The most recently identified PLC (phospholipase C) enzymes belong to the PLCη family. Their unique Ca2+-sensitivity and their specific appearance in neurons have attracted great attention since their discovery; however, their physiological role(s) in neurons are still yet to be established. PLCη enzymes are expressed in the neocortex, hippocampus and cerebellum. PLCη2 is also expressed at high levels in pituitary gland, pineal gland and in the retina. Driven by the specific localization of PLCη enzymes in different brain areas, in the present paper, we discuss the roles that they may play in neural processes, including differentiation, memory formation, circadian rhythm regulation, neurotransmitter/hormone release and the pathogenesis of neurodegenerative disorders associated with aberrant Ca2+ signalling, such as Alzheimer's disease.
PLC (phospholipase C) enzymes have pivotal roles in transducing extracellular stimuli into physiological changes. Upon their activation, they cleave the phospholipid PtdIns(4,5)P2 to generate DAG (1,2-diacylglycerol) and Ins(1,4,5)P3 . DAG remains in the membrane, where it can activate signalling molecules including PKC (protein kinase C). Ins(1,4,5)P3 accumulates in the cytosol and initiates Ca2+ release from the ER (endoplasmic reticulum) through activation of Ins(1,4,5)P3 receptors . Six classes of PLCs have been identified to date, including β-, γ-, δ-, ϵ-, ζ- and η-classes . All PLC enzymes share essentially the same enzymatic function and are classified on the basis of sequence homology and the mechanisms by which they are activated .
The present review focuses on the most recently identified class, the PLCη enzymes. The function of these enzymes remains unknown. However, they are typically found in neurons and are therefore likely to play a role in neural processes. In the last few years, the properties of these enzymes and the mechanisms by which they are activated have begun to come to light. In the present review, we provide an update on recent developments surrounding these enzymes and discuss their potential involvement in neuronal signalling pathways.
Cellular properties of PLCη enzymes
Mammals possess two PLCη enzymes, PLCη1 and PLCη2, that are highly expressed in the brain (as summarized in Figure 1), particularly in the cerebral cortex, cerebellum, olfactory bulb and hippocampus [3–6]. Interestingly, some fish species (including zebrafish) appear to have additional PLCη-encoding genes, although only the mammalian enzymes have been studied to date. In mice, PLCη1 is also expressed in the zona incerta and in the spinal cord . The expression of PLCη2 is also not restricted to the brain and is found in the retina, lung and in neuroendocrine cells [6,7]. The levels of PLCη2 gradually elevate after birth in both retina and brain, indicating a possible role for this isoform in age-related processes [4,8].
The appearance of PLCη enzymes in the brain and their possible involvement in neuronal processes
PLCη enzymes contain several conserved structural domains that are also found in other PLCs. These include the membrane-binding PH (pleckstrin homology) domain, an EF-hand domain, the catalytic site (X and Y domains) and a C2 domain which possesses Ca2+-dependent membrane-binding ability [3–6]. In addition, PLCη enzymes possess an alternatively spliced C-terminal domain. Three splice variants of PLCη1 and five of PLCη2 have been identified which differ in size and sequence [5,6]. The C-terminal regions of most variants contain a number of proline-rich motifs which may interact with SH3 (Src homology 3) domain-containing proteins, suggesting that the C-terminus is a protein interaction site on PLCη enzymes. These regions are also rich in serine/threonine residues, suggesting possible regulation of interactions by protein kinases/phosphatases. . Moreover, a PDZ motif is present at the C-terminus of certain spliceoforms .
Most vertebrate PLC enzymes are cytosolic and only undergo membrane association following activation . PLCη1 appears to be equally distributed between membrane and cytosolic fractions under resting conditions . PLCη2 has been shown to associate with intracellular membranes via the PH domain before activation [4,10]. The C2 domain of PLCη2 is important for activity, presumably due to Ca2+-dependent membrane association. Mutation of Asp920 to alanine at the predicted C2 domain Ca2+-binding site was found to dramatically reduce enzyme activity . PLCη enzymes are highly sensitive to Ca2+ and are activated by Ca2+ concentrations in the nanomolar range [4,5,10,11]. PLCη enzymes are likely to serve as amplifiers of Ca2+ signals by responding to subtle changes in Ca2+ levels to stimulate further Ins(1,4,5)P3-dependent release from intracellular stores. PLCη enzymes may therefore constitute ‘primarily reacting PLCs’, given that they are localized to membranes before stimulation and are also able to react to small changes in Ca2+ levels . Various studies support the idea that their activity could be important in controlling the amplitude or duration of a particular Ca2+ signalling event [10–12].
Kim et al.  have shown that PLCη1 amplifies GPCR (G-protein-coupled receptor)-mediated PLCβ signalling in Neuro2A cells, a phenomenon that occurs even in the absence of extracellular Ca2+. This highlights the potential for PLCη enzymes to act in synergy with other PLC subtypes. In the same study, PLCη1 (but not PLCη2) activity was found to be stimulated by ionomycin treatment. We have very recently shown that PLCη2 can be activated by monensin-induced Ca2+ efflux via the mitochondria Na+/Ca2+ exchanger in transfected COS-7 cells . These studies demonstrate that Ca2+ released from intracellular stores is likely to be sufficient for PLCη enzyme activation. PLCη2 (but not PLCη1) is also activated by Gβγ dimers and so may respond to GPCR activation by this mechanism [6,12]. Variations as to which type of signalling event each enzyme is able to respond to is likely to be due to differences in cellular localization.
Putative roles of PLCη enzymes in neurons
Neurogenesis and differentiation
For a long time, it was generally accepted that neurons are not replaced and do not regenerate after birth. Subsequent findings have revealed two regions in the brain where new cells are produced: the olfactory bulb and the dentate gyrus of the hippocampus . Both regions are enriched in PLCβ and PLCη enzymes. Interestingly, PLCβ1-knockout mice exhibit hippocampus-specific cognitive impairments that are connected to the increased level in neurogenesis in the dentate gyrus . The chemoattractant SDF-1 (stromal-derived factor 1) has been implicated in controlling granule cell migration in the dentate gyrus . SDF-1 acts upon CXCR4 (CXC chemokine receptor 4), which directs PLC-mediated signalling. Hence PLCη enzymes may act together with PLCs (including PLCβ1) to regulate cell survival, neuronal migration and maturation. Considering that PLCη2 expression increases in neurons after birth, it may in part reflect their maturation status. Ca2+ plays an important role in adult neurogenesis and migration of the olfactory inhibitory neurons as they undergo Ca2+ transients during migration .
The cellular localization of PLCη enzymes in neurons is not well established; they could therefore appear on pre- and/or post-synaptic sites of synapses (as illustrated in Figure 2). The pre-synaptic knob of the axon is enriched in mitochondria, which are important for Ca2+ sequestration and release . PLCη2 has been shown to respond to mitochondrial Ca2+ release , and so could be involved in mitochondria–ER cross-talk in nerve terminals where these organelles form a strongly co-operating unit . PLCη1 and PLCη2 have both been observed at the plasma membrane, where they could act to generate the local Ca2+ levels required to facilitate exocytosis. The Ca2+ which triggers vesicle exocytosis is partly extracellular and gated by VOCs (voltage-operated channels) but is also derived from the ER. This second process is directed, at least in part, by Ins(1,4,5)P3 receptors . In addition, PLC-derived DAG induces the translocation of munc-13, a protein which is important for vesicle priming [19,20]. We have shown that, in addition to its expression in neurons, PLCη2 is also highly expressed in mouse pituitary (P. Popovics and A.J. Stewart, unpublished work) and in neuroendocrine cell lines that include gonadotrope, somatotrope and corticotrope cell lines . A role for this enzyme in neurotransmitter release may extend to stimulus-coupled hormone secretion in neuroendocrine cells. During neurotransmitter/hormone release, the level of PtdIns(4,5)P2 at the plasma membrane is tightly regulated; high PLC activity decreases the membrane content of PtdIns(4,5)P2, causing a substantial inhibition in release [21,22]. In neurons, presynaptic transmitter receptors could also be coupled to PLCη enzymes. It was recently shown that a presynaptic mGluR (metabotropic glutamate receptor) (mGlu7) has a biphasic effect on transmitter release, depending on the length of the activation; PLC activity directs this downstream signal .
The proposed roles of PLCη enzymes in neuronal transmission
PLCη enzymes may also participate in postsynaptic events. PLCη enzymes may be activated by GPCRs via the mechanisms described above or by other extracellular signals which cause an increase in intracellular Ca2+ levels. Dendritic Ca2+ waves are generated by the synaptic activation of a dendritic spine where the accumulating Ins(1,4,5)P3 diffuses and activates the Ins(1,4,5)P3 receptors on the ER. Depending on the strength of the activation, this wave can invade the soma and the nucleus . The breakthrough of the threshold for an overall dendritic wave probably depends on the extent of the activation of the PLC pool. PLCη enzymes may co-operate with other PLC enzymes (activated by different mechanisms) to generate the dendritic wave. Interaction with other proteins could extend the physiological roles of PLCη enzymes. The PDZ-binding motif present on the C-termini of certain PLCη spliceoforms may allow them to bind pre- and post-synaptic PDZ motif containing proteins such as PSD (postsynaptic density) or SAPs (synapse-associated proteins) [6,24]. The binding of PDZ motif containing scaffolding proteins such as SHANK could enhance the receptor-stimulated intracellular Ca2+ release by bringing the PLC and the Ins(1,4,5)P3 receptor into close proximity as was shown with PLCβ .
Like PLCη enzymes, the sperm-specific PLCζ enzyme also possesses high Ca2+-sensitivity and generates intracellular Ca2+ oscillations during egg fertilization . Spontaneous repetitive Ca2+ oscillations also appear in the brain especially in the SCN (suprachiasmatic nucleus) where the circadian rhythm is generated . Data in the Allen Institute Mouse Brain Atlas (http://mouse.brain-map.org/) revealed that PLCη2 is expressed not only in SCN clock generator neurons, but also in the ancient regulator organ of circadian rhythm, the pineal gland. Other studies have revealed the presence of PLCη2 in the retina and habenula , regions which provide input to the SCN [27,28].
A role for PLCη enzymes in learning and memory has been suggested as both are expressed at high levels in regions of the brain associated with these processes, including the hippocampus, cerebellum and olfactory bulb [4,5,8]. Memory formation is driven by neuronal plasticity, which is essentially the strengthening and weakening of particular neuronal connections. Two of the main processes involved in controlling synaptic plasticity are LTP (long-term potentiation) and LTD (long-term depression) .
During LTP, postsynaptic dendrite spines exhibit local elevations in Ca2+ levels , which may be partly directed by PLCη enzymes. On the dendritic spines, the AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor gates Na+ and depolarizes the membrane in response to synaptic glutamate release . However, the most critical event is the opening of the NMDA (N-methyl-D-aspartate) receptor which only opens to let Ca2+ in if glutamate secretion and postsynaptic membrane depolarization occur simultaneously . For the activation of LTP, the neuron has to fire with high frequency . Therefore both the AMPA and the NMDA receptors have to open to evoke a strong depolarization . PLCη enzymes may be activated by Ca2+ influx via the NMDA receptor and enhance the development of a high local increase in Ca2+ level by triggering further release from the ER.
The mGluRs are GPCRs located on the dendritic spines of pyramidal cells . The mGlu1 subtype is abundant in the hippocampus, and its activation stimulates PLC signalling via Gq , to which PLCη1 (or PLCη2) may contribute. This event is likely to be a major factor in generating the Ca2+ signal necessary for the activation of the neuron. The hippocampus is also innervated by cholinergic inputs from the septal area . This innervation potentiates the enhancement of synaptic transmission by releasing Ca2+ from Ins(1,4,5)P3-sensitive stores .
The activation of PKC by DAG may also strengthen the synaptic connection by increasing the synthesis of spine ribosomes which are necessary for the production of synaptic proteins . An increase in PKCγ immunoreactivity has been shown to occur in hippocampal pyramidal cells and dentate granule cells during the process of spatial learning . LTP requires permanent changes in cell physiology, including alterations in gene transcription to synthesize proteins which are required for the enhancement of synaptic transmission. NMDA and mGluRs both activate ERK (extracellular-signal-regulated kinase) phosphorylation to regulate gene transcription . PLCη enzymes could have an important role in this process, as it was shown that the GPCR-induced ERK phosphorylation is reduced in PLCη1-knockdown Neuro2A cells .
Whereas the declarative memory is formed in the hippocampus, the procedural memory is generated in the cerebellum. Here, the main synaptic plasticity event is LTD, which occurs when the Purkinje cells are stimulated by both the parallel and the climbing fibres. The corresponding intracellular pathways also include PLC-dependent processes (to which PLCη enzymes may contribute) such as the phosphorylation and internalization of AMPA receptor by PKC  and the stimulation of TRPC3 (transient receptor potential canonical 3) by DAG, causing a slow excitatory postsynaptic potential .
PLCη enzymes may also be involved in synaptic dysfunction. AD (Alzheimer's disease) is a neurodegenerative disorder associated with memory loss and reduced cognitive function. The disease is characterized by extracellular β-amyloid deposits that alter Ca2+ homoeostasis. This results in increased Ca2+ entry via the formation of aberrant ion channels , and is exacerbated further by the down-regulation of Ca2+ buffer proteins such as calbindin D28k . This, in turn, alters the sensitivity of Ins(1,4,5)P3 and ryanodine receptors that control the release of Ca2+ from intracellular stores [42,43]. Furthermore, it appears that Ca2+ remodelling in this way leads to increased production and release of β-amyloid through stimulation of α-secretase activity, furthering the progression of the disease [44,45]. It is thought that these alterations in Ca2+ dynamics lead to changes in synaptic plasticity and consequent deficits in the neurological pathways associated with these diseases . PLC enzymes have been implicated in Ca2+ remodelling in AD. The expression levels of several isoforms are altered in AD-affected neurons , and brain tissue from individuals with AD exhibit alterations in the phosphoinositide composition of lipids . Both PLCη1 and PLCη2 are expressed in cortical neurons and, given their ability to respond to small changes in Ca2+ level, may contribute greatly to altered Ca2+ dynamics in AD-affected neurons through Ins(1,4,5)P3-dependent Ca2+ release from the ER.
PLC activity in the brain and neuroendocrine organs has pivotal roles in cellular physiology. Before the discovery of PLCη enzymes, research was focused on other PLCs such as PLCβ and PLCγ to elucidate the molecular mechanism of learning, memory formation, circadian clock mechanism and neurotransmitter release. Evidence for the additional involvement of PLCη enzymes in these processes is compelling, not only because of their expression in areas of the brain associated with memory or circadian rhythm, but also because of their exceptionally high sensitivity to changes in intracellular Ca2+ levels. It is still unresolved how the activity of PLCη enzymes contribute to neuronal signalling pathways, but it is certain that their involvement in these processes should be considered. It is hoped that the present article stimulates the necessary studies that are required to identify the specific roles PLCη enzymes play in neuronal (and neuroendocrine) physiology.
Signalling 2011: a Biochemical Society Centenary Celebration: A Biochemical Society Focused Meeting held at the University of Edinburgh, U.K., 8–10 June 2011. Organized and Edited by Nicholas Brindle (Leicester, U.K.), Simon Cook (The Babraham Institute, U.K.), Jeff McIlhinney (Oxford, U.K.), Simon Morley (University of Sussex, U.K.), Sandip Patel (University College London, U.K.), Susan Pyne (University of Strathclyde, U.K.), Colin Taylor (Cambridge, U.K.), Alan Wallace (AstraZeneca, U.K.) and Stephen Yarwood (Glasgow, U.K.).
metabotropic glutamate receptor
protein kinase C
stromal-derived factor 1
We thank Gabor Sarkany for his help in preparing the Figures.