Differential changes in phosphorylation of tau at PHF-1 and 12E8 epitopes during brain ischemia and reperfusion in gerbils.

Cortical neurons are vulnerable to ischemic insult, which may cause cytoskeletal changes and neurodegeneration. Tau is a microtubule-associated protein expressed in neuronal and glial cells. We examined the phosphorylation status of tau protein in the gerbil brain cortex during 5 min ischemia induced by bilateral common carotid artery occlusion followed by reperfusion for 20 min to 7 days. Control brain homogenates contained 63, 65 and 68 kD polypeptides of tau immunoreactive with Alz 50, Tau 14 and Tau 46 antibodies raised against non-phosphorylated tau epitopes. Gerbil tau was also immunoreactive with some (PHF-1 and 12E8) but not all (AT8, AT100, AT180 and AT270) antibodies raised against phosphorylated tau epitopes. PHF-1 recognized a single 68 kD polypeptide and 12E8 bound the 63 kD polypeptide. During 5 min ischemia, PHF-1 immunoreactivity declined to 6%, then recovered to control levels after 20 min of blood recirculation and subsequently increased above control values 3 and 7 days later. In contrast, 12E8 immunoreactivity remained stable during ischemia and reperfusion. Our results suggest that the two phosphorylated epitopes of tau are regulated by different mechanisms and may play different roles in microtubule dynamics. They may also define various pools of neuronal/glial cells vulnerable to ischemia.

Excitotoxicity-induced expression of amyloid precursor protein (beta-APP) in the hippocampus and cortex of rat brain. an electron-microscopy and biochemical study.

After stereotaxic microinjection of N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methyl isoxazole-4-propionic acid (AMPA) to CA1 of rat hippocampus, the animals were sacrificed: 0 h, 2 h, 12 h, 24 h and 3 days after the insult. Other groups of animals before microinjection of the excitotoxins received intraperitoneal injection of dizocilpine (MK-801). Expression of beta-APP was assessed by immunohistochemical and immunobiochemical methods, and the results were correlated with changes in tissue ultrastructure observed in the electron microscopy. The results of the immunochemical analyses study demonstrated that application of NMDA and AMPA resulted in the increase of the expression of beta-APP in the CA1 of hippocampus and to a less extent in the cortex. Pretreatment with MK-801 strongly suppressed this effect. Beta-amyloid release was not detected. Morphological and cytochemical study revealed that NMDA injection induced massive damage of hippocampal and cortical neurones, associated with mitochondrial calcium sequestration and unusual accumulation of neurofilaments. Ultrastructural changes after AMPA application were limited to the brain cortex. These data indicate that although excitotoxic insult induces hyperexpression of beta3-APP, there is no relation between this effect and neurodegeneration, and excitotoxicity does not induce amyloidogenic processing of beta-APP.

Induction of Alzheimer-specific Tau epitope AT100 in apoptotic human fetal astrocytes.

In Alzheimer's and other neurodegenerative diseases, hyperphosphorylated tau accumulates in affected neuronal and glial cells in the form of paired helical filaments (PHFs). This tau binds antibody AT100, which recognizes the double phosphorylation site (Thr212/Ser214) that is not present in normal biopsy tau. In primary cultures, highly enriched (>98%) in astrocytes of human fetal brain, three polypeptides of 52, 64, and 70 kD showed immunoreactivity with tau antibodies against non-phosphorylated epitopes, accounting for 88, 12, and <1%, respectively, of the total reactivity. All three polypeptides were phosphorylated at the PHF-1 epitope but not at the epitopes Tau-1, 12E8, AT8, and AT100. Treatment of cultures with okadaic acid resulted in apoptosis characterized by the blebbing of the plasma membrane, condensation of nuclear chromatin, and fragmentation of the nucleus. This treatment also resulted in a 3- to 5-fold increase in the content of both tau protein and phosphorylation. The increases were observed in all phosphorylation sites examined, and included the AT100 site. The AT100 site has been proposed to be generated by protein kinase B/Akt and Cdc2. Since okadaic acid can induce an AD-like hyperphosphorylated state of normal tau in primary cultures of human brain cells, a simple cellular model is available permitting study of self-aggregation of tau and phosphorylation events characteristic of neurodegeneration.

Conformation of paired helical filaments blocks dephosphorylation of epitopes shared with fetal tau except Ser199/202 and Ser202/Thr205.

To determine if the high phosphate content of paired helical filaments (PHFs) in Alzheimer's disease (AD) is a result of limited access to filament phosphorylation sites, we studied in vitro dephosphorylation of intact PHFs, PHFs with filamentous structure abolished by formic acid treatment (PHF(FA)) and fetal human tau protein. Samples were treated with alkaline phosphatase for up to 24 h at 37 degrees C and then immunoblotted with eight well characterized tau antibodies, that recognize two phosphorylation-insensitive sites and six phosphorylation-sensitive epitopes at Thr181, Ser199/202, Ser202/Thr205, Thr231, Ser262/356 and Ser396/404. Intact PHFs were effectively dephosphorylated only at the two N-terminal epitopes Ser199/202 and Ser202/Thr205, with little change in electrophoretic mobility. In contrast, PHF(FA) were dephosphorylated at all epitopes, with particular effectiveness at those in the C-terminus and with significant increase in electrophoretic mobility. The fetal tau epitopes were effectively dephosphorylated except at Thr181 and Thr231 with marked increase in mobility. The extent of dephosphorylation of PHF(FA) was equal or more effective than in fetal tau, except for Thr181 that was minimally dephosphorylated in both proteins. The results indicate that intact PHFs, but not PHF(FA) or fetal tau display differential dephosphorylation of the N- and C-terminal epitopes. The results confirm that the filamentous conformation may significantly contribute to hyperphosphorylation of PHFs in the C-terminus. The filamentous conformation, however, does not limit access to two N-terminal epitopes Ser199/202 and Ser202/Thr205. The access to these sites in AD may be limited by other factors, e.g., inhibition of phosphatase binding.

Differences between rats and rabbits in NMDA receptor-mediated calcium signalling in hippocampal neurones.

In vivo microdialysis combined with the measurement of (45)Ca(2+) efflux from prelabelled hippocampus demonstrated a pronounced N-methyl-D-aspartate (NMDA)-evoked (45)Ca(2+) release to the dialysate in the rat dentate gyrus (DG) and CA1, whereas in rabbit a slight release of (45)Ca(2+) was observed only in the DG. In vitro, we noticed that the NMDA-evoked increase in Fura-2 detected intracellular Ca(2+) concentration in synaptoneurosomes from the rat, but not from the rabbit hippocampus, was strongly inhibited by the ryanodine receptor (RyR) antagonists dantrolene and ryanodine. To establish the mechanism of these differences, we characterised their possible dependence on the expression of RyR and their co-localisation with the calcium binding protein calbindin D(28k). A pronounced expression of [(3)H]ryanodine binding sites in the rat DG, which is only slight in the CA1, was demonstrated whereas in rabbit they were only found in the DG. The pattern of expression of calbindin D(28k) immunoreactivity and RyR in the rat and rabbit hippocampus was similar. These results suggest that the functional role of RyR in the generation of the NMDA receptor-mediated intracellular Ca(2+) signalling in the rabbit hippocampal neurones is marginal when compared to the rat. These differences reflect a diverse expression of RyR in both species. The corresponding differences in calbindin D(28k) immunoreactivity are most probably secondary in nature.

Hydroxylamine attenuates the effects of simulated subarachnoid hemorrhage in the rat brain and improves neurological outcome.

Some of the neurological deficits that emerge after aneurysmal subarachnoid hemorrhage (SAH) in humans are presumably caused by ischemic brain damage consequential to SAH-induced delayed cerebral vasospasm. This vasospasm probably results from an imbalance among vasoactive factors released from both the clot formed by extravasated blood and adjacent tissues, and in particular from a decrease in the endothelium-derived relaxing factor nitric oxide (NO). Brain ischemia is also known to elevate brain production and deposition of beta-amyloid, and to induce a delayed increase in total NO synthase (NOS) activity due to induction of expression of so-called induced NOS isoform, phenomena that may secondarily contribute to SAH-related brain damage. The aim of this study was to investigate the effects of treatment with the intracellular NO donor hydroxylamine on: (i) basilar arterial wall that remained in a direct contact with the clot, (ii) formation of the beta-amyloid precursor protein (beta-APP), (iii) total brain NOS activity, and (iv) neurological outcome in a 'two-hemorrhage' rat SAH model. Intraperitoneal (i.p.) administration of 0.18 mmol/kg hydroxylamine hydrochloride (12.5 mg/kg) twice daily for 7 days beginning immediately after the first 'hemorrhage' (intracisternal blood injection) reduced basilar arterial wall damage and attenuated post-SAH neurological deficit. It also reduced the SAH-related increases in hippocampal and cortical beta-APP immunoreactivities and hippocampal NOS activity measured 24 h after commencement of the treatment. These results indicate that intracellular NO donors that yield NO through the action of widely distributed enzymes in brain cells (cytochromes, catalase) can attenuate detrimental effects of SAH.

Changes in oxidative stress in the rat brain during post-cardiac arrest reperfusion, and the effect of treatment with the free radical scavenger idebenone.

The study was designed to determine the effect of idebenone, an electron-trapping agent and free radical scavenger capable of crossing the blood-brain barrier, on cardiac arrest-induced oxidative brain stress. Stress indices used were the brain contents of thiobarbituric acid-reactive material (TBAR), conjugated dienes and protein and non-protein thiols. Twenty-four hours after receiving one oral dose of placebo or 100 mg kg(-1) idebenone, the rats were anaesthetized with diethyl ether and either decapitated immediately, or subjected to 7.5 min cardiac arrest induced by compression of the heart vessel bundle. The next groups of rats were sacrificed at the end of the cardiac arrest session, or resuscitated by external chest compression and artificial ventilation with air and sacrificed 15 min, 60 min, 24 h, and 72 h later while re-anesthetized with diethyl ether. Subsequent placebo or idebenone (100 mg kg(-1)) doses were given to the appropriate surviving rats once daily, beginning 8-10 min after the end of cardiac arrest session. Compared to pre-arrest values, TBAR and conjugated dienes' contents increased, respectively, by 339 and 286%, and protein and non-protein thiol contents decreased, respectively, by 69 and 85% within 60 min after the resuscitation in placebo-treated rats. Normalization of all oxidative stress indices in these rats was slow and incomplete even at 72 h. Idebenone treated rats showed no increase in TBAR contents, and a marked attenuation of changes in the other indices. These results show that oral idebenone greatly reduces oxidative brain stress following transient circulatory arrest in the rat. This effect could not be explained by simple stoichiometric scavenging of free radicals. Possible mechanisms of idebenone action are discussed.

Tau released from paired helical filaments with formic acid or guanidine is susceptible to calpain-mediated proteolysis.

Paired helical filaments (PHFs), a characteristic neuropathologic finding in Alzheimer's disease brain, are abnormal fibrillary forms of hyperphosphorylated tau (PHF-tau), which have been shown to be highly resistant to calpain digestion. Either excessive phosphorylation or fibrillary arrangement of tau proteins in PHFs may play a role in proteolytic resistance by limiting access to calpain recognition/digestion sites. To determine the contribution of the fibrillary conformation, isolated PHFs were subjected to treatment with either formic acid or guanidine. Both procedures effectively abolished the fibrillary structure of PHF but preserved PHF-tau immunoreactivity using a panel of antibodies that recognize nonphosphorylated and phosphorylated epitopes. These treatments also significantly increased the sensitivity of PHF-tau polypeptides to calpain proteolysis as shown by significant decreases in the half-life (t(1/2)) from the infinite with native PHF to 44 min and 4.4 min in formic acid- or guanidine-treated samples, respectively. In contrast, the sensitivity of normal fetal tau (3.4 min) was either decreased (5.9 min) or unaffected (3.6 min) by similar treatment. Our results indicate that after guanidine treatment, the sensitivity of PHF to calpain resembles that of fetal tau. These results strongly suggest that the fibrillary structure of PHF-tau, rather than hyperphosphorylation, is the major factor responsible for the resistance of abnormal filaments to calpain-mediated proteolysis.