Fucoidan inhibits cellular and neurotoxic effects of beta-amyloid (A beta) in rat cholinergic basal forebrain neurons

The deposition of beta-amyloid protein (A beta), a 39-43 amino acid peptide, in the brain and a loss of cholinergic neurons in the basal forebrain are pathological hallmarks of Alzheimer's disease (AD). Seaweeds consumed in Asia contain Fucoidan, a sulfated polysaccharide. Fucoidan has been known to exhibit various biological actions, such as an anti-inflammatory and antioxidant action. In this study, using whole-cell patch clamp recordings we examined the effects of Fucoidan on A beta-induced whole-cell currents in acutely dissociated rat basal forebrain neurons. We further investigated whether Fucoidan is capable of blocking A beta neurotoxicity in primary neuronal cultures. In dissociated cells, bath application of A beta(25-35) (1 microM) caused a reduction of the whole-cell currents by 16%. Fucoidan, in a dose-dependent manner, blocks the A beta(25-35) reduction of whole-cell currents. Exposure of A beta(25-35) (20 microM) or A beta(1-42) (20 microM) to rat cholinergic basal forebrain cultures for 48 h resulted in 40-60% neuronal death, which was significantly decreased by pretreatment of cultures with Fucoidan (0.1-1.0 microM). Fucoidan also attenuated A beta-induced down-regulation of phosphorylated protein kinase C. A beta(1-42)-induced generation of reactive oxygen species was blocked by prior exposure of cultures to Fucoidan. Furthermore, A beta activation of caspases 9 and 3, which are signaling pathways implicated in apoptotic cell death, is blocked by pretreatment of cultures with Fucoidan. These results show that Fucoidan is able to block A beta-induced reduction in whole-cell currents in basal forebrain neurons and has neuroprotective effects against A beta-induced neurotoxicity in basal forebrain neuronal cultures.     

Aluminum uptake and effects on transferrin mediated iron uptake in primary cultures of rat neurons, astrocytes and oligodendrocytes

Transferrin (Tf) is known primarily for its role in the transport and cellular uptake of iron (Fe). Tf is also the major serum binding protein for Al. In this study, primary rat oligodendrocyte, neuron and astrocyte cultures were found to differ in Tf mediated Fe and Al uptake and in the effect of Al-Tf on Fe-Tf uptake during 4 h incubation periods. When incubated with Al-Tf (1.25 microM), oligodendrocytes displayed a 3- to 4-fold increase (p=.0002) in Al, neurons demonstrated a much smaller (p=.06) increase, and no increase was seen for astrocytes. When incubated with equimolar Al citrate or Al chloride, no increase in cellular Al was seen in any of the three cell types. Oligodendrocytes, astrocytes and neurons all demonstrated greater 59Fe uptake from Fe-Tf than Fe chloride. This uptake could be inhibited by excess Fe-Tf in oligodendrocytes and neurons, but not astrocytes. A small but significant inhibition of 59Fe uptake from Fe-Tf was seen after addition of Al-Tf to the incubation medium of oligodendrocytes, but not neurons or astrocytes. Oligodendrocytes may be particularly vulnerable to the accumulation of excess intracellular Al, and to interference of Al with Fe uptake. Such effects could contribute to Al-induced neurotoxicity if they result in altered myelin formation or maintenance.     

Glutamate uptake disguises neurotoxic potency of glutamate agonists in cerebral cortex in dissociated cell culture.

The pharmacological properties of glutamate agonists were compared in astrocyte-rich and astrocyte-poor cultures derived from embryonic rat cerebral cortex. The object of this investigation was to determine the extent to which glutamate uptake might influence the receptor-mediated neurotoxic actions of these compounds. In astrocyte-rich cultures, using 30 min exposures, we observed that the potencies of the poorly transported agonists NMDA (35 microM) and D-glutamate (89 microM) were higher than that of L-glutamate (205 microM). In astrocyte-poor cultures, L-glutamate was much more potent, with an EC50 of 5 +/- 4 microM (3-12 microM), for a 30 min exposure, whereas the potencies of NMDA and D-glutamate were essentially unchanged. L- and D-aspartate were also more effective in astrocyte-poor cultures, again with EC50 values of approximately 6-10 microM, as compared with 130 and 108 microM, respectively, in astrocyte-rich cultures. In other experiments, blocking sodium-dependent glutamate uptake in astrocyte-rich cultures, by using a sodium-free medium, made glutamate as potent an agonist as in astrocyte-poor cultures. Finally, we directly assessed the glutamate uptake system in astrocyte-rich and astrocyte-poor cultures and found that uptake was reduced approximately 25-fold in the astrocyte-poor cultures. These results show that in the presence of abundant astrocytes the neurotoxic potencies of L-glutamate, L-aspartate, and D-aspartate are substantially under-estimated.     

Prevention of 1-methyl-4-phenylpyridinium- and 6-hydroxydopamine-induced nitration of tyrosine hydroxylase and neurotoxicity by EUK-134, a superoxide dismutase and catalase mimetic, in cultured dopaminergic neurons

Oxidative stress has been implicated in the selective degeneration of dopaminergic (DAergic) neurons in Parkinson's disease (PD). In this study, we tested the efficacy of EUK-134, a superoxide dismutase (SOD) and catalase mimetic, on the nitration of tyrosine hydroxylase (TH), a marker of oxidative stress, and neurotoxicity produced by 1-methyl-4-phenylpyridinium (MPP(+)) and 6-hydroxydopamine (6-OHDA) in primary DAergic neuron cultures. Exposure of cultures to 10 microM MPP(+) reduced dopamine (DA) uptake and the number of tyrosine hydroxylase immunoreactive (THir) neurons to 56 and 52% of control, while exposure to 30 microM 6-OHDA reduced DA uptake and the number of THir neurons to 58 and 59% of control, respectively. Pretreatment of cultures with 0.5 microM EUK-134 completely protected DAergic neurons against MPP(+)- and 6-OHDA-induced neurotoxicity. Exposure of primary neuron cultures to either MPP(+) or 6-OHDA produced nitration of tyrosine residues in TH. Pretreatment of cultures with 0.5 microM EUK-134 completely prevented MPP(+)- or 6-OHDA-induced nitration of tyrosine residues in TH. Taken together, these results support the idea that reactive oxygen species (ROS) are critically involved in MPP(+)- and 6-OHDA-induced neurotoxicity and suggest a potential therapeutic role for synthetic catalytic scavengers of ROS, such as EUK-134, in the treatment of PD.     

Ibogaine alters synaptosomal and glial glutamate release and uptake

Ibogaine has aroused expectations as a potentially innovative medication for drug addiction. It has been proposed that antagonism of the NMDA receptor by ibogaine may be one of the mechanisms underlying its antiaddictive properties; glutamate has also been implicated in ibogaine-induced neurotoxicity. We here report the effects of ibogaine on [3H]glutamate release and uptake in cortical and cerebellar synaptosomes, as well as in cortical astrocyte cultures, from mice and rats. Ibogaine (2-1000 microM) had no effects on glutamate uptake or release by rat synaptosomes. However, ibogaine (500-1000 microM) significantly inhibited the glutamate uptake and stimulated the release of glutamate by cortical (but not cerebellar) synaptosomes of mice. In addition, ibogaine (1000 microM) nearly abolished glutamate uptake by cortical astrocyte cultures from rats and mice. The data provide direct evidence of glutamate involvement in ibogaine-induced neurotoxicity.     

Neurons depend on astrocytes in a coculture system for protection from glutamate toxicity.

Glutamate can be toxic to neurons although it is a neurotransmitter. Regulation of extracellular glutamate levels is essential for prevention of glutamate neurotoxicity. Astrocytes play a major role in clearance of glutamate released by neurons. A coculture system combining cerebellar cells and astrocytes was employed to investigate the astrocytic control of glutamate toxicity. Coculture of astrocytes with cerebellar neurons enhanced uptake of glutamate by astrocytes. Inhibition of glutamate uptake in a coculture system led to death of cerebellar cells. This toxicity could be inhibited by MK801. However, in the presence of the glutamate uptake inhibitor, there was no increase in glutamate in the cultures compared to when the neurons were not cocultured. This indicated that neurons become more susceptible to glutamate toxicity in the presence of astrocytes and thus become dependent on astrocytes for prevention of glutamate toxicity. Astrocytes treated with conditioned medium from cerebellar cells did not show an increase in glutamate uptake but medium from astrocytes exposed to neuron conditioned medium was toxic to cerebellar cells. This toxicity was due to glutamate present in the medium. This suggests that a soluble factor released by neurons signals to astrocytes that neurons are present and stimulates a signal back to neurons which causes an increased sensitivity to glutamate toxicity.     

Molecular mechanisms of glutamate neurotoxicity in mixed cultures of NT2-derived neurons and astrocytes: protective effects of coenzyme Q10

Although glutamate excitotoxicity has long been implicated in neuronal cell death associated with a variety of neurological disorders, the molecular mechanisms underlying this process are not yet fully understood. In part, this is due to the lack of relevant experimental cell systems recapitulating the in vivo neuronal environment, mainly neuronal-glial interactions. To explore these mechanisms, we have analyzed the cytotoxic effects of glutamate on mixed cultures of NT2/N neurons and NT2/A astrocytes derived from human NT2/D1 cells. In these cultures, the neurons were resistant to glutamate alone (up to 2 mM for 24-48 hr), but they responded to a simultaneous exposure to 0.5 mM glutamate and 6 hr of hypoxia. Neuronal cell death occurred during subsequent periods of reoxygenation (>30% within 24 hr). This was associated with a marked decrease of intracellular ATP, a significant increase in reactive oxygen species (ROS) and downregulation of glutamate uptake by astrocytes. Thus, under energy failure and high levels of ROS production, only the neurons from these mixed cultures succumbed to glutamate neurotoxicity; the astrocytic cells remained unaffected by the treatment. Taken together, our data suggested that glutamate excitotoxicity might be due to the energy failure and oxidative stress affecting the properties of the NMDA glutamate receptors and causing impairment of glutamate transporters. Cells pretreated for 72 hr with 10 microg/ml of coenzyme Q(10) (functions both as a ROS scavenger and co-factor of mitochondrial electron transport), were protected, suggesting a useful role for coenzyme Q(10) in treatments of neurological diseases associated with glutamate excitotoxicity. A model of the complex interactions between neurons and astrocytes in regulating glutamate metabolism is presented.     

Arachidonic acid inhibits uptake of glutamate and glutamine but not of GABA in cultured cerebellar granule cells

The effects of arachidonic acid (20:4) on the uptake of glutamate were studied in primary cultures of cerebellar granule cells and were compared to cortical neurons and astrocytes. At a dose of 0.005 mM, the glutamate uptake was significantly inhibited in cerebellar granule cells. This inhibition was dose and time dependent. The uptake of glutamate was equally sensitive to 20:4 in primary cell cultures of cortical neurons, whereas the uptake in astrocytes was much less sensitive to 20:4. Glutamine uptake was inhibited by 20:4 in cultured cerebellar granule cells and cerebral cortical astrocytes but was not affected in cerebral cortical neurons. Furthermore, the uptake of gamma-aminobutyric acid was not affected by 20:4 in cerebellar granule cells.     

Involvement of endoplasmic reticulum Ca2+ release through ryanodine and inositol 1,4,5-triphosphate receptors in the neurotoxic effects induced by the amyloid-beta peptide

Studies with in-vitro-cultured neurons treated with amyloid-beta (A beta) peptides demonstrated neuronal loss by apoptosis that is due, at least in part, to the perturbation of intracellular Ca(2+) homeostasis. In addition, it was shown that an endoplasmic reticulum (ER)-specific apoptotic pathway mediated by caspase-12, which is activated upon the perturbation of ER Ca(2+) homeostasis, may contribute to A beta toxicity. To elucidate the involvement of deregulation of ER Ca(2+) homeostasis in neuronal death induced by A beta peptides, we have performed a comparative study using the synthetic peptides A beta(25-35) or A beta(1-40) and thapsigargin, a selective inhibitor of Ca(2+) uptake into the ER. Incubation of cortical neurons with thapsigargin (2.5 microM) increased the intracellular Ca(2+) levels and activated caspase-3, leading to a significant increase in the number of apoptotic cells. Similarly, upon incubation of cortical cultures with the A beta peptides (A beta(25-35), 25 microM; A beta(1-40), 0.5 microM), we observed a significant increase in [Ca(2+)](i), in caspase-3-like activity, and in number of neurons exhibiting apoptotic morphology. The role of ER Ca(2+) release through ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate receptors (IP(3)R) in A beta neurotoxicity has been also investigated. Dantrolene and xestospongin C, inhibitors of ER Ca(2+) release through RyR or IP(3)R, were able to prevent the increase in [Ca(2+)](i) and the activation of caspase-3 and to protect partially against apoptosis induced by treatment with A beta(25-35) or A beta(1-40). In conclusion, our results demonstrate that the release of Ca(2+) from the ER, mediated by both RyR and IP(3)R, is involved in A beta toxicity and can contribute, together with the activation of other intracellular neurotoxic mechanisms, to A beta-induced neuronal death. This study suggests that A beta accumulation may have a key role in the pathogenesis of AD as a result of deregulation of ER Ca(2+) homeostasis.     

beta-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity.

In Alzheimer's disease (AD), abnormal accumulations of beta-amyloid are present in the brain and degenerating neurons exhibit cytoskeletal aberrations (neurofibrillary tangles). Roles for beta-amyloid in the neuronal degeneration of AD have been suggested based on recent data obtained in rodent studies demonstrating neurotoxic actions of beta-amyloid. However, the cellular mechanism of action of beta-amyloid is unknown, and there is no direct information concerning the biological activity of beta-amyloid in human neurons. We now report on experiments in human cerebral cortical cell cultures that tested the hypothesis that beta-amyloid can destabilize neuronal calcium regulation and render neurons more vulnerable to environmental stimuli that elevate intracellular calcium levels. Synthetic beta-amyloid peptides (beta APs) corresponding to amino acids 1-38 or 25-35 of the beta-amyloid protein enhanced glutamate neurotoxicity in cortical cultures, while a peptide with a scrambled sequence was without effect. beta APs alone had no effect on neuronal survival during a 4 d exposure period. beta APs enhanced both kainate and NMDA neurotoxicity, indicating that the effect was not specific for a particular subtype of glutamate receptor. The effects of beta APs on excitatory amino acid (EAA)-induced neuronal degeneration were concentration dependent and required prolonged (days) exposures. The beta APs also rendered neurons more vulnerable to calcium ionophore neurotoxicity, indicating that beta APs compromised the ability of the neurons to reduce intracellular calcium levels to normal limits. Direct measurements of intracellular calcium levels demonstrated that beta APs elevated rest levels of calcium and enhanced calcium responses to EAAs and calcium ionophore. The neurotoxicity caused by EAAs and potentiated by beta APs was dependent upon calcium influx since it did not occur in calcium-deficient culture medium. Finally, the beta APs made neurons more vulnerable to neurofibrillary tangle-like antigenic changes induced by EAAs or calcium ionophore (i.e., increased staining with tau and ubiquitin antibodies). Taken together, these data suggest that beta-amyloid destabilizes neuronal calcium homeostasis and thereby renders neurons more vulnerable to environmental insults.     

Glutamate neurotoxicity in cortical cell culture

The central neurotoxicity of the excitatory amino acid neurotransmitter glutamate has been postulated to participate in the pathogenesis of the neuronal cell loss associated with several neurological disease states, but the complexity of the intact nervous system has impeded detailed analysis of the phenomenon. In the present study, glutamate neurotoxicity was studied with novel precision in dissociated cell cultures prepared from the fetal mouse neocortex. Brief exposure to glutamate was found to produce morphological changes in mature cortical neurons beginning as quickly as 90 sec after exposure, followed by widespread neuronal degeneration over the next hours. Quantitative dose-toxicity study suggested an ED50 of 50-100 microM for a 5 min exposure to glutamate. Immature cortical neurons and glia were not injured by such exposures to glutamate. Uptake processes probably do not limit GNT in culture, as the uptake inhibitor dihydrokainate did not potentiate GNT. Possibly reflecting the lack of uptake limitation, glutamate was found to be actually more potent than kainate as a neurotoxin in these cultures, a dramatic reversal of the in vivo potency rank order. Some neurons regularly survived brief glutamate exposure; these possibly glutamate-resistant neurons had electrophysiologic properties, including chemosensitivity to glutamate, that were grossly similar to those of the original population.     

Protein kinase C activity,translocation, and selective isoform subcellular redistribution in the rat cerebral cortex after in vitro ischemia

Protein kinase C (PKC) involvement in ischemia-induced neuronal damage has been investigated in superfused rat cerebral cortex slices submitted to 15 min of oxygen-glucose deprivation (OGD) and in primary cultures of rat cortical neurons exposed to 100 microM glutamate (GLU) for 10 min. OGD significantly increased the total PKC activity in the slices, mostly translocated in the particulate fraction. After 1 hr of reperfusion, the total PKC activity was reduced and the translocated fraction dropped by 84% with respect to the control. Western blot analysis of OGD samples showed an increase in total beta(2) and epsilon PKC isoform levels. After reperfusion, the total levels of alpha, beta(1), beta(2) and gamma isoforms were significantly reduced, whereas the epsilon isoform remained at an increased level. Endogenous GLU release from OGD slices increased to about 15 times the basal values after 15 min of oxygen-glucose deprivation, and to 25 and 35 times the basal level in the presence of the PKC inhibitors staurosporine (0.1 microM) and bisindolylmaleimide (1 microM), respectively. Western blot analysis of GLU-treated cortical neurons showed a significant decrease only in the total level of beta(2) isoforms. Cell survival was reduced to 31% in GLU-treated neuronal cultures; PKC inhibitors were not able to modify this effect. These findings demonstrate that the cell response to OGD and GLU involves PKC in a complex way. The net role played by PKC during OGD may be to reduce GLU release and, consequently, neurotoxicity. The isoforms beta(2) and epsilon are affected the most and may play a significant role in the mechanisms underlying neurotoxicity/neuroprotection.     

Accumulation of extracellular glutamate and neuronal death in astrocyte-poor cortical cultures exposed to glutamine.

The function of astrocytes in cerebral cortex may be studied by comparing the properties of conventional, astrocyte-rich cultures with astrocyte-poor cultures in which astrocyte proliferation has been stringently suppressed. Exposure of astrocyte-poor, but not astrocyte-rich, cultures to fresh medium containing 2 mM glutamine resulted in the death of most neurons within 24 h. This study was undertaken to understand the basis for the apparent toxicity of glutamine in astrocyte-poor cultures. The toxicity of glutamine was found to be mediated by glutamate, which demonstrated an LD50 as a neurotoxin in astrocyte-poor cultures of 2 microM. Exposure of astrocyte-poor (but not astrocyte-rich) cultures to fresh medium containing glutamine for 17.5-24 h resulted in the accumulation of substantial quantities of glutamate (255 +/- 158 microM; mean +/- standard deviation) coincident with the death of neurons in the cultures. Exposure of astrocyte-poor cultures to glutamate in the absence of glutamine did not result in the accumulation of extracellular glutamate. Both the neuronal death and the extracellular glutamate accumulation in astrocyte-poor cultures exposed to glutamine could be blocked by N-methyl-D-aspartate (NMDA) antagonists. These observations suggest that astrocytes as well as glutamine may play an important role in the pathogenesis of glutamate neurotoxicity in the central nervous system.     

Activation of Group II/III metabotropic glutamate receptors attenuates LPS-induced astroglial neurotoxicity via promoting glutamate uptake

Altered glial function that leads to oxidative stress and excitotoxicity may contribute to the initiation or progression of neuronal death in neurodegenerative diseases. We report the pivotal role of astroglial Group II and III metabotropic glutamate receptors (mGluR) against neurotoxicity. Activation of Group II or III mGluR on astrocytes with selective agonists DCG-IV or L-AP4 respectively inhibited astroglial lipopolysaccharide (LPS)-conditioned medium induced apoptosis of primary cultured mesencephalic neurons. Specific Group II or III mGluR antagonists APICA or MSOP completely abolished the neuroprotective effects of DCG-IV and L-AP4. Morphologic analysis showed that DCG-IV or L-AP4 could also attenuate the astroglial neurotoxicity to dopaminergic neurons. Measurement of extracellular glutamate concentration and [(3)H]-glutamate uptake showed that the restoration of glutamate uptake capability in LPS-treated astrocytes might be involved in the neuroprotective effects of activating astroglial Group II or III mGluR. Furthermore, we found that the repression of astroglial uptake function could be revived by GSH, and both Group II and III mGluR agonists could recover the endogenous reduced glutathione (GSH) level in LPS-treated astrocytes. These results suggested that the possible mechanisms of neuroprotection by either Type II or Type III mGluR activation may involve restoration of endogenous GSH, in turn affording recovery of astroglial capability to take up glutamate.     

Blockade of astrocytic glutamate uptake in the prefrontal cortex induces anhedonia

Major depression is associated with both dysregulated glutamatergic neurotransmission and fewer astrocytes in limbic areas including the prefrontal cortex (PFC). These deficits may be functionally related. Notably, astrocytes regulate glutamate levels by removing glutamate from the synapse via the glutamate transporter (GLT-1). Previously, we demonstrated that central blockade of GLT-1 induces anhedonia and c-Fos expression in the PFC. Given the role of the PFC in regulating mood, we hypothesized that GLT-1 blockade in the PFC alone would be sufficient to induce anhedonia in rats. We microinjected the GLT-1 inhibitor, dihydrokainic acid (DHK), into the PFC and examined the effects on mood using intracranial self-stimulation (ICSS). At lower doses, intra-PFC DHK produced modest increases in ICSS thresholds, reflecting a depressive-like effect. At higher doses, intra-PFC DHK resulted in cessation of responding. We conducted further tests to clarify whether this total cessation of responding was related to an anhedonic state (tested by sucrose intake), a nonspecific result of motor impairment (measured by the tape test), or seizure activity (measured with electroencephalogram (EEG)). The highest dose of DHK increased latency to begin drinking without altering total sucrose intake. Furthermore, neither motor impairment nor evidence of seizure activity was observed in the tape test or EEG recordings. A decrease in reward value followed by complete cessation of ICSS responding suggests an anhedonic-like effect of intra-PFC DHK; a conclusion that was substantiated by an increased latency to begin sucrose drinking. Overall, these results suggest that blockade of astrocytic glutamate uptake in the PFC is sufficient to produce anhedonia, a core symptom of depression.     

Methylmercury inhibits cysteine uptake in cultured primary astrocytes, but not in neurons

The maintenance of adequate intracellular glutathione (GSH) concentrations is dependent on the availability and transport of the rate-limiting substrate, cysteine. A suggested mechanism of methylmercury (MeHg) neurotoxicity in brain involves the formation of oxygen radicals, and a decrease in intracellular levels of GSH. Recently, we have characterized various cysteine transport systems (both Na(+)-dependent and -independent) in cerebrocortical astrocytes and hippocampal neurons. The present study was carried out to investigate the effect of MeHg on cysteine uptake in both astrocytes and neurons, and to determine whether cysteine transport is differentially affected in the two cell types by MeHg treatment. Sixty-minute pretreatment with MeHg caused significant concentration-dependent inhibition in cysteine uptake in astrocytes, but not in neurons. As most of the cysteine transport is Na(+)-dependent (80-90% of total), additional studies focused on MeHg's effect on the Na(+)-dependent cysteine transporters X(AG(-)) and ASC. An additive inhibitory effect on cysteine uptake was observed in astrocytes treated with MeHg (5 microM) plus sub-maximal inhibitory concentrations (0.1 and 0.5 mM) of threo-beta-hydroxy-aspartate (THA), a specific inhibitor of the Na(+)-dependent transporter, X(AG(-)), compared to astrocytes treated with MeHg (P<0.001) or THA alone (P<0.05). There was no additive effect of MeHg and maximal inhibitory concentrations of THA (1.0 and 5.0 mM) on astrocytic cysteine uptake inhibition. Additional studies examined the sensitivity of the Na(+)-dependent ASC transport system to MeHg treatment. Maximal inhibitory concentration of L-serine (10 mM) alone had a rather modest inhibitory effect on cysteine uptake, and when applied in the presence of MeHg there was no additive effect. These results suggest that the inhibition of cysteine uptake by MeHg in astrocytes occurs through specific inhibition of both the X(AG(-)) as well as the ASC transport system.     

A beta 25-35 induces presynaptic changes in organotypic hippocampal slice cultures

Memory loss in Alzheimer's disease (AD) may be related to synaptic defects in damaged hippocampal neurons. We investigated the relationship between amyloid peptide A beta 25-35-induced neuronal death pattern and presynaptic changes in organotypic hippocampal slice cultures. In propidium iodide (PI) uptake and annexin V labeling, A beta 25-35-induced neuronal damage dramatically increased in a concentration dependent manner, indicating both types of cell death. In ultrastructural analysis, apoptotic features in CA1 and CA3 area and synaptic disruption in stratum lucidum were detected in A beta 25-35-treated slices. Immunofluorescence and Western blot analysis for caspase-3 showed A beta 25-35 concentration dependently induced caspase-3 activation. Immunofluorescence and Western blot analysis to determine changes in presynaptic marker proteins demonstrated that expression of synaptosomal-associated protein-25 (SNAP-25) and synaptophysin were reduced by A beta 25-35 in CA1, CA3 and DG area at concentrations >2.5 microM. In conclusion, A beta 25-35-induced apoptotic cell death and caspase-3 activation at relatively low concentration, and induced synaptic disruption and loss of synaptic marker protein at concentrations >2.5 microM in organotypic hippocampal slice cultures. These suggest that A beta 25-35-induced apoptosis via triggering caspase-3 activation and lead to synaptic dysfunction in organotypic hippocampal slice cultures.     

Beta-amyloid fragment 25-35 causes mitochondrial dysfunction in primary cortical neurons

Beta-amyloid deposition and compromised energy metabolism both occur in vulnerable brain regions in Alzheimer's disease. It is not known whether beta-amyloid is the cause of impairment of energy metabolism, nor whether impaired energy metabolism is specific to neurons. Our results, using primary neuronal cultures, show that 24-h incubation with A beta(25-35) caused a generalized decrease in the specific activity of mitochondrial enzymes per milligram of cellular protein, induced mitochondrial swelling, and decreased total mitochondrial number. Incubation with A beta(25-35) decreased ATP concentration to 58% of control in neurons and 71% of control in astrocytes. Levels of reduced glutathione were also lowered by A beta(25-35) in both neurons (from 5.1 to 2.9 nmol/mg protein) and astrocytes (from 25.2 to 14.9 nmol/mg protein). We conclude that 24-h treatment with extracellular A beta(25-35) causes mitochondrial dysfunction in both astrocytes and neurons, the latter being more seriously affected. In astrocytes mitochondrial impairment was confined to complex I inhibition, whereas in neurons a generalized loss of mitochondria was seen.     

Gliotoxicity in hippocampal cultures is induced by transportable, but not by nontransportable, glutamate uptake inhibitors

Extracellular glutamate is kept below a toxic level by glial and neuronal glutamate transporters. Here we show that the transportable glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (t-PDC) induced cell death in mature, but not in immature, hippocampal neuron-enriched cultures. The cell death produced by a 24-hr treatment with t-PDC was dose-dependent and reached 85% of the cell population at a 250 microM concentration at 23 days in vitro (DIV). Immunocytochemistry experiments showed that, under these experimental conditions, t-PDC killed not only neurons as expected but also glial cells. The N-methyl-D-aspartate (NMDA) antagonist D-2-aminophosphonovalerate (D-APV; 250 microM) only partially reversed this toxicity, completely protecting the neuronal cell population but not the glial population. The antioxidant compounds alpha-tocopherol or Trolox, used at concentrations that reverse the oxidative stress-induced toxicity, did not block the gliotoxicity specifically produced by t-PDC in the presence of D-APV. The nontransportable glutamate uptake inhibitor DL-threo-beta-benzyloxyaspartate (TBOA) elicited cell death only in mature, but not in immature, hippocampal cultures. The TBOA toxic effect was dose dependent and reached a plateau at 100 microM in 23-DIV cultures. About 50% of the cell population died. TBOA affected essentially the neuronal population. D-APV (250 microM) completely reversed this toxicity. It is concluded that nontransportable glutamate uptake inhibitors are neurotoxic via overactivation of NMDA receptors, whereas transportable glutamate uptake inhibitors induce both an NMDA-dependent neurotoxicity and an NMDA- and oxidative stress-independent gliotoxicity, but only in mature hippocampal cultures.     

The effect of an uptake inhibitor (dihydrokainate) on endogenous excitatory amino acids in the lamprey spinal cord as revealed by microdialysis

Microdialysis was utilized to test the effects of the uptake inhibitor dihydrokainate (DHK) on endogenous amino acid levels in the lamprey spinal cord in vitro. The level of L-glutamate increased markedly (165%) in the presence of DHK, whereas the level of the glutamate precursor L-glutamine decreased (53%). Since DHK can potentiate or evoke fictive locomotion in the lamprey spinal cord, it is suggested that L-glutamate is released by neurons which take part in the activation of the spinal locomotor network.