This study’s primary purpose was to test the hypothesis that FFAs released by oxidative stress-induced PLA2 activity are a factor in learning and memory dysfunction in L. stagnalis. Our primary observations are: 1- Increasing the level of extracellular PLA2 activity through injection of venom-derived extracellular PLA2 evoked LTM impairment. 2- Experimental induction of oxidative stress with the lipid peroxidation-inducing free radical generator AAPH triggers an increase in circulating FFA levels that peaks within 24–48 hrs and attenuates LTM performance. 3- Experimental elevation of circulating arachidonic acid through intracoelomic injection of exogenous arachidonic acid does not affect LTM performance. 4. Experimentally induced oxidative stress-associated LTM impairment can be rescued with the general PLA2 inhibitor aristolochic acid but not by normalizing the level of circulating FFAs with defatted BSA.
The present data substantiate previous evidence where we link lipid peroxidation and PLA2 activation to age- and oxidative stress-associated LTM impairment in another, aversive operant conditioning learning and memory paradigm
. In both our learning models age- and oxidative stress associated LTM failure is associated with reduced electrical excitability of key interneurons in the circuits underlying the respective behaviours
[6, 7, 22, 30]. Both behavioural and electrophysiological facets of age-associated respiratory and appetitive LTM impairment can be reproduced in young animals through treatment with AAPH, a water-soluble free radical generator commonly used to induce FA peroxidation (present study and
). In addition, in both our two models, all behavioural, electrophysiological and biochemical symptoms of aging and experimental oxidative stress can be reversed by treatment with aristolochic acid, a broad spectrum PLA2 inhibitor (present study and
[6, 7, 11]). No evidence was found of significant age-associated impairment or experimental oxidative stress-induced repression of short/intermediate term forms in either of the two behavioural conditioning paradigms
[7, 27], suggesting that transcription-independent forms of memory are relatively impervious to aging or oxidative stress. Intriguingly, selective appetitive LTM impairment could also be induced in young Lymnaea through systemic activation of their cellular immune system and, as before in aged and oxidation-stressed young animals, LTM could be rescued by means of PLA2 inhibition
. Thus, although, the possibility exists that the cellular and molecular mechanisms underlying oxidative stress induced PLA2 activation dependent LTM impairments in our two learning models are different, this seems unlikely. The brain and its neurons are due to their high polyunsaturated fatty acids (PUFA) content inordinately sensitive to free radical attack, oxidative stress and subsequent peroxidation, a process that if not properly contained can severely disrupt membrane architecture and lipid signaling processes
[31–33]. Neurons defend themselves against lipid-peroxidation through various mechanisms, one that involves excision of (per)oxidized FA by PLA2[12, 15]. As will be discussed below, PLA2, (per)oxidized FA and their various metabolites all can alter signal transduction, various neuronal signaling pathways, ion channel functioning and gene transcription processes and ultimately behavioral plasticity such as learning and memory processes. Thus, although to our knowledge our studies are the first to show these aspects in molluscs, it is not surprisingly that similar conclusions are drawn with increasing frequency in studies linking oxidative stress related PLA2 activation with cognitive impairment, neuronal dysfunction and disease not only in other, non-molluscan, invertebrate species but also in mammals and humans
[12, 16, 34–38]. Together this suggests that Lymnaea’s LTM functions in general are sensitive to lipid peroxidation and oxidative membrane damage, and that lipid peroxidation-dependent activation of PLA2 is a fundamental and evolutionary conserved factor in the decline in behavioral and neuronal plasticity widely observed across the animal kingdom.
Activation of PLA2 by reactive oxygen species in L. stagnalis results in the release of extracellular (per)oxidized FFAs (present study and
). Many FFAs, including AA and DHA the two FAs most commonly found at the glycerophospholipid sn-2 position in neurons, have various demonstrated biological activity in the nervous system. For instance, under normal conditions they can regulate membrane fluidity and other aspects of phospholipid membrane microarchitecture, may engage in modulatory interactions with various ion channels, affect alterations in membrane protein clustering, modify receptor sensitivity and signal transduction pathways as well as affect gene transcription processes
[17–21, 39–43]. However, under conditions of oxidative stress, causes (over) activation of PLA2 resulting in peroxidized FA excision from the lipid bilayer matrix
[12, 15]. Recent evidence increasingly associates deregulation of lipid metabolism due to PLA2 (over) activation as a cause of nervous system dysfunction and cognitive impairment
[7, 12, 22, 23]. In addition, AA is the substrate for many potentially neuroactive lipids metabolized including those generated by the eicosanoid metabolic pathways. Each of the three main branches of eicosanoid metabolism, the epoxygenase or CYP-450, the lipoxygenase (LOX) and the cyclooxygenase (COX) pathways, has the potential to generate a variety of lipid-derived neuromodulatory substances that may engage with either intracellular or extracellular targets
[16, 18, 20, 41, 44]. In addition, (per)oxidized FFAs can undergo further metabolic processing resulting in the production of highly cytotoxic aldehydes such as 4-hydroxynonenal and malondialdehyde
Interestingly, we recently showed that challenging the immune system impairs LTM function in L. stagnalis that seems to involve PLA2 and COX activity
. In addition, non-enzymatic lipid peroxidation (including AAPH induced), has been shown to result in the production of AA and DHA derived iso- and neuroprostanes including the LOX series
[45, 46]. PLA2- and/or LOX-dependent arachidonic-acid modulated background K+ channels have been described in Lymnaea and various other molluscs
[47–51]. Opening of these 4TM2P channels generates an outward current. It is therefore conceivable that elevated levels of PLA2-activity associated with inflammation, oxidative stress and aging induce a decline in neuronal excitability through activation of these 4TM2P background K+ channels. In this respect the 12-LOX product 12-HPETE, first described in the gastropod Aplysia californica, is quite interesting. 12-HPETE has long been known as an activator of Aplysia’s S-type K+-channels
. These K+ channels, later identified to belong to the TREK-1 family, are instrumental in non-synaptic forms of plasticity underlying behavioral modification of Aplysia’s gill withdrawal reflex
 and are implicated as synaptic modulator in Aplysia and various vertebrate model systems
[54–58]. Alterations in receptor sensitivity, ion channels and signal transduction pathways as well as affect gene transcription by PLA2, it’s products and their metabolites will most likely affect activity,- transcription- and protein synthesis-dependent processes such as LTM formation.
Based on the evidence described above it seems therefore very plausible that lipid-peroxidation evoked elevated levels of FFAs through activation of PLA2 are the source of the observed LTM dysfunction in L. stagnalis (present study and
[7, 22]). However, in the present study we show that increasing extracellular levels of AA does not affect LTM performance in L. stagnalis. Moreover, we also show in the current study that removing AAPH-induced elevated levels of circulating peroxidized FFA with BSA does not rescue LTM failure in our model system.
So, if not extracellular FFAs then what might be a source of the lipid-peroxidation dependent LTM failure observed in L. stagnalis? The first possibility is that PLA2 itself is the main culprit. The PLA2 family is a family of heterogeneous enzymes acting in different cellular locations and to some extent different activation profiles usually classified into intracellular located cytosolic PLA2 (cPLA2), calcium-independent PLA2 (iPLA2), extracellular acting secretory PLA2 (sPLA2) and the recently identified lysosomal and adipose-specific PLA2’s
[15, 34, 59]. Upon activation, the various PLA2 family members such as cPLA2 and sPLA2 can induce FA release and eicosanoid production by themselves
[60–62]. In addition, recent evidence also indicates the existence of cross-talk and trans-activation between the various intracellular and cytosolic PLA2 family members
[63–65]. Thus it is quite conceivable that AAPH, a water-soluble extracellular free radical generator used to induce lipid peroxidation in the present study activates various intracellular and extracellular PLA2 enzymes.
Besides being present and acting on the cellular plasmamembrane both cPLA2 and iPLA2 can localize and/or target organellar membranes including mitochondrial and nuclear membranes
[34, 66–68]. For instance, it has been shown that activation of PLA2 can induce mitochondrial dysfunction due to a loss of mitochondrial membrane potential, swelling of mitochondria and/or the production of superoxide from mitochondria
. Furthermore, interactions between cPLA2 and NADPH-oxidase complexes in the plasmamembrane have been implicated in the redox-pathology of various neurodegenerative diseases
[34, 70]. In addition to their potential interference with numerous signaling processes, PLA2 enzymatic activity may impact various membrane architecture-sensitive processes. For example, PLA2 may alter phospholipid-packing causing membranes to become more molecularly ordered and affect processes involved in vesicle fusion, exocytosis and actin-dependent processes
[71–75]. Moreover, there is evidence that architectural changes induced by PLA2 may alter gating states for a variety of ion channels including members of the K2P two-pore “leak” potassium family
[76, 77]. At this point in time we cannot say if and which of these scenarios underpins LTM impairment in our model system. Nor can we pinpoint which PLA2 is involved. However, the observation in the current and one of our previous studies
, that treatment with aristolochic acid, commonly considered a general PLA2 inhibitor and inhibiting various classes of PLA2[68, 78–81], reverses the AAPH-induced LTM failure is consistent with PLA2’s involvement.
Another intriguing potential scenario to consider as explanation for the current results is the possibility that other products released extracellular after PLA2 activation are causing LTM failure in L. stagnalis. For instance, hydrolysis of oxidized membrane phospholipids by PLA2 will not only liberate FFAs but also produce lysophospholipids (LPLs). Involvement of LPLs in membrane-associated processes such as membrane budding, ruffle formation, protein complex assembly and ion channel gating has been reported
[34, 59, 82–85]. However, extracellular LPLs, like FFAs, can bind to BSA
[86–88]. Therefore, we currently interpret BSA’s inability to reverse AAPH-induced LTM deficiency as evidence against the idea that circulating LPLs play a major role in the current LTM failure model. Alternatively, extracellular (per)oxidized FAs can undergo further metabolic processing resulting in the production of highly cytotoxic aldehydes such as 4-hydroxynonenal and malondialdehyde
[1, 14]. It is important to note that BSA is not cell permeable. Thus, indirect activation of intracellular PLA2 might cause elevated levels of intracellular FFAs and LPLs thereby affecting various intracellular targets
[14, 89, 90]. For instance, it has been demonstrated that increased levels of intracellular FFAs can directly affect NADPH-oxidase
[90, 91] resulting in a further increase of ROS and lipid peroxidation
In addition to FFAs and LPLs, PLA2 activation results in the extracellular release of lyso-platelet-activating factor (lyso-PAF), the platelet-activating factor (PAF) precursor
[92–94]. PAF is a bioactive phospholipid that under normal conditions is thought to be involved in the regulation of synaptic plasticity, memory and neuronal protection
[92–94]. In addition, PAF is a transcriptional activator of the cyclooxygenase-2 (COX-2) gene
. As noted before, AA is the primary substrate for the COX branch of the eicosanoid (inflammatory) pathway. Importantly, ROS can also be generated as a by-product of COX activity, thus creating a positive feedback loop that potentially can cause escalation of PLA2, PAF and COX-activity thereby causing substantive deregulation of lipid homeostasis
[92–94]. In this respect it is interesting to note that we recently showed that systemic immune challenges in L. stagnalis induces a PLA2-dependent LTM failure that could be rescued with treatment of indomethacin, a putative COX inhibitor
One of the intriguing questions still remaining is why inhibition of PLA2 24 hrs after its activation is sufficient to reverse the oxidative stress induced LTM failure? We show in the present study that circulating FFA levels are their highest level 24–48 hrs after PLA2 activation suggesting that some of the PLA2 and FFA-dependent and associated pathways are initiated in the first 24 hrs. after induction of oxidative stress. At present we cannot definitively provide a mechanism to what causes the PLA2-dependent LTM failure within this time frame. However based on the actions of PLA2, FFA and their various products as outlined above there are some potential explanations. For instance, it is conceivable that by inhibiting PLA2 activity, even though FFAs are already released for some time, we stop the positive feedback loop discussed above, thereby preventing further escalation of PLA2, its products and their pathways. Notwithstanding, further investigations are clearly needed to resolve this issue.