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Cerebral reperfusion injury in stroke, stemming from interconnected thrombotic and inflammatory signatures, often involves platelet activation, aggregation and its interaction with various immune cells, contributing to microvascular dysfunction. However, the regulatory mechanisms behind this platelet activation and the resulting inflammation are not well understood, complicating the development of effective stroke therapies. Utilizing animal models and platelets from hemorrhagic stroke patients, our research demonstrates that human cerebral dopamine neurotrophic factor (CDNF) acts as an endogenous antagonist, mitigating platelet aggregation and associated neuroinflammation. CDNF moderates mitochondrial membrane potential, reactive oxygen species production, and intracellular calcium in activated platelets by interfering with GTP binding to Rap1b, thereby reducing Rap1b activation and downregulating the Rap1b-MAPK-PLA2 signaling pathway, which decreases release of the pro-inflammatory mediator thromboxane A2. In addition, CDNF reduces the inflammatory response in BV2 microglial cells co-cultured with activated platelets. Consistent with ex vivo findings, subcutaneous administration of CDNF in a rat model of ischemic stroke significantly reduces platelet activation, aggregation, lipid mediator production, infarct volume, and neurological deficits. In summary, our study highlights CDNF as a promising therapeutic target for mitigating platelet-induced inflammation and enhancing recovery in stroke. Harnessing the CDNF pathway may offer a novel therapeutic strategy for stroke intervention., Competing Interests: Declaration of interests M.S. is one of inventors of the CDNF-related patent (7452969), which is owned by the Herantis Pharma Company (Espoo, Finland)., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cerebral dopamine neurotrophic factor (CDNF) is an endogenous protein in humans and other vertebrates, and it has been shown to have protective and restorative effects on cells in various disease models. Although it is named as a neurotrophic factor, its actions are drastically different from classical neurotrophic factors such as neurotrophins or the glial cell line-derived neurotrophic family of proteins. Like all secreted proteins, CDNF has a signal sequence at the N-terminus, but unlike common growth factors it has a KDEL-receptor retrieval sequence at the C-terminus. Thus, CDNF is mainly located in the ER. In response to adverse effects, such as ER stress, the expression of CDNF is upregulated and can alleviate ER stress. Also different from other neurotrophic factors, CDNF reduces protein aggregation and inflammation in disease models. Although it is an ER luminal protein, it can surprisingly directly interact with alpha-synuclein, a protein involved in the pathogenesis of synucleinopathies e.g., Parkinson's disease. Pleiotropic CDNF has therapeutic potential and has been tested as a recombinant human protein and gene therapy. The neuroprotective and neurorestorative effects have been described in a number of preclinical studies of Parkinson's disease, stroke and amyotrophic lateral sclerosis. Currently, it was successfully evaluated for safety in a phase 1/2 clinical trial for Parkinson's disease. Collectively, based on recent findings on the mode of action and therapeutic potential of CDNF, its use as a drug could be expanded to other ER stress-related diseases., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest. MS and MA are inventors of CDNF- and MANF-related patents owned by Herantis Pharma Plc and MS is the shareholder and the founder of the company., (Copyright © 2024. Published by Elsevier Inc.)

Ischemic cerebral stroke is a severe medical condition that affects about 15 million people every year and is the second leading cause of death and disability globally. Ischemic stroke results in neuronal cell death and neurological impairment. Current therapies may not adequately address the deleterious metabolic changes and may increase neurological damage. Oxygen and nutrient depletion along with the tissue damage result in endoplasmic reticulum (ER) stress, including the Unfolded Protein Response (UPR), and neuroinflammation in the affected area and cause cell death in the lesion core. The spatio-temporal production of lipid mediators, either pro-inflammatory or pro-resolving, decides the course and outcome of stroke. The modulation of the UPR as well as the resolution of inflammation promotes post-stroke cellular viability and neuroprotection. However, studies about the interplay between the UPR and bioactive lipid mediators remain elusive and this review gives insights about the crosstalk between lipid mediators and the UPR in ischemic stroke. Overall, the treatment of ischemic stroke is often inadequate due to lack of effective drugs, thus, this review will provide novel therapeutical strategies that could promote the functional recovery from ischemic stroke., Competing Interests: Conflict of Interest Authors declare the absence of any conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Stroke is a devastating medical condition with no treatment to hasten recovery. Its abrupt nature results in cataclysmic changes in the affected tissues. Resident cells fail to cope with the cellular stress resulting in massive cell death, which cannot be endogenously repaired. A potential strategy to improve stroke outcomes is to boost endogenous pro-survival pathways. The unfolded protein response (UPR), an evolutionarily conserved stress response, provides a promising opportunity to ameliorate the survival of stressed cells. Recent studies from us and others have pointed toward mesencephalic astrocyte-derived neurotrophic factor (MANF) being a UPR responsive gene with an active role in maintaining proteostasis. Its pro-survival effects have been demonstrated in several disease models such as diabetes, neurodegeneration, and stroke. MANF has an ER-signal peptide and an ER-retention signal; it is secreted by ER calcium depletion and exits cells upon cell death. Although its functions remain elusive, conducted experiments suggest that the endogenous MANF in the ER lumen and exogenously administered MANF protein have different mechanisms of action. Here, we will revisit recent and older bodies of literature aiming to delineate the expression profile of MANF. We will focus on its neuroprotective roles in regulating neurogenesis and inflammation upon post-stroke administration. At the same time, we will investigate commonalities and differences with another UPR responsive gene, X-box binding protein 1 (XBP1), which has recently been associated with MANF's function. This will be the first systematic comparison of these two UPR responsive genes aiming at revealing previously uncovered associations between them. Overall, understanding the mode of action of these UPR responsive genes could provide novel approaches to promote cell survival., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lõhelaid, Anttila, Liew, Tseng, Teppo, Stratoulias and Airavaara.)

A catalase-related allene oxide synthase (cAOS) or a hydroperoxide lyase (cHPL) fused together with an 8R-lipoxygenase is involved in the stress signaling of corals via an arachidonic acid pathway. cAOS gives rise to α-ketol and cyclopentenone, while cHPL catalyzes the cleavage of 8R-hydroperoxyeicosatetraenoic acid (8R-HpETE) to C8-oxo acid and C12 aldehyde. In silico analysis of the substrate entry sites of highly identical coral cAOS and cHPL indicated that two positively charged residues of cAOS, K60 and K107, and the corresponding residues of cHPL, E60 and K107, may be involved in the anchoring of the carboxy group of polyunsaturated fatty acid (PUFA) hydroperoxides. A mutational analysis of cAOS and cHPL revealed that K60 or E60 and K107 were not necessary in the tethering of 8R-HpETE, however, the E60 of cHPL was essential in the productive binding of PUFA hydroperoxides. The substrate preferences of cAOS and cHPL were determined with hydroperoxy derivatives of C18, C20, C22 PUFAs, anandamide (AEA), 1-arachidonoyl glycerol (1-AG) and selected methylated substrates. Although cAOS and cHPL were able to metabolize different free PUFA substrates and arachidonoyl derivatives, only cHPL catalyzed the reaction with methylated PUFA hydroperoxides. The differences in the substrate binding and preferences between cAOS and cHPL can be explained by the distinct properties of their substrate entry sites. The current study demonstrated that homologous PUFA metabolizing enzymes may contribute to the versatile usage of the substrate pool., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Oxylipins are well-established lipid mediators in plants and animals. In mammals, arachidonic acid (AA)-derived eicosanoids control inflammation, fever, blood coagulation, pain perception and labor, and, accordingly, are used as drugs, while lipoxygenases (LOX), as well as cyclooxygenases (COX) serve as therapeutic targets for drug development. In soft corals, eicosanoids are synthesized on demand from AA by LOX, COX, and catalase-related allene oxide synthase-lipoxygenase (cAOS-LOX) and hydroperoxide lyase-lipoxygenase (cHPL-LOX) fusion proteins. Reef-building stony corals are used as model organisms for the stress-related genomic studies of corals. Yet, the eicosanoid synthesis capability and AA-derived lipid mediator profiles of stony corals have not been determined. In the current study, the genomic and transcriptomic data about stony coral LOXs, AOS-LOXs, and COXs were analyzed and the eicosanoid profiles and AA metabolites of three stony corals, Acropora millepora , A. cervicornis , and Galaxea fascicularis , were determined by reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with MS-MS and a radiometric detector. Our results confirm that the active LOX and AOS-LOX pathways are present in Acropora sp., which correspond to the genomic/sequence data reported earlier. In addition, LOX, AOS-LOX, and COX products were detected in the closely related species G. fascicularis . In conclusion, the functional 8 R -LOX and/or AOS-LOX pathways are abundant among corals, while COXs are restricted to certain soft and stony coral lineages., Competing Interests: Authors declare no conflict of interest.

Two highly identical fusion proteins, an allene oxide synthase-lipoxygenase (AOS-LOX) and a hydroperoxide lyase-lipoxygenase (HPL-LOX), were identified in the soft coral Capnella imbricata. Both enzymes initially catalyze the formation of 8R-hydroperoxy-eicosatetraenoic acid (8R-HpETE) from arachidonic acid by the C-terminal lipoxygenase (LOX) domain. Despite the fact that the defined catalytically important residues of N-terminal catalase-related allene oxide synthase (cAOS) domain are also conserved in C. imbricata hydroperoxide lyase (cHPL), their reaction specificities differ. In the present study, we tested which of the amino acid substitutions around the active site of cHPL are responsible for a control in the reaction specificity. The possible candidates were determined via comparative sequence and structural analysis of the substrate channel and the heme region of coral cAOSs and C. imbricata cHPL. The amino acid replacements in cHPL-R56G, ME59-60LK, P65A, F150L, YS176-177NL, I357V, and SSSAGE155-160PVKEGD-with the corresponding residues of cAOS were conducted by site-directed mutagenesis. Although all these mutations influenced the catalytic efficiency of cHPL, only F150L and YS176-177NL substitutions caused a shift in the reaction specificity from HPL to AOS. The docking analysis of P. homomalla cAOS with 8R-HpETE substrate revealed that the Leu150 of cAOS interacts with the C5-C6 double bond and the Leu177 with the hydrophobic tail of 8R-HpETE. We propose that the corresponding residues in cHPL, Phe150 and Ser177, are involved in a proper coordination of the epoxy allylic radical intermediate necessary for aldehyde formation in the hydroperoxide lyase reaction.

In corals a catalase-lipoxygenase fusion protein transforms arachidonic acid to the allene oxide 8R,9-epoxy-5,9,11,14-eicosatetraenoic acid from which arise cyclopentenones such as the prostanoid-related clavulones. Recently we cloned two catalase-lipoxygenase fusion protein genes (a and b) from the coral Capnella imbricata, form a being an allene oxide synthase and form b giving uncharacterized polar products (Lõhelaid, H., Teder, T., Tõldsepp, K., Ekins, M., and Samel, N. (2014) PloS ONE 9, e89215). Here, using HPLC-UV, LC-MS, and NMR methods, we identify a novel activity of fusion protein b, establishing its role in cleaving the lipoxygenase product 8R-hydroperoxy-eicosatetraenoic acid into the short-chain aldehydes (5Z)-8-oxo-octenoic acid and (3Z,6Z)-dodecadienal; these primary products readily isomerize in an aqueous medium to the corresponding 6E- and 2E,6Z derivatives. This type of enzymatic cleavage, splitting the carbon chain within the conjugated diene of the hydroperoxide substrate, is known only in plant cytochrome P450 hydroperoxide lyases. In mechanistic studies using (18)O-labeled substrate and incubations in H2(18)O, we established synthesis of the C8-oxo acid and C12 aldehyde with the retention of the hydroperoxy oxygens, consistent with synthesis of a short-lived hemiacetal intermediate that breaks down spontaneously into the two aldehydes. Taken together with our initial studies indicating differing gene regulation of the allene oxide synthase and the newly identified catalase-related hydroperoxide lyase and given the role of aldehydes in plant defense, this work uncovers a potential pathway in coral stress signaling and a novel enzymatic activity in the animal kingdom., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

In octocorals, a catalase-like allene oxide synthase (AOS) and an 8R-lipoxygenase (LOX) gene are fused together encoding for a single AOS-LOX fusion protein. Although the AOS-LOX pathway is central to the arachidonate metabolism in corals, its biological function in coral homeostasis is unclear. Using an acute incision wound model in the soft coral Capnella imbricata, we here test whether LOX pathway, similar to its role in plants, can contribute to the coral damage response and regeneration. Analysis of metabolites formed from exogenous arachidonate before and after fixed time intervals following wounding indicated a significant increase in AOS-LOX activity in response to mechanical injury. Two AOS-LOX isoforms, AOS-LOXa and AOS-LOXb, were cloned and expressed in bacterial expression system as active fusion proteins. Transcription levels of corresponding genes were measured in normal and stressed coral by qPCR. After wounding, AOS-LOXa was markedly up-regulated in both, the tissue adjacent to the incision and distal parts of a coral colony (with the maximum reached at 1 h and 6 h post wounding, respectively), while AOS-LOXb was stable. According to mRNA expression analysis, combined with detection of eicosanoid product formation for the first time, the AOS-LOX was identified as an early stress response gene which is induced by mechanical injury in coral.

Prostaglandins, well-known lipid mediators in vertebrate animals, have also shown to play certain regulatory roles in insects and other arthropods acting on reproduction, immune system and ion transport. However, knowledge of their biosynthetic pathways in arthropods is lacking. In the present study, we report the cloning and expression of cyclooxygenase (COX) from amphipod crustaceans Gammarus spp and Caprella spp. The amphipod COX proteins contain key residues shown to be important for cyclooxygenase and peroxidase activities. Differently from all other known cyclooxygenases the N-terminal signal sequence of amphipod enzymes is not cleaved during protein expression in mammalian cells. The C-terminus of amphipod COX is shorter than that of mammalian isoforms and lacks the KDEL(STEL)-type endoplasmic reticulum retention/retrieval signal. Despite that, amphipod COX proteins are N-glycosylated and locate similarly to the vertebrate COX on the endoplasmic reticulum and nuclear envelope. Both amphipod COX mRNAs encode functional cyclooxygenases that catalyze the transformation of arachidonic acid into prostaglandins. Using bioinformatic analysis we identified a COX-like gene from the human body louse Pediculus humanus corporis genome that encodes a protein with about 30% sequence identity with human COX-1 and COX-2. Although the COX gene is known to be absent from genomes of Drosophila sp., Aedes aegypti, Bombyx mori, and other insects, our studies establish the existence of the COX gene in certain lineages within the insect world., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The conversion of fatty acid hydroperoxides to allene epoxides is catalysed by a cytochrome P450 in plants. In contrast, in the coral Plexaura homomalla, a catalase-related hemoprotein fused to the lipoxygenase (LOX) was found to function as an allene oxide synthase. This work reports the homology-based RT-PCR cloning and functional expression of a Gersemia fruticosa analogue of the allene oxide synthase-lipoxygenase (AOS-LOX) fusion protein. The G. fruticosa mRNA codes for a protein with 84% sequence identity to the P. homomalla AOS-LOX. Our data indicate that the AOS-LOX fusion protein pathway is used by another coral and P. homomalla represents no exception.