Inhibition of cytosolic Phospholipase A2prevents prion peptide-induced neuronal damage and co-localisation with Beta III Tubulin

Victoria Last,Dirk Werling,Alun Williams

doi:10.1186/1471-2202-13-106

Victoria Last, Dirk Werling + Show 1 more

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https://doi.org/10.1186/1471-2202-13-106

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Abstract

BackgroundActivation of phospholipase A2 (PLA2) and the subsequent metabolism of arachidonic acid (AA) to prostaglandins have been shown to play an important role in neuronal death in neurodegenerative disease. Here we report the effects of the prion peptide fragment HuPrP106-126 on the PLA2 cascade in primary cortical neurons and translocation of cPLA2 to neurites.ResultsExposure of primary cortical neurons to HuPrP106-126 increased the levels of phosphorylated cPLA2 and caused phosphorylated cPLA2 to relocate from the cell body to the cellular neurite in a PrP-dependent manner, a previously unreported observation. HuPrP106-126 also induced significant AA release, an indicator of cPLA2 activation; this preceded synapse damage and subsequent cellular death. The novel translocation of p-cPLA2 postulated the potential for exposure to HuPrP106-126 to result in a re-arrangement of the cellular cytoskeleton. However p-cPLA2 did not colocalise significantly with F-actin, intermediate filaments, or microtubule-associated proteins. Conversely, p-cPLA2 did significantly colocalise with the cytoskeletal protein beta III tubulin. Pre-treatment with the PLA2 inhibitor, palmitoyl trifluoromethyl ketone (PACOCF3) reduced cPLA2 activation, AA release and damage to the neuronal synapse. Furthermore, PACOCF3 reduced expression of p-cPLA2 in neurites and inhibited colocalisation with beta III tubulin, resulting in protection against PrP-induced cell death.ConclusionsCollectively, these findings suggest that cPLA2 plays a vital role in the action of HuPrP106-126 and that the colocalisation of p-cPLA2 with beta III tubulin could be central to the progress of neurodegeneration caused by prion peptides. Further work is needed to define exactly how PLA2 inhibitors protect neurons from peptide-induced toxicity and how this relates to intracellular structural changes occurring in neurodegeneration.

Highlights

Activation of phospholipase A2 (PLA2) and the subsequent metabolism of arachidonic acid (AA) to prostaglandins have been shown to play an important role in neuronal death in neurodegenerative disease
It is known that cytosolic PLA2 (cPLA2) is promptly activated within 1 hour by agonists including phorbol 12-myristate 13-acetate (PMA) A23187 and ionomycin [24,25,26], this was confirmed in murine primary cortical neurons via preliminary experiments (Additional file 1: Figure S1), and neurons were initially treated for 30 minutes. phosphospecific anti-cPLA2 polyclonal (p-cPLA2) was visualised by confocal microscopy using an anti-phospho cPLA2 antibody against the serine-505 residue
In untreated neurons a low basal level of p-cPLA2 labelling in the nuclear region could be seen, exposure to 40 μM HuPrP106-126 resulted in a significant increase in the intensity of p-cPLA2 labelling (P < 0.001), indicating amplified levels of the enzyme (Figure 1A)

Summary

Introduction

Activation of phospholipase A2 (PLA2) and the subsequent metabolism of arachidonic acid (AA) to prostaglandins have been shown to play an important role in neuronal death in neurodegenerative disease. We report the effects of the prion peptide fragment HuPrP106-126 on the PLA2 cascade in primary cortical neurons and translocation of cPLA2 to neurites. Effects of HuPrP106-126 on cells include aggregation of PrPC in neuroblastoma cells [7], copper uptake inhibition in cerebellar neurons [8], p38 MAPK activation in correlation with cell death in SHSY5Y cells [9] and an increase in intracellular Ca2+ coupled with membrane viscosity in leucocytes [10]

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