease

Protein phosphatase 2B (PP2B) is one of the major brain phosphatases and can dephosphorylate tau at several phosphorylation sites in vitro. Previous studies that measured PP2B activity in human brain crude extracts showed that PP2B activity was either unchanged or decreased in Alzheimer's disease (AD) brain. These results led to the speculation that PP2B might regulate tau phosphorylation and that a down-regulation of PP2B might contribute to abnormal hyperphosphorylation of tau. In this study, we immunoprecipitated PP2B from brains of six AD subjects and seven postmortem- and age-matched controls and then measured the phosphatase activity. We found a three-fold increase in PP2B activity in AD brain as compared with control brains. The activation was due to the partial cleavage of PP2B by calpain I that was activated in AD brain. The truncation of PP2B appeared to alter its intracellular distribution in the brain. In human brains, PP2B activity correlated positively, rather than negatively, to the levels of tau phosphorylation at several sites that can be dephosphorylated by PP2B in vitro. Truncation of PP2B in the frontal cortex was more than in the temporal cortex, and tau phosphorylation was also more in the frontal cortex. Taken together, these results indicate that truncation of PP2B by calpain I elevates its activity but does not counteract the abnormal hyperphosphorylation tau in AD brain.     

IGF‐I neuroprotection in the immature brain after hypoxia‐ischemia,involvement of Akt and GSK3β?

Insulin-like growth factor I (IGF-I) is a neurotrophic factor that promotes neuronal growth, differentiation and survival. Neuroprotective effects of IGF-I have previously been shown in adult and juvenile rat models of brain injury. We wanted to investigate the neuroprotective effect of IGF-I after hypoxia-ischemia (HI) in 7-day-old neonatal rats and the mechanisms of IGF-I actions in vivo. We also wanted to study effects of HI and/or IGF-I on the serine/threonine kinases Akt and glycogen synthase kinase 3beta (GSK3beta) in the phophatidylinositol-3 kinase (PI3K) pathway. Immediately after HI, phosphorylated Akt (pAkt) and phosphorylated GSK3beta (pGSK3beta) immunoreactivity was lost in the ipsilateral and reduced in the contralateral hemisphere. After 45 min, pAkt levels were restored to control values, whereas pGSK3beta remained low 4 h after HI. Administration of IGF-I (50 microg i.c.v.) after HI resulted in a 40% reduction in brain damage (loss of microtubule-associated protein) compared with vehicle-treated animals. IGF-I treatment without HI was shown to increase pAkt whereas pGSK3beta decreased in the cytosol, but increased in the nuclear fraction. IGF-I treatment after HI increased pAkt in the cytosol and pGSK3beta in both the cytosol and the nuclear fraction in the ipsilateral hemisphere compared with vehicle-treated rats, concomitant with a reduced caspase-3- and caspase-9-like activity. In conclusion, IGF-I induces activation of Akt during recovery after HI which, in combination with inactivation of GSK3beta, may explain the attenuated activation of caspases and reduction of injury in the immature brain.     

Microtubule assembly competence analysis of freshly-biopsied human tau,dephosphorylated tau,and Alzheimer tau

Phosphorylation of the microtubule-associated protein tau regulates its binding to microtubules; highly phosphorylated tau is also a prime component of paired helical filaments (PHFs) of Alzheimer's disease (AD). Tau from freshly biopsied human, monkey, and rat brain share similar electrophoretic mobility patterns and overlapping phosphorylated epitopes when compared to AD tau isolated from AD brain. We compared the microtubule reassembly competence of fresh isolates of phosphorylated tau to that of maximally dephosphorylated tau and tau from AD brain. A rapid procedure was developed which permitted the enrichment of phosphorylated and dephosphorylated tau from human biopsies in the absence of protein kinase and phosphatase activity. Microtubule assembly assays, using a spectrophotometric measure and purified bovine brain tubulin, were used to correlate assembly competence with states of tau electrophoretic mobility. Maximally dephosphorylated human biopsy-derived tau and monkey tau were assembly competent; tau from AD brain was virtually unable to direct microtubule assembly. Unmodified, biopsy-derived tau from non-AD brain was intermediate in assembly competence relative to AD tau and dephosphorylated tau. Several lines of evidence were used to correlate phosphorylation states of tau with microtubule assembly. First, in vitro dephosphorylation of human biopsy-derived tau with either PP2A or PP2B alone or in combination led to increasing assembly competence as the electrophoretic mobility of tau increased. Second, addition of the protein phosphatase inhibitor okadaic acid (10 μM) to brain-slice preparations slowed electrophoretic mobility of tau and decreased binding competence. We suggest that tau derived from freshly-biopsied brain exists in a range of phosphorylated states, and that dephosphorylation by PP2A and/or PP2B increases microtubules assembly competence. © 1996 Wiley-Liss, Inc.