It has previously been shown that PLA2 could play a key role in the neurodegenerative processes in prion disease pathogenesis
[11–13]. The aim of the present study was to examine the effects of neurotoxic PrP peptide on cPLA2 activation and location in murine primary cortical neurons. Exposure of primary cortical neurons to HuPrP106-126 significantly increased levels of p-cPLA2 with subsequent release of AA prior to synapse loss and subsequent cell death. It was also shown that cPLA2 translocated to a novel region of the neuron in a PrP-dependent manner when stimulated by HuPrP106-126 this was not seen when stimulated by other PLA2 agonists PMA and A21387 suggesting that this abnormal translocation was PrP-disease related. The effects of HuPrP106-126 were prevented by an inhibitor of cPLA2.
The regulation of cPLA2 is complex and involves a variety of cellular processes including phosphorylation of serine residues and an increase in intracellular calcium to induce membrane translocation
[20, 23]. It is not clear how cPLA2 translocates although shuttle proteins and attraction to lipids or proteins within the membranes have been suggested
Here confocal microscopy showed an increase in p-cPLA2 after 30 minutes incubation with HuPrP106-126 but without apparent translocation to the ER or nuclear membrane as described in previous reports
[34, 35] and as found using PMA and A23187 in this study. One explanation for this could be that PrP peptides may be interacting with intracellular PrP rather than glycosylphosphatidylinositol (GPI)-linked PrP at the cell surface and this may promote p-cPLA2 to localise in neurites. PrPC is usually anchored to the cell membrane via a GPI anchor
, however transmembrane forms of PrP, CtmPrP and NtmPrP exist that are localised to the endoplasmic reticulum membrane
. Indeed, the peptides used in this study contain part of the transmembrane CtmPrP region
. Alternatively, the HuPrP106-126 may interact with PrPC to cause clustering of PrP-GPIs
 and create altered intracellular signalling resulting in p-cPLA2 locating to neurites.
In this study colocalisation of p-cPLA2 and beta III tubulin after exposure to HuPrP(106–126) was demonstrated. As HuPrP106-126 has been shown to bind directly to tubulin
, the data presented here potentially indicate a direct interaction of HuPrP106-126 and p-cPLA2 involved beta III tubulin filaments. In line with that observation, PrPC has been shown to associate with tubulin in porcine and Syrian hamster brain extracts
[41–43]. This is in contrast to a previous study
, which concluded that cPLA2 co-immunopreciptated with actin.
Interaction of PrP peptide and p-cPLA2 with tubulin could facilitate their transport to neurite and axon terminals to mediate the disease-associated synapse degeneration as axonal membrane proteins are transported via microtubules
. The interaction of PrP with tubulin has been related to the active transport of PrP in cells, by anterograde and retrograde movement via a kinesin/dyenin mechanism in N2a cells
, in hamster peripheral nerves
 and in the entorhinal cortex of rats
. Therefore, any association of cPLA2 with tubulin could bring it into close association with PrP. Indeed, p-cPLA2 co-immunoprecipitated with PrPSc in ScGT1 cells
 and cPLA2 colocalises transiently with the murine PrP peptide MoPrP(105–132) in untreated neuroblastoma cells
. This supports the hypothesis that there is a direct interaction of cPLA2 with tubulin in the presence of HuPrP106-126 and endogenous PrP.
C Any subsequent disruption of the microtubules could lead to induction of apoptosis
The present study also supports previous reports that activation of cPLA2 is associated with PrP peptide-induced neurotoxicity
. Indeed the PLA2 inhibitor PACOCF3 inhibited cPLA2 phosphorylation in primary cortical neurons even after 24 hours of HuPrP106-126 exposure. Furthermore, only low levels of p-cPLA2 were seen by confocal microscopy and colocalisation with beta III tubulin was lost. This correlated with prevention of 3 H]-AA release and ultimately cells were protected against synaptic synaptophysin loss and neuronal death. This is consistent with published data where levels of synaptophysin have been found to decrease in brains of CJD patients
 and murine scrapie models
. The finding that PACOCF3 did not appear to affect 3 H]-AA release induced by PMA and A23187 are consistent with a report that PMA induced activation of cPLA2 may not involve the Ser505 phosphorylation site therefore the mechanism of PACOCF3 may not effect PMA and A23187-induced cPLA2 phosphorylation
. Alternatively the agonist A23187 has been shown to have membrane perturbing effects leading to non labelled fatty acid release, therefore the lack of effect of PACOCF3 on 3 H]-AA release induced by PMA and A23187 could be due to a high amount of unlabelled material in the culture medium
. PACOCF3 pre-treatment inhibited the loss of synaptophysin, indicating that PLA2 could be implicated in synapse degeneration and loss of synaptophysin. It also suggests that PLA2 changes occur as an early event prior to neuronal death, again consistent with in vivo data
[11, 56] in which prostaglandin E2, an end-product of the PLA2 pathway was elevated in specific brain regions before the onset of detectable neuronal loss. Activated microglia also precedes neuronal loss and is a pathological hallmark of prion disease
. The neurotoxicity of PrP peptides are greatly enhanced in the presence of microglia
, however microglial cell contamination of neuronal cultures as measured by GFAP labelling was negligible in this study therefore this is unlikely to have affected results.
The implication that PLA2 changes are a preliminary occurrence prior to cell death is also compounded by the toxicity data seen in this study cortical neurons show a significant decrease in metabolic activity after 5 days whereas an increase in p-cPLA2 is seen after 30 minutes. It is likely that the increase in cPLA2 phosphorylation encourage the cell into a cascade of intracellular signalling events which eventually lead to cell death. This is the first report of PACOCF3 protecting neurons against PrP-induced toxicity however the PACOCF3 analogue arachidonyl trifluoromethyl ketone (AACOCF3) has previously been reported to reduce PrPSc levels in neuroblastoma cells
. The results presented here are also consistent with earlier data showing an increased survival of SHSY-5Y cells treated with the PACOCF3 analogue AACOCF3 before exposure to HuPrP106-126
Thus cPLA2 inhibition by AACOCF3 and PACOCF3 indicates a vital role for cPLA2 activation in neuronal death, and indicates a potential role for the use of cPLA2 inhibitors for neuroprotection in neurodegenerative diseases. PACOCF3 and AACOCF3 are both trifloromethyl ketone analogues of fatty acids
; AACOCF3 inhibits cPLA2 by binding to its active site
 and it is likely that its analogue PACOCF3 acts in a similar way. As PLA2-induced AA release causes free oxygen radical release membrane disruption
 and subsequent cellular damage, the neuroprotective action of PACOCF3 and other PLA2 inhibitors could be through inhibition of these events, and/or through disrupting colocalisation of PrPSc, p-cPLA2 and beta III tubulin, by attaching to the active site of cPLA2. Whether cPLA2 is activated via direct cell exposure to neurodegenerative peptides or is due to a secondary event remains to be investigated.