VIA iPLA Research Articles

β-Lactones: A Novel Class of Ca2+-Independent Phospholipase A2 (Group VIA iPLA2) Inhibitors with the Ability To Inhibit β-Cell Apoptosis.

Ca2+-independent phospholipase A2 (GVIA iPLA2) has gained increasing interest recently as it has been recognized as a participant in biological processes underlying diabetes development and autoimmune-based neurological disorders. The development of potent GVIA iPLA2 inhibitors is of great importance because only a few have been reported so far. We present a novel class of GVIA iPLA2 inhibitors based on the β-lactone ring. This functionality in combination with a four-carbon chain carrying a phenyl group at position-3 and a linear propyl group at position-4 of the lactone ring confers excellent potency. trans-3-(4-Phenylbutyl)-4-propyloxetan-2-one (GK563) was identified as being the most potent GVIA iPLA2 inhibitor ever reported ( XI(50) 0.0000021, IC50 1 nM) and also one that is 22 000 times more active against GVIA iPLA2 than GIVA cPLA2. It was found to reduce β-cell apoptosis induced by proinflammatory cytokines, raising the possibility that it can be beneficial in countering autoimmune diseases, such as type 1 diabetes.

Neurons and astrocytes in an infantile neuroaxonal dystrophy (INAD) mouse model show characteristic alterations in glutamate-induced Ca2+ signaling

INAD (infantile neuroaxonal dystrophy, OMIM#256600), an autosomal recessive inherited degenerative disease, is associated with PLA2G6 mutations. PLA2G6 encodes Ca2+-independent phospholipase A2 (VIA iPLA2). However, it is unclear how the PLA2G6-mutations lead to disease. Non-canonical functions, which were suggested for VIA iPLA2, such as regulation of cellular and mitochondrial Ca2+ are promising candidates. Therefore, we investigate glutamate (Glu)-evoked Ca2+ signals in neurons and astrocytes in co-culture obtained from three INAD mouse model strains with Pla2g6 mutations, (i) hypomorphic Pla2g6 allele with reduced transcript levels, (ii) knocked-out Pla2g6, and (iii) (G373R)-point mutation with inactive VIA iPLA2 enzyme. Homozygous offspring from these strains develop pathology similar to that observed in INAD patients. We found that in mouse neurons the Pla2g6 mutation disrupted the dependency of Glu-induced extracellular Ca2+ influx on mitochondrial Ca2+ uptake. Thus, in neurons with Pla2g6 mutation we did not detect the characteristic reduction in Glu-induced Ca2+ influx upon treatment with Ru360, a blocker of mitochondrial Ca2+ uniporter, or with rotenone. In contrast to neurons, in astrocytes, both with Pla2g6 mutation or wild-type cells, the treatment with Ru360 or rotenone reduced the rate of Glu-induced Ca2+ influx ∼2-fold. This Ca2+ influx in astrocytes represents capacitative Ca2+ entry. In astrocytes with Pla2g6 mutation, the Glu-induced Ca2+ influx was ∼2-fold lower than in wild-type controls. We suggest that this is the mechanism for strongly decreased durations of Glu-induced Ca2+ responses in astrocytes with Pla2g6 mutation. We could mimic the mutation by pharmacological inhibition of iPLA2 using S-BEL. Thus, lack of VIA iPLA2 activity caused effects in astrocytes. In summary, three INAD mouse models show comparable changes in Glu-induced Ca2+ signaling, but specific for neurons or astrocytes. This finding helps to identify pathways altered during INAD and highlights non-canonical VIA iPLA2 functions, like regulation of cellular Ca2+ fluxes by mitochondria or capacitative Ca2+-entry.

Mitochondria from a mouse model of the human infantile neuroaxonal dystrophy (INAD) with genetic defects in VIA iPLA2 have disturbed Ca2+ regulation with reduction in Ca2+ capacity

Mutations in the PLA2G6 gene which encodes Ca2+-independent phospholipase A2 (VIA iPLA2) were detected in 85% of cases of the inherited degenerative nervous system disorder INAD (infantile neuroaxonal dystrophy, OMIM #256600). However, molecular mechanisms linking these mutations to the disease progression are unclear. VIA iPLA2 is expressed also in mitochondria. Here, we investigate Ca2+ handling by brain mitochondria derived from mice with hypomorph Pla2g6 allele. These animals with reduced transcript levels (5% of wild type) represent a suitable model for INAD. We demonstrated significant reduction of Ca2+ uptake rate and Ca2+ retention capacity in brain mitochondria isolated from this mutant. This phenotype could be mimicked when in wild-type controls VIA iPLA2 was inhibited by S-BEL. Importantly, the reduction could be ameliorated partly by addition of the VIA iPLA2 product, sn-2 lysophosphatidyl-choline. Furthermore, we demonstrated in situ a reduced mitochondrial potential in neurons from mice deficient in VIA iPLA2, which could cause the reduced Ca2+ uptake rate via the potential-dependent mitochondrial Ca2+ uniporter. Thus, the disturbances in mitochondrial potential and the changes in Ca2+ handling were dependent on VIA iPLA2 activity. Reduced mitochondrial Ca2+ uptake rate and Ca2+ retention capacity might result in increased vulnerability of mitochondria to the Ca2+ overload and in disturbed cellular Ca2+ signaling during INAD. For VIA iPLA2, non-canonical functions beyond sole phospholipid turnover seem to be important, such as regulation of store-operated Ca2+ entry in cells. Thus, our findings bring new insight into molecular mechanism affected in INAD and highlight the non-canonical function of VIA iPLA2 in regulation of mitochondrial Ca2+ handling.

Development of Potent and Selective Inhibitors for Group VIA Calcium-Independent Phospholipase A2 Guided by Molecular Dynamics and Structure-Activity Relationships.

The development of inhibitors for phospholipase A2 (PLA2) is important in elucidating the enzymes implication in various biological pathways. PLA2 enzymes are an important pharmacological target implicated in various inflammatory diseases. Computational chemistry, organic synthesis, and in vitro assays were employed to develop potent and selective inhibitors for group VIA calcium-independent PLA2. A set of fluoroketone inhibitors was studied for their binding mode with two human cytosolic PLA2 enzymes: group IVA cPLA2 and group VIA iPLA2. New compounds were synthesized and assayed toward three major PLA2s. This study led to the development of four potent and selective thioether fluoroketone inhibitors as well as a thioether keto-1,2,4-oxadiazole inhibitor for GVIA iPLA2, which will serve as lead compounds for future development and studies. The keto-1,2,4-oxadiazole functionality with a thioether is a novel structure, and it will be used as a lead to develop inhibitors with higher potency and selectivity toward GVIA iPLA2.

In the neurodegenerative disease INAD, infantile neuroaxonal dystrophy, glutamate‐ and ATP‐induced Ca2+ signaling is disturbed in neurons and astrocytes, with mitochondrial dysfunction

About 85% of cases of INAD (infantile neuroaxonal dystrophy, OMIM #256600), an autosom al recessive inherited degenerative disease with early onset can be associated with mutations of the PLA2G6 gene . Ca2+‐independent phospholipase A2 (VIA iPLA2) is encoded in PLA2G6 . However, connections of these mutations to disease are unclear. For the mainly phospholipid metabolizing VIA iPLA2 also so‐called non‐canonical functions were discussed and reported. These are regulation of store‐regulated Ca2+ entry in cells and change in mitochondrial Ca2+ homeostasis. In this study, we investigate glutamate‐evoked Ca2+ signals in neurons and astrocytes in co‐culture from three INAD mouse models with Pla2g6 gene mutations. The strain‐1 has a hypomorphic Pla2g6 allele with reduced transcript levels (5% of wild type), the strain‐2 has knocked‐out Pla2g6, and strain‐3 has a Pla2g6 point mutation with the VIA iPLA2 inactive. It is shown that homozygous offspring from all strains develop pathology similar to that observed in INAD patients. We demonstrate in our present study a strongly reduced glutamate‐induced Ca2+ response in mutants in astrocytes compared to control cells. When we elucidate the mechanism we find that reduced Ca2+ responses are due to about 2‐fold‐reduced Glu‐induced capacitative Ca2+ entry in astrocytes in all three mouse strains. This effect is based on the lack in VIA iPLA2 activity since we could mimic the decrease by pharmacologically inhibiting iPLA2 with the compound S‐BEL enantiomer. On the contrary, in neurons the Pla2g6 mutation completely disrupted the influence of mitochondrial Ca2+ uptake on the extracellular Glu‐induced Ca2+ influx . We see it in neurons treated with Ru360, a blocker of mitochondrial Ca2+ uniporter or treated with rotenone. The dependency of Glu‐induced Ca2+ influx on mitochondrial Ca2+ uptake was wiped out in neurons with Pla2g6 mutation, whereas a strong reduction was seen in wild‐type cells. Thus, all INAD models have comparable changes, but they are different for astrocytes and neurons, respectively, in Glu‐induced Ca2+ signaling. These modifications in Ca2+ signaling in cells with mutated Pla2g6 are critical for adequate neuron‐astrocyte communication. Thus our data help to understand molecular mechanisms of INAD pathology.Support or Funding InformationDeutsche Forschungsgemeinschaft (Re 563‐22‐1)

Membranes serve as allosteric activators of phospholipase A2, enabling it to extract, bind, and hydrolyze phospholipid substrates

Defining the molecular details and consequences of the association of water-soluble proteins with membranes is fundamental to understanding protein-lipid interactions and membrane functioning. Phospholipase A2 (PLA2) enzymes, which catalyze the hydrolysis of phospholipid substrates that compose the membrane bilayers, provide the ideal system for studying protein-lipid interactions. Our study focuses on understanding the catalytic cycle of two different human PLA2s: the cytosolic Group IVA cPLA2 and calcium-independent Group VIA iPLA2. Computer-aided techniques guided by deuterium exchange mass spectrometry data, were used to create structural complexes of each enzyme with a single phospholipid substrate molecule, whereas the substrate extraction process was studied using steered molecular dynamics simulations. Molecular dynamic simulations of the enzyme-substrate-membrane systems revealed important information about the mechanisms by which these enzymes associate with the membrane and then extract and bind their phospholipid substrate. Our data support the hypothesis that the membrane acts as an allosteric ligand that binds at the allosteric site of the enzyme's interfacial surface, shifting its conformation from a closed (inactive) state in water to an open (active) state at the membrane interface.

Severe disturbance in the Ca2+ signaling in astrocytes from mouse models of human infantile neuroaxonal dystrophy with mutated Pla2g6

Infantile neuroaxonal dystrophy (INAD; OMIM #no. 256600) is an inherited degenerative nervous system disorder characterized by nerve abnormalities in brain, spinal cord and peripheral nerves. About 85% of INAD patients carry mutations in the PLA2G6 gene that encodes for a Ca(2+)-independent phospholipase A(2) (VIA iPLA(2)), but how these mutations lead to disease is unknown. Besides regulating phospholipid homeostasis, VIA iPLA(2) is emerging with additional non-canonical functions, such as modulating store-regulated Ca(2+) entry into cells, and mitochondrial functions. In turn, defective Ca(2+) regulation could contribute to the development of INAD. Here, we studied possible changes in ATP-induced Ca(2+) signaling in astrocytes derived from two mutant strains of mice. The first strain carries a hypomorphic allele of the Pla2g6 that reduces transcript levels to 5-10% of that observed in wild-type mice. The second strain carries a point mutation in Pla2g6 that results in inactive VIA iPLA(2) protein with postulated gain in toxicity. Homozygous mice from both strains develop pathology analogous to that observed in INAD patients. The nucleotide ATP is the most important transmitter inducing Ca(2+) signals in astroglial networks. We demonstrate here a severe disturbance in Ca(2+) responses to ATP in astrocytes derived from both mutant mouse strains. The duration of the Ca(2+) responses in mutant astrocytes was significantly reduced when compared with values observed in control cells. We also show that the reduced Ca(2+) responses are probably due to a reduction in capacitative Ca(2+) entry (2.3-fold). Results suggest that altered Ca(2+) signaling could be a central mechanism in the development of INAD pathology.

Proinflammatory treatment of astrocytes with lipopolysaccharide results in augmented Ca2+ signaling through increased expression of VIA phospholipase A2 (iPLA2)

Many Ca(2+)-regulated intracellular processes are involved in the development of neuroinflammation. However, the changes of Ca(2+) signaling in the brain under inflammatory conditions were hardly studied. ATP-induced Ca(2+) signaling is a central event of signal transmission in astrocytic networks. We investigated primary astrocytes after proinflammatory stimulation with lipopolysaccharide (LPS; 100 ng/ml) for 6-24 h. We reveal that Ca(2+) responses to purinergic ATP stimulation are significantly increased in amplitude and duration after stimulation with LPS. We detected that increased amplitudes of Ca(2+) responses to ATP in LPS-treated astrocytes can be explained by substantial increase of Ca(2+) load in stores in endoplasmic reticulum. The mechanism implies enhanced Ca(2+) store refilling due to the amplification of capacitative Ca(2+) entry. The reason for the increased duration of Ca(2+) responses in LPS-treated cells is also the amplified capacitative Ca(2+) entry. Next, we established that the molecular mechanism for the LPS-induced amplification of Ca(2+) responses in astrocytes is increased expression and activity of VIA phospholipase A(2) (VIA iPLA(2)). Indeed, both gene silencing with specific small interfering RNA and pharmacological inhibition of VIA iPLA(2) with S-bromoenol lactone reduced the load of the Ca(2+) stores and caused a decrease in the amplitudes of Ca(2+) responses in LPS-treated astrocytes to values, which were comparable with those in untreated cells. Our findings highlight a novel regulatory role of VIA iPLA(2) in development of inflammation in brain. We suggest that this enzyme might be a possible target for treatment of pathologies related to brain inflammation.

A link between endoplasmic reticulum stress‐induced β‐cell apoptosis and the group VIA Ca2+‐independent phospholipase A2 (iPLA2β)

Endoplasmic reticulum (ER) stress is becoming recognized as an important contributing factor in various diseases, including diabetes mellitus. Prolonged ER stress can cause β-cell apoptosis; however, the underlying mechanism(s) that contribute to this process are not well understood. Early reports suggested that arachidonic acid metabolites and a Ca(2+)-independent phospholipase A(2) (iPLA(2)) activity play a role in β-cell apoptosis. The PLA(2) family of enzymes catalyse the hydrolysis of the sn-2 substituent (i.e. arachidonic acid) of membrane phospholipids. In light of our findings that the pancreatic islet β-cells are enriched in arachidonate-containing phospholipids and express the group VIA iPLA(2)β, we considered the possibility that iPLA(2)β participates in ER stress-induced β-cell apoptosis. Our work revealed a novel mechanism, involving ceramide generation and triggering of mitochondrial abnormalities, by which iPLA(2)β participates in the β-cell apoptosis process. Here, we review our evidence linking ER stress, β-cell apoptosis and iPLA(2)β. Continued studies in this area will increase our understanding of the contribution of iPLA(2)β to the evolution of diabetes mellitus and will further our knowledge of factors that influence β-cell health in diabetes mellitus and identify potential targets for future therapeutic interventions to prevent β-cell death.

Role of Phospholipase A2 in the Induction of Drip Loss in Porcine Muscle

The role of phospholipase A2 in the induction of drip loss from pig muscle has been investigated. In samples from porcine M. longissimus dorsi, total PLA2 activity as well as mRNA and protein levels of the group VIA iPLA2 (iPLA2-VIA) increased during the initial 4 h post-mortem period. Morphological studies of porcine muscle showed that at 4 h post-mortem, gaps had formed between muscle fibers and that the sarcolemma membrane borders appeared blurred. At the same time iPLA2-VIA protein levels were increased inside muscle fibers and at the sarcolemma. iPLA2-VIA mRNA abundance in samples from different breeds of pigs with variations in drip loss revealed no clear correlation between drip loss level and iPLA2-VIA expression. Together, these data indicate that during the post-mortem period, iPLA2-VIA expression and activity is increased at the muscle fiber membranes. PLA2 activity may affect membrane permeability and consequently the progression of drip formation in porcine muscle.

Systemic and Intrathecal Effects of a Novel Series of Phospholipase A2 Inhibitors on Hyperalgesia and Spinal Prostaglandin E2 Release

Phospholipase A(2) (PLA(2)) forms are expressed in spinal cord, and inhibiting spinal PLA(2) induces a potent antihyperalgesia. Here, we examined the antihyperalgesic effects after systemic and i.t. delivery of four compounds constructed with a common motif consisting of a 2-oxoamide with a hydrocarbon tail and a four-carbon tether. These molecules were characterized for their ability to block group IVA calcium-dependent PLA(2) (cPLA(2)) and group VIA calcium-independent PLA(2) (iPLA(2)) in inhibition assays using human recombinant enzyme. The rank ordering of potency in blocking group IVA cPLA(2) was AX048 (ethyl 4-[(2-oxohexadecanoyl)amino]butanoate), AX006 (4-[(2-oxohexadecanoyl)amino]butanoic acid), and AX057 (tert-butyl 4-[(2-oxohexadecanoyl)amino]butanoate) > AX010 (methyl 4-[(2-oxohexadecanoyl)amino]butanoate) and for inhibiting group VIA iPLA(2) was AX048, AX057 > AX006, and AX010. No agent altered recombinant cyclooxygenase activity. In vivo, i.t. (30 mug) and systemic (0.2-3 mg/kg i.p.) AX048 blocked carrageenan hyperalgesia and after systemic delivery in a model of spinally mediated hyperalgesia induced by i.t. substance P (SP). The other agents were without activity. In rats prepared with lumbar i.t. loop dialysis catheters, SP evoked spinal prostaglandin E(2) (PGE(2)) release. AX048 alone inhibited PGE(2) release. Intrathecal SR141617, a cannabinoid CB1 inhibitor at doses that blocked the effects of i.t. anandamide had no effect upon i.t. AX048. These results suggest that AX048 is the first systemically bioavailable compound with a significant affinity for group IVA cPLA(2), which produces a potent antihyperalgesia. The other agents, although demonstrating enzymatic activity in cell-free assays, appear unable to gain access to the intracellular PLA(2) toward which their action is targeted.