The symptoms of Alzheimer's disease (AD), or the transmissible spongiform encephalopathies, otherwise known as the prion diseases, are thought to arise after the dysfunction or degeneration of neurons. In these diseases, extracellular aggregates of insoluble, misfolded, fibrillar proteins are thought to cause the neuronal damage. In AD, fibrils consisting of amyloid-β peptides are formed following the cleavage of the amyloid precursor protein by γ-secretases 1. In the prion diseases the cellular prion protein (designated PrP^C) is converted into a disease-related isoforms (PrP^d), in a process whereby a portion of the α-helix and random coil structure in PrP^C is refolded into a β-pleated sheet 2.

Standard techniques to study the mechanisms of neuronal loss in vitro include incubating neuronal cells with peptides derived from the PrP protein 3 or from amyloid-β 4. The neuronal injury induced by these peptides include events characteristic of apoptosis such as surface blebbing, chromatin condensation and DNA fragmentation 3. In the present study we examined the neurotoxicity of fibrillar peptides by exposing the SH-SY5Y neuroblastoma cell line, or murine primary cortical neurons, to amyloid-β_1-42, to a peptide derived from the human PrP protein (HuPrP82-146), or to a synthetic murine "miniprion" (sPrP106) 5. Further studies examined the interactions between amyloid-β_1-42 or sPrP106 damaged neurons and microglia.

Extracts from the leaves of the Ginkgo biloba tree are becoming increasingly popular as a treatment that is claimed to reduce memory loss and the symptoms of mild cognitive disorders including AD. However, there remains considerable debate regarding the mechanisms of action of these preparations, or even whether such preparations have any clinical benefit. While some published studies conclude that the use of a standardized extract of the leaves of the Ginkgo biloba tree (EGb 761) reduces the symptoms of mild cognitive disorders including AD 678, more recent studies have failed to show clinical benefit with other preparations 9. In tissue culture studies the EGb 761 extract reduces amyloid-β aggregation and caspase-3 activity 10, and protects hippocampal neurons against amyloid-β 11. Since the EGb 761 extract contains many compounds including ginkgolides and the flavonoglycosides myricetin or quercetin, it is not clear which of these compounds provide the protective effect. In the present study, the main components of the EGb 761 extract were examined for their effects on SH-SY5Y neuroblastoma cells, primary cortical neurons and microglia. Low concentrations of the ginkgolides A or B were shown to protect neurons against PrP peptides or amyloid-β_1-42, and to reduce microglial killing of damaged neurons.

In the present study the affects of individual components of the EGb 761 extract on some of the pathological processes that occur during AD or prion diseases were examined. Although previous studies suggest that the flavonoglycosides had protective properties against oxidative stress in vitro 18, we were unable to demonstrate a protective effect of myricetin or quercetin against PrP peptides or amyloid-β_1-42. Moreover, recent studies reported that these flavonoglycosides have limited bioavailability after oral administration 19 raising doubts as to whether such compounds are responsible for the protective effects of the EGb 761 extract. In contrast, both ginkgolides A and B prevented neuronal death in response to sPrP106, PrP peptides or to amyloid-β_1-42. The protective effects of the ginkgolides were dose-dependent, and both the ginkgolides protected neurons at nanomolar concentrations. Moreover, we found no evidence that the protective effect of the ginkgolide B could be overcome by increasing the concentration of sPrP106 or amyloid-β_1-42. In AD and prion diseases the loss of neurons in vivo occurs though apoptosis 320, and although fibrillar PrP/amyloid-β peptides kill neurons in vitro the mechanisms that activate neuronal apoptosis remain unknown. In the present study both sPrP106 and amyloid-β_1-42 induced caspase-3, a marker of apoptosis that is increased in murine scrapie 21 and AD 22. The presence of ginkgolides greatly reduced the activation of apoptotic pathways in these cells. The presence of ginkgolides also reduced microglial killing of neurons damaged by sPrP106 or amyloid-β_1-42. Microglia respond to changes in neurons induced by PrP peptides 12 and our data are compatible with the hypothesis that pre-treatment with the ginkgolides prevents the PrP-induced changes that activate microglia.

It is worth noting that for many Ginkgo biloba extracts, extraction procedures are used that optimise the flavonoglycoside content. However, these procedures may result in extracts that contain different amounts of ginkgolides. Variations in the ginkgolide content of different extracts may be a factor in the variability of results obtained in clinical studies. The ginkgolides inhibit the activity of PAF 23 that is produced in neurons via the remodeling pathway 13, and a recent study showed that PAF antagonists protect neurons against the toxicity of PrP or amyloid-β peptides 24. In the present study ginkgolide treated cells were resistant to the toxicity of PAF as well as that of PrP peptides or amyloid-β_1-42. It was noteworthy that ginkgolide B was consistently stronger than that of ginkgolide A, consistent with prior reports that ginkgolide B has greater PAF antagonism than ginkgolide A 25.

Further studies were designed to determine how antagonism of PAF might prevent the toxicity of PrP or amyloid-β peptides. Levels of prostaglandin E₂ (a measure of COX activity) are increased in the cerebrospinal fluid of patients with either Creutzfeldt-Jakob disease 26 or AD 27. A causative role for prostaglandins in neurotoxicity was strengthened by in vitro observations that neurons treated with COX inhibitors, the enzymes that convert arachidonic acid to prostaglandins, are also resistant to prions 14 or amyloid-β_1-42 15. These results are compatible with epidemiological data that show that the use of COX inhibitors reduces the onset and severity of AD 28. In the present study the addition of PAF stimulated the production of prostaglandin E₂ from neuronal cells suggesting that it activates neuronal COX. Furthermore, the ginkgolides reduced prostaglandin E₂ production after the addition of arachidonic acid or PAF, as well as in response to PrP or amyloid-β peptides, and cells pre-treated with ginkgolide B were partially resistant to the toxicity of arachidonic acid. These results are compatible with the hypothesis that ginkgolides modulate the COX related production of toxic prostaglandins. It is worth noting that in cell free systems the ginkgolides did not affect the conversion of arachidonic acid to prostaglandins showing they did not have a direct effect on neuronal COX (data not shown).

The present study showed that neurons treated with nanomolar concentrations ginkgolides A or B are resistant to the otherwise toxic effects PrP peptides or amyloid-β_1-42. This protective effect coincided with reduced neuronal prostaglandin E₂ production indicating that neuronal COX was impaired, compatible with previous observations that COX inhibitors protected neurons against PrP peptides 14 or amyloid-β_1-42 15. However, unlike aspirin or ibuprofen, the ginkgolides did not have a direct effect on COX enzymes. The protective effect of the ginkgolides in vitro correlated with their ability to inhibit PAF 25. These compounds also prevented amyloid-β_1-42 or sPrP106 treated neurons activating microglia resulting in increased neuronal survival in co-cultures. Recent studies showed that the bioavailability of ginkgolides after oral administration is high 19, although studies to determine if the ginkgolides cross the blood brain barrier and penetrate the central nervous system are still forthcoming. These results suggest that the ginkgolides have the potential to reduce neuronal loss in prion diseases or AD. However, the processes that underlie neuronal loss in prion or Alzheimer's diseases are varied and more complex than the simple model of neurodegeneration presented here, and further study is required to evaluate the benefits of these compounds.

Alzheimer's disease (AD), cyclo-oxygenases (COX), a standardized extract of the leaves of the Ginkgo biloba tree (EGb 761), fetal calf serum (FCS), flourometric immunosorbent enzyme assay (FIENA), a peptide containing amino acid residues 82 to 146 of the human PrP protein (HuPrP82-146), platelet activating factor (PAF), cellular prion protein (PrP^C), disease-associated isoform of the prion protein (PrP^d), synthetic miniprion (sPrP106).

None declared.

CB was responsible for the conception, planning of performance of experiments, and for writing this manuscript. Both MS and AW contributed to the planning of experiments, interpretation of results and the writing of the manuscript.