Tau protein expression in frontotemporal dementias.

The syndrome of frontotemporal dementia represents a diverse group of diseases presenting with behavioral and cognitive disturbances. The expression of the microtubule-associated protein tau was studied in postmortem samples of frontal cortex of 19 cases (12 Pick's disease A, B, C; 4 dementia lacking distinct histology; 3 motor neuron disease type) by Western blotting with a phosphorylation-independent anti-tau antibody. The presence of tau protein was detected in all cases evaluated, including the 11 brains classified as frontotemporal lobe degeneration (diagnostic categories Pick's disease B, C and dementia lacking distinct histology). These findings indicate that the recently reported decreased expression of tau protein in frontotemporal lobe degeneration represents pathogenic mechanism of neurodegeneration detectable only in a special subset of these disorders.

Multiple-label immunocytochemistry for the evaluation of nature of cell death in experimental models of neurodegeneration.

A prominent feature of neurodegenerative diseases is a loss of specific neuronal populations. The pathophysiological mechanisms responsible are, however, poorly understood. Primary cultures of rodent embryonic neurons represent a useful experimental system for investigation of molecular pathways of neurodegeneration and mechanisms of cell death. Here, we report a technique utilizing triple-label immunocytochemistry with confocal immunofluorescence detection designed to simultaneously assess multiple parameters of cell injury in individual hippocampal neurons in primary culture. This method combines detection of DNA damage (TUNEL or Klenow assay) with double-label immunocytochemistry for the activated form of caspase-3 or, alternatively, caspase-cleaved actin (fractin), and microtubule-associated protein-2 (MAP-2) or beta-tubulin. The combined evaluation of the form of nuclear damage (karyorrhexis, pyknosis), the presence or absence of activated caspase-3, and the extent of the damage to cell cytoskeleton, allows for precise assessment of the extent of injury and the mode of cell death (apoptosis, oncosis) for individual neurons.

Up-regulation of the lysosomal system in experimental models of neuronal injury: implications for Alzheimer's disease.

Previous studies established that the populations of neurons that frequently degenerate in Alzheimer's disease exhibit robust up-regulation of the lysosomal system. In this study, we investigated alterations of the lysosomal system during different forms of experimental injury in rat hippocampal neurons in culture, utilizing a combination of immunocytochemical and biochemical methods. Using triple-label immnocytochemistry for activated caspase-3, fragmentation of DNA and the microtubule-associated protein-2, we characterized treatment paradigms as models of the apoptotic (staurosporine, camptothecin), the oncotic (high-dose menadione, glutamate), and the mixed apoptotic and oncotic (low-dose menadione) pathways of neuronal death. Slowly developing apoptotic or slowly developing mixed apoptotic and oncotic forms of neuronal injury were associated with substantial increases in the number and size of cathepsin D-positive vesicles (late endosomes and mature lysosomes) as determined by immunocytochemistry, and elevated levels of cathepsin D by western blotting. In agreement with our previous findings in Alzheimer's disease, where lysosomal system activation was not restricted to overtly degenerating neurons, up-regulation of this system was also detected quite early during the course of experimental neuronal injury, preceding the development of dystrophic neurites, nuclear segmentation or fragmentation of DNA. These findings implicate lysosomal system activation, both in Alzheimer's disease and in experimental models of neuronal injury, as an important event associated with early stages of neurodegeneration.

DNA strand breaks in Alzheimer's disease.

The goal of this study was to investigate the presence of DNA damage in Alzheimer's disease (AD) utilizing independent assays for three different types of DNA strand breaks. Sections from hippocampi of AD brains, brains with Alzheimer neurofibrillary changes (Ch) from non-demented individuals, and controls (C) were labeled with (1) the TUNEL assay to identify blunt-ended and 3' protruding termini of breaks in double-stranded DNA, (2) the Klenow assay to detect single-stranded and double-stranded breaks with protruding 5' termini, and (3) the Apostain assay which utilizes a monoclonal antibody to single-stranded DNA and is based on the decreased stability of apoptotic DNA to thermal denaturation caused by DNA breaks. The highest incidence of nuclei positive for either molecular form of DNA strand breaks was detected in AD, followed by Ch, and controls (C). In either AD and Ch, the incidence of TUNEL- or Klenow-positive nuclei did not differ significantly, but was higher than the incidence of Apostain-positive nuclei. With all three assays, the highest incidence of positive nuclei was in the molecular layer of CA1. In the majority of nuclei positive for either the Klenow or the Apostain assay, the product of the labeling reaction was localized either to the periphery of the nucleus or to distinct clumps of chromatin (or both). With the TUNEL assay, the majority of positive nuclei were diffusely labeled. In both AD and Ch, the individual positive nuclei were labeled with both the Klenow and the TUNEL assays. The results indicate high incidence of nuclei with either double-stranded or single-stranded DNA breaks in AD, which, for the forms detectable with the Klenow or TUNEL assays, were colocalized.

Isoform-specific translocation of protein kinase C following glutamate administration in primary hippocampal neurons.

High concentrations of glutamate, the major excitatory neurotransmitter in the mammalian brain, lead to intracellular calcium overload resulting in excitotoxic damage and death of neurons. Since protein kinase C (PKC) is involved in neuronal degeneration resulting from cerebral ischemia and from glutamate excitotoxicity, we investigated the effect of glutamate on changes in the cellular distribution of various PKC isoforms in cultured hippocampal neurons in comparison with the effects elicited by the PKC activator phorbol ester. Out of the expressed PKC isoforms alpha, gamma, epsilon, zeta and lambda only the conventional isoforms PKC alpha and gamma responded to glutamate. Using subcellular fractionation and Western blotting with isoform-specific antibodies and immunocytochemical localization with confocal laser scanning microscopy, we observed that phorbol ester and glutamate have different effects on PKC isoform redistribution: Whereas phorbol ester resulted in translocation of PKC alpha and PKC gamma toward a membrane fraction, the glutamate-mediated rise in intracellular calcium concentration induced a translocation mainly toward a detergent-insoluble, cytoskeletal fraction. Immunocytochemical analysis revealed an isoform-specific translocation following glutamate treatment: PKC gamma was translocated mainly to cytoplasmic, organelle-like structures, whereas PKC alpha redistributed to the plasma membrane and into the cell nucleus. The latter result is of special interest, as it indicates that nuclear PKC may play a role in processes of excitotoxic cell damage.

Developmental regulation of the recovery process following glutamate-induced calcium rise in rodent primary neuronal cultures.

CNS neurons exhibit a profound, maturation-dependent increase in the vulnerability to injury. Little is, however, known about the cellular mechanisms involved. This study investigated the developmental influence on the ability to recover the resting concentration of free cytoplasmic Ca2+ ([Ca2+]i) following stimulation with 100 microM glutamate in hippocampal and cerebellar granule cells in culture. Primary neurons were exposed to glutamate for either 1 min or 10 min. Hippocampal neurons were evaluated at 7, 12-14, and 17-19 days in vitro (DIV), and cerebellar granule cells were tested at 8-9 or 15-16 DIV. In hippocampal neurons, either an increased age in culture or longer drug exposure were both associated with less efficient [Ca2+]i recovery. Additionally, for both 1-min and 10-min drug exposure, increased age in culture was the primary determinant of the development of secondary [Ca2+]i destabilization followed by a very variable recovery patterns. Similar to hippocampal neurons, older cerebellar granule cells also recovered less efficiently from glutamate-mediated [Ca2+]i rise. The difference in the extent of recovery was not directly influenced by the magnitude of the [Ca2+]i rise, since cerebellar granule cells recovered from both high or low [Ca2+]i rise with similar kinetic profiles. Overall, the results presented in this study implicate the age in culture as an important influencing factor of both the less efficient recovery from glutamate-induced Ca2+ load and the development of secondary [Ca2+]i destabilizations. The progressive, maturation-dependent, decrease in the ability to recover from Ca2+ load might represent a potentially important mechanism contributing to the increased vulnerability of fully developed neurons to injury.

Calpain I activation in rat hippocampal neurons in culture is NMDA receptor selective and not essential for excitotoxic cell death.

Administration of glutamate (100 microM) to primary cultures of rat hippocampal neurons for 1 h led to calpain I activation as determined by monitoring the extent of spectrin breakdown with the antibodies designed to specifically recognize the calpain I-mediated spectrin breakdown products. Based on the studies with subtype selective antagonists of glutamate receptors, glutamate caused calpain I activation specifically through the activation of the NMDA receptor. In parallel experiments, the magnitude and the temporal profiles of Ca2+ rise were determined by Fura-2 microfluorimetry. Ca2+ influx through voltage-sensitive Ca2+ channels, even though leading to substantial Ca2+ rise, did not by itself activate calpain I. These results indicate that for calpain I activation, the source of Ca1+ influx is more important than the magnitude of Ca2+ rise. Glutamate-mediated calpain I activation was fully blocked by preincubation (30 min) of the cultures with calpain inhibitor I, calpain inhibitor II, or calpeptin (all 10 microM). The presence of calpain inhibitors did not, however, in any way ameliorate the massive excitotoxicity resulting from 16 h exposure to glutamate, indicating that calpain I activation and excitotoxicity are not causally related events. Similarly, preincubation with any of the tested calpain inhibitors was detrimental to the clearance of neuritic from a 10-min exposure to glutamate. Additionally, the presence of calpain inhibitors was detrimental to the clearance of neuritic varicosities resulting from a short-term sublethal exposure to glutamate, suggesting that a physiological level of calpain I activation might actually play an important homeostatic role in the restoration of normal cytoskeletal organization.

Acute rise in the concentration of free cytoplasmic calcium leads to dephosphorylation of the microtubule-associated protein tau.

The objective of this study was to asses the response of the microtubule-associated protein tau to acute rise in the concentration of free cytoplasmic calcium ([Ca2+]i) in rat cortical neurons and mouse cerebellar granule cells in culture. One-hour exposure to glutamate (100 microM), N-methyl-D-aspartate (100 microM), KCl (50 mM), and ionomycin (5 microM) led to tau protein dephosphorylation as indicated by an appearance of additional faster moving bands on Western immunoblots with a phosphorylation-independent antibody and an increase in the tau-1 immunoreactivity associated with the appearance of an additional faster moving band. Lowering the extracellular concentration of Ca2+ to less than 1 microM fully prevented the drug-induced tau protein dephosphorylation indicating a dependence on Ca2+ influx from the extracellular environment. Administration of okadaic acid (inhibitor of phosphatase 1/2A) simultaneously with the above mentioned drugs decreased the drug-mediated dephosphorylation. Pre-incubation with okadaic acid fully prevented the dephosphorylation. Treatment with cypermethrin (inhibitor of phosphatase 2B) was without effect when administered either alone, simultaneously with the drugs, or pre-incubated. These findings indicate that, independently of the influx pathway, [Ca2+]i elevation leads to dephosphorylation of the microtubule-associated protein tau and implicate phosphatase 1 and/or 2A in the process.

DNA strand breaks induced by sustained glutamate excitotoxicity in primary neuronal cultures.

We developed a new approach to study single- and double-stranded DNA breaks during chronic, moderate excitotoxicity resulting from the inhibition of the glutamate transporter in cerebellar granule cell primary cultures. A 24 hr treatment of 2-week-old cultures with L-alpha-amino adipate (LAA), an inhibitor of the cerebellar glutamate uptake transporter, caused a gradual extracellular accumulation of endogenous glutamate that induced reversible morphological change of granule neurons but no neuronal cell death despite sustained, but moderate, elevations of the free intracellular calcium concentrations. Nick translation experiments on isolated nuclei or cells from cerebellar cultures chronically exposed to LAA revealed increased radioactive nucleotide incorporation indicative of DNA nicking. This LAA effect was dose-dependent and suppressed by NMDA receptor antagonists. Cultures treated for 24 hr with LAA and subjected to in situ nick translation showed an intense nuclear labeling of neurons but not glia, which could be abolished by MK801. A similar labeling was also observed in altered nuclei of granule neurons acutely exposed to high glutamate concentrations or undergoing an apoptotic cell death. Although the TUNEL labeling method detected no DNA double-strand breaks in LAA-treated cerebellar cultures, it displayed clear evidence of DNA damage during acute glutamate excitotoxicity or during apoptosis. However, Southern blot analysis of nuclear DNA revealed a DNA laddering only in apoptotic cell death. Our results demonstrate that DNA damage, characterized by DNA single-strand breaks, is an early event in chronic, moderate excitotoxicity. This type of DNA degradation, which appears before any nuclear morphological changes, is distinct from the massive DNA single- and/or double-strand damages observed during acute glutamate excitotoxicity or apoptosis.

The effects of chronic haloperidol administration on GABA-immunoreactive axon terminals in rat medial prefrontal cortex.

Several reports have suggested that chronic haloperidol (HAL) treatment induces ultrastructural changes in synapses of substantia nigra, corpus striatum, and medial prefrontal cortex (mPFC) of rat brain. The effects of HAL on specific cortical transmitter systems, however, are not well characterized. Recent studies have indicated that there may be a loss of gamma-aminobutyric acid (GABA)ergic cells in anterior cingulate cortex of schizophrenic subjects and this hypothesis has prompted interest in the question of whether dopamine receptor antagonists, such as HAL, may influence the activity of this transmitter system. This current report describes a quantitative light microscopic analysis of GABA-immunolabeled axosomatic terminals in mPFC of rats treated with HAL decanoate (0.5 mg/kg/day, i.m.) for a period of 4 months. GABA-containing terminals were visualized with an avidin-biotin immunoperoxidase method for localizing anti-GABA antibodies. Computer-assisted image processing was employed to determine the total number of pixels representing GABA-immunoreaction product in axon terminals that were in direct apposition to pyramidal cell bodies. Drug-treated animals showed a significant increase in the number of pixels representing GABA-immunoreaction product in axosomatic terminals of layers II, III, VI, and VI (93%, 63%, 31%, and 43%, respectively). These data are consistent with the idea that chronic HAL administration may be associated with a significant increase in the amount of GABA present in terminals surrounding pyramidal neurons of rat mPFC. The fact that GABA-containing terminals showed the greatest increase in layer II is not consistent with the known distribution of dopamine afferents to this region which is lowest in superficial laminae. Based on the laminar distribution of non-dopaminergic receptor types that have a high affinity for HAL, the effect of this drug on GABAergic transmission could potentially involve changes that are mediated through mechanisms in which 5-HT2 or sigma opiate receptors play a role.

Rapid kinetics of potassium-evoked release of acetylcholine from rat brain synaptosomes: analysis by rapid superfusion.

The rapid kinetics of spontaneous and evoked [3H]acetylcholine efflux from synaptosomes was investigated using the technique of rapid superfusion. Synaptosomes were isolated from whole rat brain and the intraterminal pool of acetylcholine was radiolabeled by preincubation with [3H]choline. Synaptosomes were retained within the superfusion system on filter disks and superfused with Krebs-bicarbonate buffer, pH 7.4, at flow rates of 0.3-0.5 ml/s. These experimental conditions provided a mixing half-life of 119 ms and efficiency of superfusion of greater than 85%. The kinetics of tritium efflux was followed on the second and subsecond time scales by collection of serial 4.8-s and 50-ms samples for a total of 67.2 and 1.0 s, respectively. Superfusion for 48 s with isoosmotic Krebs buffer containing 10, 20, 30, 50, 75, and 100 mM potassium ion stimulated concentration-dependent tritium release. All of potassium-evoked release, but only 17% of spontaneous release, was calcium-dependent. Kinetic analysis of net (total minus spontaneous) potassium-stimulated release revealed a single calcium-dependent component of release that fit a single exponential function with a half-life of 12.7 s. Analysis of the area under the tritium efflux curves observed on the millisecond time scale revealed that 0.111, 0.550, and 0.614% net tritium release was evoked by superfusion for 750 ms with isoosmotic buffer containing 20, 50, and 100 mM KCl, respectively. Consistent with the results observed on the second time scale, a small fraction of spontaneous release and all of potassium-evoked release observed on the millisecond time scale were calcium-dependent. These data indicate that the technique of rapid superfusion can be utilized for the direct investigation of spontaneous and evoked [3H]acetylcholine release, as well as the factors that regulate this release from brain synaptosomes on the second and millisecond time scales.