Phospholipase A2 inhibitors protect against prion and Abeta mediated synapse degeneration

Abstract

Background: An early event in the neuropathology of prion and Alzheimer's diseases is the loss of synapses and a corresponding reduction in the level of synaptophysin, a pre-synaptic membrane protein essential for neurotransmission. The molecular mechanisms involved in synapse degeneration in these diseases are poorly understood. In this study the process of synapse degeneration was investigated by measuring the synaptophysin content of cultured neurones incubated with the prion derived peptide (PrP82-146) or with Abeta1-42, a peptide thought to trigger pathogenesis in Alzheimer's disease. A pharmacological approach was used to screen cell signalling pathways involved in synapse degeneration.

Results: Pre-treatment with phospholipase A2 inhibitors (AACOCF3, MAFP and aristolochic acids) protected against synapse degeneration in cultured cortical and hippocampal neurones incubated with PrP82-146 or Abeta1-42. Synapse degeneration was also observed following the addition of a specific phospholipase A2 activating peptide (PLAP) and the addition of PrP82-146 or Abeta1-42 activated cytoplasmic phospholipase A2 within synapses. Activation of phospholipase A2 is the first step in the generation of platelet-activating factor (PAF) and PAF receptor antagonists (ginkgolide B, Hexa-PAF and CV6029) protected against synapse degeneration induced by PrP82-146, Abeta1-42 and PLAP. PAF facilitated the production of prostaglandin E2, which also caused synapse degeneration and pre-treatment with the prostanoid E receptor antagonist AH13205 protected against PrP82-146, Abeta1-42 and PAF induced synapse degeneration.

Conclusions: Our results are consistent with the hypothesis that PrP82-146 and Abeta1-42trigger abnormal activation of cytoplasmic phospholipase A2 resident within synapses, resulting in elevated levels of PAF and prostaglandin E2that cause synapse degeneration. Inhibitors of this pathway that can cross the blood brain barrier may protect against the synapse degeneration seen during Alzheimer's or prion diseases.

Figure 1

PLA₂ inhibitors protected cortical neurones against PrP82-146 induced synapse degeneration. (A) The synaptophysin content of cortical neurones pre-treated for with a vehicle control (■), 1 μM AACOCF₃ (○), 1 μM MAFP (●), 5 μg/ml aristolochic acid (□), 10 μM U73122 (▲) or 10 μM ethyl-18-OCH₃ (△) and subsequently incubated with varying concentrations of PrP82-146 for 24 hours. Values shown are the mean average amount of synaptophysin ± standard deviation (SD), n = 15. (B) Immunoblots showing the amount of synaptophysin and β-actin in cell extracts from neurones incubated with PrP82-146 (1.25 - 0.05 μM).

Figure 2

PLA₂ inhibitors protected hippocampal neurones against PrP82-146 induced synapse degeneration. The synaptophysin content of cultured hippocampal neurones pre-treated with a vehicle control (■), 1 μM AACOCF₃ (○) or 1 μM MAFP (●) and incubated with varying concentrations of PrP82-146 for 24 hours. Values shown are the mean average amount of synaptophysin ± SD, n = 9.

Figure 3

PLA₂ inhibitors protected against Aβ_1-42 induced synapse degeneration. (A) The synaptophysin content of cortical neurones pre-treated with a vehicle control (■), 1 μM AACOCF₃ (○) or 1 μM MAFP (●) and incubated with varying concentrations of Aβ_1-42 for 24 hours. Values shown are the mean average amount of synaptophysin ± SD, n = 12. (B) The synaptophysin content of hippocampal neurones pre-treated with a vehicle control (■) or 1 μM AACOCF₃ (○) and incubated with varying concentrations of Aβ_1-42 for 24 hours. Values shown are the mean average amount of synaptophysin ± SD, n = 6.

Figure 4

PLA₂ inhibitors protected against 7PA2-CM induced synapse degeneration. (A) The synaptophysin content of cortical neurones incubated for 24 hours with 7PA2-CM (□) or CHO-CM (■). Values shown are the mean average amount of synaptophysin ± SD, n = 12. (B) The synaptophysin content of cortical neurones incubated for pre-treated with a vehicle control (□), 1 μM AACOCF₃ (■) or 1 μM MAFP (striped bars) and incubated with doubling dilutions of 7PA2-CM for 24 hours. Values shown are the mean average amount of synaptophysin ± SD, n = 12.

Figure 5

PLA₂ inhibitors did not affect the accumulation of PrP82-146 in synapses. The amount of PrP82-146 detected in synaptosomes isolated from cortical neurones incubated with a vehicle control (■), 1 μM AACOCF₃ (□) or 1 μM MAFP (striped bars). Treated cortical neurones were incubated with 100 nM PrP82-146 for the time periods shown. Synaptosomes were isolated from neuronal cultures and the amount of PrP82-146 was measured by ELISA. Values shown are the mean average amount of PrP82-146 (nM) ± SD, n = 9.

Figure 6

PLA₂ inhibitors protected against PLAP induced synapse degeneration. The synaptophysin content of cortical neurones pre-treated with a vehicle control (■), 1 μM AACOCF₃ (○) or 1 μM MAFP (●) and incubated with varying concentrations of PLAP for 24 hours. Values shown are the mean average amount of synaptophysin ± SD, n = 12.

Figure 7

PrP82-146 increased activation of cPLA₂ in synapses. Correlation between the amounts of activated cPLA₂ in synaptosomes isolated from primary cortical neurones 1 hour after the addition of varying concentrations of PrP82-146 and the amount of synaptophysin in cell extracts from primary cortical neurones incubated for 24 hours with the same concentrations of PrP82-146.

Figure 8

PAF receptor antagonists protected against PrP82-146 induced synapse degeneration. The synaptophysin content of cortical neurones pre-treated with a vehicle control (■), 1 μM ginkgolide B (○), 2 μM Hexa-PAF (●) or 2 μM CV6209 (□) and subsequently incubated with varying concentrations of PrP82-146 for 24 hours. Values shown are the mean average amount of synaptophysin ± SD, n = 12.

Figure 9

Prostaglandin E₂ caused synapse degeneration. The synaptophysin content of cortical neurones treated with varying concentrations of prostaglandins E₂ (●) D₂ (○), F_2α(□), I₂ (■) or 15d-J₂ (▲) for 24 hours. Values shown are the mean average amount of synaptophysin ± SD, n = 9.