Mechanisms of Musculoskeletal Frailty in People Living with HIV.

People over age 50 living with HIV experience frailty including functional declines and illnesses usually attributed to aging, more frequently and ten years earlier than people without HIV. As the number of people living with HIV over age 50 is expected to triple by the year 2040, those experiencing early frailty will continue to grow. This review synthesizes the known correlates and contributors to musculoskeletal frailty in people living with HIV. A conceptual model of musculoskeletal frailty in HIV that outlines chronic inflammation, altered energy metabolism, immune activation, and endocrine alterations as mechanisms associated with frailty development is presented. Additionally, the potential ability of aerobic exercise to modify the risk of frailty is highlighted as an important intervention.

Sex-dependent mitophagy and neuronal death following rat neonatal hypoxia-ischemia.

Males are more susceptible than females to long-term cognitive deficits following neonatal hypoxic-ischemic encephalopathy (HIE). Mitochondrial dysfunction is implicated in the pathophysiology of cerebral hypoxia-ischemia (HI), but the influence of sex on mitochondrial quality control (MQC) after HI is unknown. Therefore, we tested the hypothesis that mitophagy is sexually dimorphic and neuroprotective 20-24h following the Rice-Vannucci model of rat neonatal HI at postnatal day 7 (PN7). Mitochondrial and lysosomal morphology and degree of co-localization were determined by immunofluorescence in the cerebral cortex. No difference in mitochondrial abundance was detected in the cortex after HI. However, net mitochondrial fission increased in both hemispheres of female brain, but was most extensive in the ipsilateral hemisphere of male brain following HI. Basal autophagy, assessed by immunoblot for the autophagosome marker LC3BI/II, was greater in males suggesting less intrinsic reserve capacity for autophagy following HI. Autophagosome formation, lysosome size, and TOM20/LAMP2 co-localization were increased in the contralateral hemisphere following HI in female, but not male brain. An accumulation of ubiquitinated mitochondrial protein was observed in male, but not female brain following HI. Moreover, neuronal cell death with NeuN/TUNEL co-staining occurred in both hemispheres of male brain, but only in the ipsilateral hemisphere of female brain after HI. In summary, mitophagy induction and neuronal cell death are sex dependent following HI. The deficit in elimination of damaged/dysfunctional mitochondria in the male brain following HI may contribute to male vulnerability to neuronal death and long-term neurobehavioral deficits following HIE.

Anaesthetic neurotoxicity and neuroplasticity: an expert group report and statement based on the BJA Salzburg Seminar.

Although previously considered entirely reversible, general anaesthesia is now being viewed as a potentially significant risk to cognitive performance at both extremes of age. A large body of preclinical as well as some retrospective clinical evidence suggest that exposure to general anaesthesia could be detrimental to cognitive development in young subjects, and might also contribute to accelerated cognitive decline in the elderly. A group of experts in anaesthetic neuropharmacology and neurotoxicity convened in Salzburg, Austria for the BJA Salzburg Seminar on Anaesthetic Neurotoxicity and Neuroplasticity. This focused workshop was sponsored by the British Journal of Anaesthesia to review and critically assess currently available evidence from animal and human studies, and to consider the direction of future research. It was concluded that mounting evidence from preclinical studies reveals general anaesthetics to be powerful modulators of neuronal development and function, which could contribute to detrimental behavioural outcomes. However, definitive clinical data remain elusive. Since general anaesthesia often cannot be avoided regardless of patient age, it is important to understand the complex mechanisms and effects involved in anaesthesia-induced neurotoxicity, and to develop strategies for avoiding or limiting potential brain injury through evidence-based approaches.

Mitochondrial detachment of hexokinase 1 in mood and psychotic disorders: implications for brain energy metabolism and neurotrophic signaling.

The pathophysiology of mood and psychotic disorders, including unipolar depression (UPD), bipolar disorder (BPD) and schizophrenia (SCHZ), is largely unknown. Numerous studies, from molecular to neuroimaging, indicate that some individuals with these disorders have impaired brain energy metabolism evidenced by abnormal glucose metabolism and mitochondrial dysfunction. However, underlying mechanisms are unclear. A critical feature of brain energy metabolism is attachment to the outer mitochondrial membrane (OMM) of hexokinase 1 (HK1), an initial and rate-limiting enzyme of glycolysis. HK1 attachment to the OMM greatly enhances HK1 enzyme activity and couples cytosolic glycolysis to mitochondrial oxidative phosphorylation, through which the cell produces most of its adenosine triphosphate (ATP). HK1 mitochondrial attachment is also important to the survival of neurons and other cells through prevention of apoptosis and oxidative damage. Here we show, for the first time, a decrease in HK1 attachment to the OMM in postmortem parietal cortex brain tissue of individuals with UPD, BPD and SCHZ compared to tissue from controls without psychiatric illness. Furthermore, we show that HK1 mitochondrial detachment is associated with increased activity of the polyol pathway, an alternative, anaerobic pathway of glucose metabolism. These findings were observed in samples from both medicated and medication-free individuals. We propose that HK1 mitochondrial detachment could be linked to these disorders through impaired energy metabolism, increased vulnerability to oxidative stress, and impaired brain growth and development.

Mitochondrial dysfunction in mouse trisomy 16 brain.

Mitochondrial function in the brain of mouse trisomy 16, an animal model of Down syndrome with accelerated neuron death, was studied in isolated cortex mitochondria. Using an oxygen-sensitive Clarke electrode, a selective 16% decrease in respiration was detected with the Complex I substrates malate and glutamate but not with the Complex II substrate succinate. Western blotting revealed a 20% decrease in the 20 kDa subunit of Complex I in Ts16 brain cortex homogenates with no significant decrease in marker proteins for the other complexes of the electron transport chain. Although no differences in H(2)O(2) production or maximal calcium uptake were detected in the Ts16 mitochondria, there was an 18% decrease in pyruvate dehydrogenase levels, a change associated with oxidative stress in ischemia. These results are similar to those found in Parkinson's disease suggesting some neurodegenerative diseases may have mitochondrial pathology as a common step.

Methoxychlor causes mitochondrial dysfunction and oxidative damage in the mouse ovary.

Methoxychlor (MXC) is an organochlorine pesticide that reduces fertility in female rodents by causing ovarian atrophy, persistent estrous cyclicity, and antral follicle atresia (apoptotic cell death). Oxidative damage resulting from reactive oxygen species (ROS) generation has been demonstrated to lead to toxicant-induced cell death. Thus, this work tested the hypothesis that MXC causes oxidative damage to the mouse ovary and affects mitochondrial respiration in a manner that stimulates ROS production. For the in vitro experiments, mitochondria were collected from adult cycling mouse ovaries, treated with vehicle (dimethyl sulfoxide; DMSO) or MXC, and subjected to polarographic measurements of respiration. For the in vivo experiments, adult cycling CD-1 mice were dosed with either vehicle (sesame oil) or MXC for 20 days. After treatment, ovarian mitochondria were isolated and subjected to measurements of respiration and fluorimetric measurements of H2O2 production. Some ovaries were also fixed and processed for immunohistochemistry using antibodies for ROS production markers: nitrotyrosine and 8-hydroxy-2'-deoxyguanosine (8-OHG). Ovaries from in vivo experiments were also used to measure the mRNA expression and activity of antioxidants such as Cu/Zn superoxide dismutase (SOD1), glutathione peroxidase (GPX), and catalase (CAT). The results indicate that MXC significantly impairs mitochondrial respiration, increases production of H2O2, causes more staining for nitrotyrosine and 8-OHG in antral follicles, and decreases the expression and activity of SOD1, GPX, and CAT as compared to controls. Collectively, these data indicate that MXC inhibits mitochondrial respiration, causes ROS production, and decreases antioxidant expression and activity in the ovary, specifically in the antral follicles. Therefore, it is possible that MXC causes atresia of ovarian antral follicles by inducing oxidative stress through mitochondrial production of ROS.

Distribution of hypothalamic, hippocampal and other limbic peptidergic neuronal cell bodies giving rise to retinopetal fibers: anterograde and retrograde tracing and neuropeptide immunohistochemical studies.

In our present work utilizing the retrograde or anterograde transport of tracers (biotinylated dextran amine and Fluorogold, respectively) we have provided direct evidence for the cells of origin of the limboretinal pathway in rats and their termination in the retina using light microscopic approach. Administration of biotinylated dextran amine into the vitreous body resulted in nerve cell body labeling in several structures: the supraoptic and paraventricular nuclei, the hippocampus (CA1, CA3), the dentate gyrus, the indusium griseum, the olfactory tubercle, and the medial habenula, all of them belong to the limbic system. We estimated that the total number of retrogradely labeled cells is 1495+/-516. We have seen fiber labeling in the retinorecipient suprachiasmatic nucleus and in the primary visual center, the lateral geniculate body, but labeled nerve cell bodies in these structures were never seen. Iontophoretic application of Fluorogold into the hippocampal formation, where the major part of the biotinylated dextran amine-labeled cell bodies was observed, resulted in labeled fibers in the optic nerve and in the retina indicating that the retrogradely labeled cells in the hippocampus and the dentate gyrus among others are the cells of origin of the centrifugal visual fibers. Sections showing biotinylated dextran amine labeling were stained for vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactivity using immunohistochemistry. Some biotinylated dextran amine-labeled cells also showed vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactivity. We conclude that the limboretinal pathway exists and that the cells of origin are partially vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactive.

Neuroprotective effects of bilobalide, a component of Ginkgo biloba extract (EGb 761) in global brain ischemia and in excitotoxicity-induced neuronal death.

In this study, we compared the protective effect of bilobalide, a purified terpene lactone component of ginkgo biloba extract EGb 761, (definition see editorial) and EGb 761 against ischemic injury and against glutamate-induced excitotoxic neuronal death. In ischemic injury, we measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In rat cerebellar neuronal cultures, addition of bilobalide or EGb 761 protected in a dose-dependent manner against glutamate-induced excitotoxic neuronal death (effective concentration [EC (50)] = 5 microg/ml (12 microM) for bilobalide and 100 microg/ml for EGb 761. These results suggest that both EGb 761 and bilobalide are protective against ischemia-induced neuronal death in vivo and glutamate-induced neuronal death in vitro by synergistic mechanisms involving anti-excitotoxicity, inhibition of free radical generation, scavenging of reactive oxygen species, and regulation of mitochondrial gene expression.

Ovarian steroid modulation of seizure severity and hippocampal cell death after kainic acid treatment.

To determine whether maintained estrogen or progesterone levels affect kainic acid (KA) seizure patterns or the susceptibility of hippocampal neurons to death from seizures, ovariectomized Sprague-Dawley rats were implanted with estrogen pellets, 0.1 or 0.5 mg, that generated serum levels of 42.4 +/- 6.6 (mean +/- SEM) and 242.4 +/- 32.6 pg/ml or one to six capsules of progesterone that generated serum levels of 11.00 +/-.72 to 48.62 +/- 9.4 ng/ml. Seven days later, the rats were administered KA (8.5mg/kg, ip) and scored for seizure activity; 96 h later, the rats were killed and their brains processed for localization of neuron nuclear antigen (NeuN), a general neuronal marker. The hippocampus was scored for spread (the number of separate regions showing cell loss), and the area within the CA fields occupied by NeuN immunoreactivity was measured (indicating surviving neurons). Administration of estrogen or progesterone (independent of dose) significantly reduced mortality from KA seizures. Progesterone reduced seizure severity in animals that received one to four implants; compared with controls, no difference in seizure severity was noted for animals with six progesterone implants. The reduced seizures in progesterone-treated animals were accompanied by a reduction in the spread of hippocampal damage (r(2) = 0.87; P < 0.05). Likewise, in progesterone-treated rats, neuron survival and reduction in seizure scores were correlated (r(2) = 0.76; P < 0.0001). Estrogen had no effect on seizure severity (P > 0.05), but reduced both the spread (P < 0.05) and degree of neuronal loss (P < 0.05). Indeed, in the estrogen-treated rats, neuronal death was significantly lower than that observed in progesterone-treated animals with equally severe seizures (P < 0.05). These data are consistent with the hypothesis that progesterone produces its effects by reducing seizures, whereas estrogen has little beneficial effect on seizure behavior but protects the hippocampus from the damage seizures produce.

Postnatal brain development and neural cell differentiation modulate mitochondrial Bax and BH3 peptide-induced cytochrome c release.

Bax mediates cytochrome c release and apoptosis during neurodevelopment. Brain mitochondria that were isolated from 8-day, 17-day, and adult rats displayed decreasing levels of mitochondrial Bax. The amount of cytochrome c released from brain mitochondria by a peptide containing the BH3 cell death domain decreased with increasing age. However, approximately 60% of cytochrome c in adult brain mitochondria could be released by the BH3 peptide in the presence of exogenous human recombinant Bax. Mitochondrial Bax was downregulated in PC12S neural cells differentiated with nerve growth factor, and mitochondria isolated from these cells demonstrated decreased sensitivity to BH3-peptide-induced cytochrome c release. These results demonstrate that immature brain mitochondria and mitochondria from undifferentiated neural cells are particularly sensitive to cytochrome c release mediated by endogenous Bax and a BH3 death domain peptide. Postnatal developmental changes in mitochondrial Bax levels may contribute to the increased susceptibility of neurons to pathological apoptosis in immature animals.

Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia.

The neuroprotective effect of Ginkgo biloba extract (EGb 761) against ischemic injury has been demonstrated in animal models. In this study, we compared the protective effect of bilobalide, a purified terpene lactone from EGb 761, and EGb 761 against ischemic injury. We measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global forebrain ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In addition, both bilobalide and EGb 761 protected against ischemia-induced reductions in COX III mRNA in CA1 neurons prior to their death, at 1 day of reperfusion. These results suggest that oral administration of bilobalide and EGb 761 protect against ischemia-induced neuron death and reductions in mitochondrial gene expression.

BH3 death domain peptide induces cell type-selective mitochondrial outer membrane permeability.

The BH3 domain is essential for the release of cytochrome c from mitochondria by pro-apoptotic Bcl-2 family proteins during apoptosis. This study tested the hypothesis that a Bax peptide that includes the BH3 domain can permeabilize the mitochondrial outer membrane and release cytochrome c in the absence of a permeability transition at the mitochondrial inner membrane. BH3 peptide (0.1-60 microm) released cytochrome c from mitochondria in the presence of physiological concentrations of ions in a cell type-selective manner, whereas a BH3 peptide with a single amino acid substitution was ineffective. The release of cytochrome c by BH3 peptide correlated with the presence of endogenous Bax at the mitochondria and its integral membrane insertion. Cytochrome c release was accompanied by adenylate kinase release, was not associated with mitochondrial swelling or substantial loss of electrical potential across the inner membrane, and was unaffected by inhibitors of the permeability transition pore. Cytochrome c release was, however, inhibited by Bcl-2. Although energy-coupled respiration was inhibited after the release of cytochrome c, mitochondria maintained membrane potential in the presence of ATP due to the reversal of the ATP synthase. Overall, results support the hypothesis that BH3 peptide releases cytochrome c by a Bax-dependent process that is independent of the mitochondrial permeability transition pore but regulated by Bcl-2.

ATP synthesis is coupled to rat liver mitochondrial RNA synthesis.

Rat liver mitochondria respond to changes in energy demand by modulating the amount of RNA synthesized. Coupled rat liver mitochondria were used to determine the relationship between mitochondrial respiration, ATP levels, and mitochondrial transcription. This system included oxidizable substrates (malate and glutamate) and constituents that could support both mitochondrial respiration and transcription. The respiratory inhibitor rotenone, phosphorylation inhibitor oligomycin, and the uncoupler of oxidative phosphorylation carbonyl-cyanide p-triflouromethoxyphehylhydrazone inhibited RNA synthesis. Addition of ADP stimulated mitochondrial transcription and peak RNA synthesis was observed at 1-2 mM ADP. At ADP concentrations above 2 mM, RNA synthesis decreased. These results demonstrate that mitochondrial transcription is tightly coupled to ATP levels.

Mitochondrial precursor signal peptide induces a unique permeability transition and release of cytochrome c from liver and brain mitochondria.

This study tested the hypothesis that mitochondrial precursor targeting peptides can elicit the release of cytochrome c from both liver and brain mitochondria by a mechanism distinct from that mediated by the classical, Ca2+-activated permeability transition pore. Human cytochrome oxidase subunit IV signal peptide (hCOXIV1-22) at concentrations from 15 to 100 microM induced swelling, a decrease in membrane potential, and cytochrome c release in both types of mitochondria. Although cyclosporin A and bongkrekic acid were without effect, dibucaine, propanolol, dextran, and the uncoupler FCCP were each able to inhibit signal peptide-induced swelling and cytochrome c release. Adenylate kinase was coreleased with cytochrome c, arguing against a signal peptide-induced cytochrome c-specific pathway of efflux across the outer membrane. Taken together, the data indicate that a human mitochondrial signal peptide can evoke the release of cytochrome c from both liver and brain mitochondria by a unique permeability transition that differs in several characteristics from the classical mitochondrial permeability transition.

Myxothiazol induces H(2)O(2) production from mitochondrial respiratory chain.

Interruption of electron flow at the quinone-reducing center (Q(i)) of complex III of the mitochondrial respiratory chain results in superoxide production. Unstable semiquinone bound in quinol-oxidizing center (Q(o)) of complex III is thought to be the sole source of electrons for oxygen reduction; however, the unambiguous evidence is lacking. We investigated the effects of complex III inhibitors antimycin, myxothiazol, and stigmatellin on generation of H(2)O(2) in rat heart and brain mitochondria. In the absence of antimycin A, myxothiazol stimulated H(2)O(2) production by mitochondria oxidizing malate, succinate, or alpha-glycerophosphate. Stigmatellin inhibited H(2)O(2) production induced by myxothiazol. Myxothiazol-induced H(2)O(2) production was dependent on the succinate/fumarate ratio but in a manner different from H(2)O(2) generation induced by antimycin A. We conclude that myxothiazol-induced H(2)O(2) originates from a site located in the complex III Q(o) center but different from the site of H(2)O(2) production inducible by antimycin A.

Chronic exposure of neural cells to elevated intracellular sodium decreases mitochondrial mRNA expression.

Regulation of expression of mitochondrial DNA- (mtDNA-) encoded genes of oxidative phosphorylation can occur rapidly in neural cells subjected to a variety of physiological and pathological conditions. However, the intracellular signal(s) involved in regulating these processes remain unknown. Using mtDNA-encoded cytochrome oxidase subunit III (COX III), we show that its mRNA expression in a differentiated rat pheochromocytoma cell line PC12S is decreased by chronic exposure to agents that increase intracellular sodium. Treatment of differentiated PC12S cells either with ouabain, an inhibitor of Na/K-ATPase, or with monensin, a sodium ionophore, decreased the steady-state levels of COX III mRNA by 50%, 3-4 h after addition of the drugs. No significant reduction in mtDNA-encoded 12S rRNA or nuclear DNA-encoded beta-actin mRNA were observed. Removal of the drugs restored the normal levels of COX III mRNA. Determination of half-lives of COX III mRNA, 12S rRNA, and beta-actin mRNA revealed a selective decrease in the half-life of COX III mRNA from 3.3 h in control cells to 1.6 h in ouabain-treated cells, and to 1 h in monensin-treated cells. These results suggest the existence of a mechanism of posttranscriptional regulation of mitochondrial gene expression that is independent of the energetic status of the cell and may operate under pathological conditions.

Mitochondrial participation in ischemic and traumatic neural cell death.

Mitochondria play critical roles in cerebral energy metabolism and in the regulation of cellular Ca2+ homeostasis. They are also the primary intracellular source of reactive oxygen species, due to the tremendous number of oxidation-reduction reactions and the massive utilization of O2 that occur there. Metabolic trafficking among cells is also highly dependent upon normal, well-controlled mitochondrial activities. Alterations of any of these functions can cause cell death directly or precipitate death indirectly by compromising the ability of cells to withstand stressful stimuli. Abnormal accumulation of Ca2+ by mitochondria in response to exposure of neurons to excitotoxic levels of excitatory neurotransmitters, for example, glutamate, is a primary mediator of mitochondrial dysfunction and delayed cell death. Excitoxicity, along with inflammatory reactions, mechanical stress, and altered trophic signal transduction, all likely contribute to mitochondrial damage observed during the evolution of traumatic brain injury. The release of apoptogenic proteins from mitochondria into the cytosol serves as a primary mechanism responsible for inducing apoptosis, a form of cell death that contributes significantly to neurologic impairment following neurotrauma. Although several signals for the release of mitochondrial cell death proteins have been identified, the mechanisms by which these signals increase the permeability of the mitochondrial outer membrane to apoptogenic proteins is controversial. Elucidation of the precise biochemical mechanisms responsible for mitochondrial dysfunction during neurotrauma and the roles that mitochondria play in both necrotic and apoptotic cell death should provide new molecular targets for neuroprotective interventions.

Elevation of resting mitochondrial membrane potential of neural cells by cyclosporin A, BAPTA-AM, and bcl-2.

This study tested the hypothesis that the activity of the mitochondrial membrane permeability transition pore (PTP) affects the resting mitochondrial membrane potential (DeltaPsi) of normal, healthy cells and that the anti-apoptotic gene product Bcl-2 inhibits the basal activity of the PTP. DeltaPsi was measured by both fluorometric and nonfluorometric methods with SY5Y human neuroblastoma cells and with GT1-7 hypothalamic cells and PC12 pheochromocytoma cells in the absence and presence of Bcl-2 gene overexpression. The resting DeltaPsi of Bcl-2 nonexpressing PC12 and wild-type SY5Y cells was increased significantly by the presence of the PTP inhibitor cyclosporin A (CsA) or by intracellular Ca(2+) chelation through exposure to the acetoxymethyl ester of 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). The DeltaPsi of Bcl-2-overexpressing PC12 cells was larger than that of Bcl-2-negative cells and not significantly increased by CsA or by Ca(2+) chelation. CsA did not present a significant effect on the DeltaPsi monitored in unstressed GT1-7 cells but did inhibit the decrease in DeltaPsi elicited by the addition of t-butyl hydroperoxide, an oxidative inducer of the mitochondrial permeability transition. These results support the hypothesis that an endogenous PTP activity can contribute to lowering the basal DeltaPsi of some cells and that Bcl-2 can regulate the endogenous activity of the mitochondrial PTP.

Rapid tau protein dephosphorylation and differential rephosphorylation during cardiac arrest-induced cerebral ischemia and reperfusion.

The effects of cerebral ischemia/reperfusion on phosphorylation of microtubule-associated tau proteins were assessed in a canine model of cardiac arrest. As tau proteins are phosphorylated by kinases involved in different transduction signal pathways, their phosphorylation state is an excellent marker of neuronal homeostasis and microtubule dynamics. Canine brain tau proteins were characterized by immunoblotting using phosphorylation-dependent antibodies and antisera raised against different amino- and carboxy-terminal tau sequences. The present study reports a complete dephosphorylation of tau proteins during ischemia, which is shown by a higher electrophoretic mobility and the almost (if not total) disappearance of phosphorylation-dependent monoclonal antibody labeling. After 2-hour restoration of spontaneous circulation, a decrease in the electrophoretic mobility was observed, and after 24 hours of reperfusion, a full restoration of the phosphorylation was visualized using phosphorylation-dependent monoclonal antibodies directed against Ser/Thr-Pro sites. However, one particular phosphorylation site involved in tau binding to microtubules, located on Ser262/356, was never fully significantly rephosphorylated, suggesting that microtubule metabolism was still affected after 24 hours of reperfusion. Thus, the sequential and differential recovery of tau phosphorylation after ischemia followed by reperfusion is a useful marker with which to monitor neuronal integrity after brain ischemia.