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.

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 Alzheimer's 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.

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.

beta-Amyloid-Induced Tau Phosphorylation does not Correlate with Degeneration in Cultured Neurons.

Treatment of cultured neurons with beta-amyloid (Abeta) evokes multiple consequences, including calcium influx, production of reactive oxygen species (ROS), hyperphosphorylation of tau. Which of these events is the major cause of Abeta-induced neurodegeneration has been the subject of controversy. We undertook to determine whether or not the accumulation of hyperphosphorylated tau mediated neurodegeneration. Murine cortical neurons demonstrated increased phospho-tau immunoreactivity between 2-8 hr after treatment of murine cortical neurons with Abeta_25-35. Cultures underwent overall neurodegeneration between 8-16 hr as ascertained by phase-contrast microscopy, a commercial "live/dead" assay and externalization of phosphatidyl serine. Unexpectedly, however, the healthiest-appearing neurons in Abeta-treated cultures contained relatively more phospho-tau immunoreactivity, while obviously degenerating neurons contained less; degenerating neurons often contained less phospho-tau immunoreactivity than did non-Abeta-treated control neurons. By contrast, accumulation of reactive oxygen species, previously demonstrated to mediate Abeta-induced neurodegeneration, was most prominent within visibly-degenerating neurons. These studies do not address the long-term consequences of PHF formation; however, they indicate that tau hyperphosphorylation, although a consequence of Abeta treatment, does not directly contribute to acute degeneration of cultured neurons.

Biphasic Effect of Calcium Influx on Tau Phosphorylation: Phosphorylation: Biphasic Effect of Calcium Influx on Tau Phosphorylation: Involvement of Calcium-Dependent Phosphatase and Kinase Activities.

Conflicting data has emerged documenting decreased and increased levels of phospho-tau following calcium influx. Calcium influx achieved by treatment of SH-SY-5Y human neuroblastoma with 1 micro M calcium ionophore A23187 in the presence of 0.1 mM extracellular calcium depleted phospho-tau levels within 30 min. However, extending ionophore treatment to 60 min raised phospho-tau levels beyond that of control levels. Total tau levels were unchanged throughout these treatments, indicating that the reduction in PHF-1 reflected sequential alterations in tau phosphorylation rather than total tau. More rapid accumulation of phospho-tau accompanied treatment with increased concentrations of ionophore (3 micro M) and extracellular calcium (0.9 mM). An inhibitor active against calcium-dependent kinase(s) prevented the increase in phospho-tau following calcium influx. These data underscore that phospho-tau levels represent the summation of kinase and phosphatase activities and indicate that net dephosphorylation or phosphorylation is dependent upon the extent and/or rate of calcium influx