Fatma J. Ekinci

Activation of the L Voltage-sensitive Calcium Channel by Mitogen-activated Protein (MAP) Kinase following Exposure of Neuronal Cells to β-Amyloid MAP KINASE MEDIATES β-AMYLOID-INDUCED NEURODEGENERATION

Neuronal degeneration in Alzheimer’s disease (AD) has been variously attributed to increases in cytosolic calcium, reactive oxygen species, and phosphorylated forms of the microtubule-associated protein tau. β-Amyloid (βA), which accumulates extracellularly in AD brain, induces calcium influx in culture via the L voltage-sensitive calcium channel. Since this channel is normally activated by protein kinase A-mediated phosphorylation, we examined kinase activities recruited following βA treatment of cortical neurons and SH-SY-5Y neuroblastoma. βA increased channel phosphorylation; this increase was unaffected by the protein kinase A inhibitor H89 but was reduced by the mitogen-activated protein (MAP) kinase inhibitor PD98059. Pharmacological and antisense oligonucleotide-mediated reduction of MAP kinase activity also reduced βA-induced accumulation of calcium, reactive oxygen species, phospho-tau immunoreactivity, and apoptosis. These findings indicate that MAP kinase mediates multiple aspects of βA-induced neurotoxicity and indicates that calcium influx initiates neurodegeneration in AD. βA increased MAP kinase-mediated phosphorylation of membrane-associated proteins and reduced phosphorylation of cytosolic proteins without increasing overall MAP kinase activity. Increasing MAP kinase activity with epidermal growth factor did not increase channel phosphorylation. These findings indicate that redirection, rather than increased activation, of MAP kinase activity mediates βA-induced neurotoxicity.

β-Amyloid-induced calcium influx induces apoptosis in culture by oxidative stress rather than tau phosphorylation

Beta-amyloid (betaA) toxicity in culture is accompanied by multiple events culminating in apoptosis. Calcium influx may represent the initial event, since calcium chelation prevents all subsequent events, while subsequent events include increased generation of reactive oxygen species (ROS) and hyperphosphorylation of tau. In the present study, we undertook to determine whether ROS generation or tau hyperphosphorylation mediate betaA-induced apoptosis. The anti-oxidant vitamin E or the kinase inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenslfonamide (W7) was added following brief treatment of differentiated SH-SY-5Y human neuroblastoma cells with 22 microM betaA. Under these conditions, vitamin E prevented ROS generation and apoptosis, but did not prevent intracellular calcium accumulation or tau phosphorylation. W7 prevented tau phosphorylation but did not block betaA-induced calcium influx, ROS generation or apoptosis. While these studies do not address the long-term consequences of PHF formation, they indicate that ROS generation, rather than tau hyperphosphorylation, leads to apoptosis following betaA-induced calcium influx into cultured cells.

Monitoring thiobarbituric acid-reactive substances (TBARs) as an assay for oxidative damage in neuronal cultures and central nervous system

Oxidative stress is a pivotal factor in neuronal degeneration. A simple method to quantify oxidative damage in culture and in situ is therefore important for studies of neurodegeneration. We present herein modifications of the standard assay for thiobarbituric acid-reactive substances (TBARs) for analyses of both cell cultures and brain tissue homogenates. Since the TBAR assay measures end-point oxidative damage, it is useful to assess the overall impact of oxidative stress-inducing and neuroprotective agents; interpretation is not potentially confounded by the presence or absence of transient products of oxidative damage.

β‐amyloid and ionophore A23187 evoke tau hyperphosphorylation by distinct intracellular pathways: Differential involvement of the calpain/protein kinase C system

SH‐SY‐5Y human neuroblastoma cells were treated with 22 μM of a synthetic peptide corresponding to amino acid residues 25–35 of β‐amyloid (βA) or 3 μM calcium ionophore A23187 in culture medium containing 1.8 mM extracellular calcium. Both agents increased tau immunoreactivity towards antibodies (PHF‐1, ALZ‐50) that recognize epitopes common with paired helical filaments (PHFs) and towards an antibody (5E2) that recognized a phosphate‐independent tau epitope. However, only ionophore increased immunoreactivity with an additional phosphate‐dependent antibody (AT‐8) that recognized an epitope of tau when phosphorylated, and induced a corresponding decrease in immunoreactivity towards an additional antibody (Tau‐1) that recognizes the same site when that site is not phosphorylated. Moreover, the ionophore‐mediated increase in PHF‐1 was blocked by EGTA, by the calpain inhibitor calpeptin and by the PKC inhibitor H7, while that evoked by βA treatment was not inhibited by any of these treatments. Since ionophore‐mediated calpain activation induces proteolytic PKC activation, we further examined the influence of PKC inhibition on βA and ionophore‐mediated PHF‐1 induction. Antisense oligonucleotide‐mediated downregulation of PKCϵ in a stable transfectant SH‐SY‐5Y subclone diminished the ionophore‐mediated, but not the βA‐mediated, increase in PHF‐1 immunoreactivity. These data indicate specific differences in the intracellular cascade of events invoked by βA and ionophore A23187. Moreover, although βA invoked calcium influx in these cells, our findings further suggest that the induction of tau hyperphosphorylation by βA may not be due to calcium influx. J. Neurosci. Res. 49:759–768, 1997.

Phosphorylation of tau alters its association with the plasma membrane.

Abstract1. The potential functions of the microtubule-associated protein tau have been expanded by the recent demonstration of its interaction with the plasma membrane. Since the association of tau with microtubules is regulated by phosphorylation, herein we examine whether or not the association of tau with the plasma membrane is also regulated by phosphorylation.2. A range of tau isoforms migrating from 46 to 64 kDa was associated with crude particulate fractions derived from SH-SY-5Y human neuroblastoma cells, and were retained during the initial stages of plasma membrane purification. During the extensive washing utilized in purification of the plasma membrane, portions of each of these isoforms were depleted from the resultant purified membrane. Immunoblot analysis with phospho-dependent and -independent antibodies revealed selective depletion of phospho isoforms during membrane washing. This effect was more pronounced for the slowest-migrating (64-kDa) tau isoform.3. This putative influence of phosphorylation on the association of tau with the plasma membrane was further probed by transfection of SH-SY-5Y human neuroblastoma cells with a tau construct that could associate with the plasma membrane but not with microtubules. Treatment with phorbol ester or calcium ionophore, both of which increased phospho-tau levels within the cytosol and plasma membrane, was accompanied by the dissociation of this tau construct from the membrane.4. These data indicate that phosphorylation regulates the association with the plasma membrane. Dissociation from the membrane by phosphorylation may place tau at risk for hyperphosphorylation and ultimate PHF formation in a manner previously considered for tau dissociated from microtubules.

Hyperactivation of Mitogen-Activated Protein Kinase Increases Phospho-Tau Immunoreactivity Within Human Neuroblastoma: Additive and Synergistic Influence of Alteration of Additional Kinase Activities

Mitogen-activated protein (MAP) kinase phosphorylates tau in cell-free analyses, but whether or not it does so within intact cells remains controversial. In the present study, microinjection of MAP kinase into SH-SY-5Y human neuroblastoma cells increased tau immunoreactivity toward the phosphodependent antibodies PHF-1 and AT-8. In contrast, treatment with a specific inhibitor of MAP kinase (PD98059) did not diminish “basal” levels of these immunoreactivities in otherwise untreated cells. These findings indicate that hyperactivation of MAP kinase increases phospho-tau levels within cells, despite that MAP kinase apparently does not substantially influence intracellular tau phosphorylation under normal conditions. These findings underscore that results obtained following inhibition of kinase activities do not necessarily provide an indication of the consequences accompanying hyperactivation of that same kinase. Several studies conducted in cell-free systems indicate that exposure of tau to multiple kinases can have synergistic effects on the nature and extent of tau phosphorylation. We therefore examined whether or not such effects could be demonstrated within these cells. Site-specific phospho-tau immunoreactivity was increased in additive and synergistic manners by treatment of injected cells with TPA (which activates PKC), calcium ionophore (which activates calcium-dependent kinases), and wortmannin (which inhibits PIP3 kinase). Alteration in total tau levels was insufficient to account for the full extent of the increase in phospho-tau immunoreactivity. These additional results indicate that multiple kinase activities modulate the influence of MAP kinase on tau within intact cells.

EFFICACY OF VITAMIN E, PHOSPHATIDYL CHOLINE, AND PYRUVATE ON BUFFERING NEURONAL DEGENERATION AND OXIDATIVE STRESS IN CULTURED CORTICAL NEURONS AND IN CENTRAL NERVOUS TISSUE OF APOLIPOPROTEIN E-DEFICIENT MICE

Oxidative stress is a pivotal factor in neuronal degeneration. However, vitamin E was only marginally effective in clinical trials. We examined whether or not a mixture of vitamin E (as alpha-tocopherol), sodium pyruvate and phosphatidyl choline (PC), a mixture that promotes wound healing in non-neuronal systems, would provide neuroprotection beyond that observed with vitamin E alone. Combined treatment with these agents improved survival and neuritic spouting of murine embryonic cortical neurons in culture, and provided neuroprotection against oxidative damage following treatment with hydrogen peroxide. Dietary treatment with these three agents also compensated for the diminished oxidative buffering capacity of brains of apolipoprotein E-deficient mice, while vitamin E alone failed to do so. These data underscore the possibility that critical nutritional deficiencies may modulate the impact of genetic compromise on neurodegeneration.

Okadaic acid mediates tau phosphorylation via sustained activation of the L-voltage-sensitive calcium channel

Accumulation of phosphorylated isoforms of the microtubule-associated protein tau is one hallmark of affected neurons in Alzheimers disease (AD). This increase has been attributed to increased kinase or decreased phosphatase activity. Prior studies indicate that one of the kinases that phosphorylates tau (mitogen-activated protein kinase, or MAP kinase) does so at least in part indirectly within intact neuronal cells by phosphorylating and activating the L-voltage-sensitive calcium channel. Resultant calcium influx then fosters tau phosphorylation via one or more calcium-activated kinases. We demonstrate herein that treatment of differentiated SH-SY-5Y human neuroblastoma with the phosphatase inhibitor okadaic acid (OA) similarly may increase tau phosphorylation via sustained activation of the L-voltage-sensitive calcium channel. OA increased phospho-tau as indicated by increased immunoreactivity towards an antibody (PHF-1) directed against paired helical filaments from AD brain. This increase was blocked by co-treatment with the channel antagonist nimodipine. OA treatment increased channel phosphorylation. The increases in calcium influx, PHF-1 immunoreactivity and channel phosphorylation were all attenuated by co-treatment with PD98059, which inhibits MAP kinase activity, suggesting that OA mediates these effects at least in part via sustained activation of MAP kinase. These findings underscore that divergent and convergent kinase and phosphatase activities regulate tau phosphorylation.

Selective activation by bryostatin-1 demonstrates unique roles for PKCε in neurite extension and tau phosphorylation

Phorbol esters such as 12‐O‐tetradeonyl phorbol‐13 acetate (TPA) induce a time‐dependent biphasic effect on protein kinase C (PKC)‐mediated events by fostering translocation of cytosolic (latent) PKC to the plasma membrane (where it is activated). Continued treatment, however, depletes the cells entire PKC complement and induces a functional stake of PKC inhibition. Previous studies from several laboratories have demonstrated that long‐term TPA treatment, like treatment with PKC inhibitors, induces neuronal differentiation. Bryostatin‐1 also induces translocation and overall downregulation of PKC following long‐term treatment, yet, unlike TPA or PKC inhibitors, does not induce neuronal differentiation, promoting controversy regarding the role of PKC inhibition in neuronal differentiation. We demonstrate herein that, despite overall downregulation in human neuroblastoma cells, membrane‐associated levels of one PKC isoform (PKCε) are actually increased following long‐term bryostatin‐1 treatment. Since previous studies have implicated this PKC isoform in phosphorylation of the microtubule‐associated protein tau and in neuritogenesis, we examined the consequences of long‐term bryostatin treatment on these phenomena. Treatment with 25 n‐100 M bryostatin‐1 for 72 h increased tau phosphorylation and inhibited neuritogenesis. By contrast, treatment with either TPA or the PKC inhibitor staurosporine did not induce tau phosphorylation and induced neurite elaboration. Bryostatin‐1 antagonized neurite induction by staurosporine. These findings provide additional evidence for a unique role of PKCε in the regulation of tau phosphorylation and neuronal differentiation, and demonstrate that bryostatin‐1 can function under certain conditions as a selective PKCε activator even following long‐term treatment.

amyloid induced hyperphosphorylation of tau in human neuroblastoma cells involves map kinase

Treatment of retinoic acid-differentiated SH-SY-5Y human neuroblastoma cells with a sublethal concentration (22μM) β-amyloid (βA) in the presence of 1.8mM extracellular calcium induced a marked increase in PHF-1 immunoreactivity. The βA-induced increase in PHF-1 immunoreactivity was prevented by the MAP kinase kinase inhibitor PD 89059. These data indicate that βA-induced tau hyperphosphorylation is mediated via activation of the MAP kinase pathway.