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Diadenosine tetraphosphate (Ap4A) inhibits ATP-induced excitotoxicity: a neuroprotective strategy for traumatic spinal cord injury treatment.

ABSTRACT: Reducing cell death during the secondary injury is a major priority in the development of a cure for traumatic spinal cord injury (SCI). One of the earliest processes that follow SCI is the excitotoxicity resulting from the massive release of excitotoxicity mediators, including ATP, which induce an excessive and/or prolonged activation of their receptors and a deregulation of the calcium homeostasis. Diadenosine tetraphosphate (Ap4A) is an endogenous purinergic agonist, present in both extracellular and intracellular fluids, with promising cytoprotective effects in different diseases including neurodegenerative processes. In a search for efficient neuroprotective strategies for SCI, we have tested the capability of Ap4A to reduce the excitotoxic death mediated by the ATP-induced deregulation of calcium homeostasis and its consequences on tissue preservation and functional recovery in a mouse model of moderate contusive SCI. Our analyses with the murine neural cell line Neuro2a demonstrate that treatment with Ap4A reduces ATP-dependent excitotoxic death by both lowering the intracellular calcium response and decreasing the expression of specific purinergic receptors. Follow-up analyses in a mouse model of contusive SCI showed that acute administration of Ap4A following SCI reduces tissue damage and improves motor function recovery. These results suggest that Ap4A cytoprotection results from a decrease of the purinergic tone preventing the effects of a massive release of ATP after SCI, probably together with a direct induction of anti-apoptotic and pro-survival pathways via activation of P2Y2 proposed in previous studies. In conclusion, Ap4A may be a good candidate for an SCI therapy, particularly to reduce excitotoxicity in combination with other modulators and/or inhibitors of the excitotoxic process that are being tested.

SUBMITTER: Reigada D

PROVIDER: S-EPMC5334201 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Diadenosine tetraphosphate (Ap<sub>4</sub>A) inhibits ATP-induced excitotoxicity: a neuroprotective strategy for traumatic spinal cord injury treatment.

Reigada David D Navarro-Ruiz Rosa María RM Caballero-López Marcos Javier MJ Del Águila Ángela Á Muñoz-Galdeano Teresa T Maza Rodrigo M RM Nieto-Díaz Manuel M

Purinergic signalling 20161019 1

Reducing cell death during the secondary injury is a major priority in the development of a cure for traumatic spinal cord injury (SCI). One of the earliest processes that follow SCI is the excitotoxicity resulting from the massive release of excitotoxicity mediators, including ATP, which induce an excessive and/or prolonged activation of their receptors and a deregulation of the calcium homeostasis. Diadenosine tetraphosphate (Ap<sub>4</sub>A) is an endogenous purinergic agonist, present in bot ...[more]

PMID: 27761681

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Project description:The opportunistic bacterial pathogen Pseudomonas aeruginosa causes a wide range of infections that are difficult to treat, largely because of the spread of antibiotic-resistant isolates. Antivirulence therapy, í.e. the use of drugs that inhibit the expression or activity of virulence factors, is currently considered an attractive strategy to reduce P. aeruginosa pathogenicity and complement antibiotic treatments. Because of the multifactorial nature of P. aeruginosa virulence and the broad arsenal of virulence factors this bacterium can produce, the regulatory networks that control the expression of multiple virulence traits have been extensively explored as potential targets for antivirulence drug development. The intracellular signaling molecule diadenosine tetraphosphate (Ap4A) has been reported to control stress resistance and virulence-related traits in some bacteria, but its role has not been investigated in P. aeruginosa so far. To fill this gap, we generated a mutant of the reference strain P. aeruginosa PAO1 that lacks the Ap4A-hydrolysing enzyme ApaH and, consequently, accumulates high intracellular levels of Ap4A. Phenotypic and transcriptomic analyses revealed that the lack of ApaH causes a drastic reduction in the expression of several virulence factors, including extracellular proteases, elastases, siderophores, and quorum sensing signal molecules. Accordingly, infection assays in plant and animal models demonstrated that ApaH-deficient cells are significantly impaired in infectivity and persistence in different hosts, including mice. Finally, deletion of apaH in P. aeruginosa clinical isolates demonstrated that the positive effect of ApaH on the production of virulence-related traits and on infectivity is conserved in P. aeruginosa. This study provides the first evidence that the Ap4A-hydrolysing enzyme ApaH is important for P. aeruginosa virulence, highlighting this protein as a novel potential target for antivirulence therapies against P. aeruginosa.

Project description:Diadenosine tetraphosphate (Ap4A) is a dinucleotide found in both prokaryotes and eukaryotes. In bacteria, its cellular levels increase following exposure to various stress signals and stimuli, and its accumulation is generally correlated with increased sensitivity to a stressor(s), decreased pathogenicity, and enhanced antibiotic susceptibility. Ap4A is produced as a by-product of tRNA aminoacylation, and is cleaved to ADP molecules by hydrolases of the ApaH and Nudix families and/or by specific phosphorylases. Here, considering evidence that the recombinant protein YqeK from Staphylococcus aureus copurified with ADP, and aided by thermal shift and kinetic analyses, we identified the YqeK family of proteins (COG1713) as an unprecedented class of symmetrically cleaving Ap4A hydrolases. We validated the functional assignment by confirming the ability of YqeK to affect in vivo levels of Ap4A in B. subtilis YqeK shows a catalytic efficiency toward Ap4A similar to that of the symmetrically cleaving Ap4A hydrolases of the known ApaH family, although it displays a distinct fold that is typical of proteins of the HD domain superfamily harboring a diiron cluster. Analysis of the available 3D structures of three members of the YqeK family provided hints to the mode of substrate binding. Phylogenetic analysis revealed the occurrence of YqeK proteins in a consistent group of Gram-positive bacteria that lack ApaH enzymes. Comparative genomics highlighted that yqeK and apaH genes share a similar genomic context, where they are frequently found in operons involved in integrated responses to stress signals.IMPORTANCE Elevation of Ap4A level in bacteria is associated with increased sensitivity to heat and oxidative stress, reduced antibiotic tolerance, and decreased pathogenicity. ApaH is the major Ap4A hydrolase in gamma- and betaproteobacteria and has been recently proposed as a novel target to weaken the bacterial resistance to antibiotics. Here, we identified the orphan YqeK protein family (COG1713) as a highly efficient Ap4A hydrolase family, with members distributed in a consistent group of bacterial species that lack the ApaH enzyme. Among them are the pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Mycoplasma pneumoniae By identifying the player contributing to Ap4A homeostasis in these bacteria, we disclose a novel target to develop innovative antibacterial strategies.

Dataset Information

Diadenosine tetraphosphate (Ap4A) inhibits ATP-induced excitotoxicity: a neuroprotective strategy for traumatic spinal cord injury treatment.

Publications

Diadenosine tetraphosphate (Ap<sub>4</sub>A) inhibits ATP-induced excitotoxicity: a neuroprotective strategy for traumatic spinal cord injury treatment.

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