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. 2017 Mar;13(1):75-87.
doi: 10.1007/s11302-016-9541-4. Epub 2016 Oct 19.

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

David Reigada  1 , Rosa María Navarro-Ruiz  2 , Marcos Javier Caballero-López  2 , Ángela Del Águila  2 , Teresa Muñoz-Galdeano  2 , Rodrigo M Maza  2 , Manuel Nieto-Díaz  2
Affiliations

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

David Reigada et al. Purinergic Signal. 2017 Mar.

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.

Keywords: Apoptosis; Diadenosine; Excitotoxicity; Intracellular calcium; Mouse model; Neuro-2a; Neuroprotection; Secondary injury; Spinal cord injury; Tissue damage.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Excitotoxic-induced cell death pathways. Massive release of glutamate and ATP from necrotic and broken cells at trauma level activates NMDA and purinergic receptors expressed in neighbor cells. The subsequent increase in intracellular calcium concentration induces the activation of several cell death pathways implying endoplasmic reticulum and oxidative stress, autophagy, pro-apoptotic caspase cleavage, etc. All these mechanisms are part of the secondary wave of neural death activated minutes to few days after injury in the spinal cord
Fig. 2
Fig. 2
Reduction of ATP-induced apoptotic cell death by Ap4A in Neuro-2a cells. Neuroprotective effect of Ap4A was analyzed by flow cytometry in Neuro-2a cultures stimulated with ATP (300 μM, 24 h). Panel a shows representative flow cytometry histogram of cell cycle of propidium iodide-stained Neuro-2a under ATP treatment, with (gray trace) or without (black trace) Ap4A pre-treatment (100 μM, 24 h previous to ATP treatment). Insert in a shows the sub-G0/G1 region (apoptotic cells with condensed nuclei) of the cell cycle. The bar graph in b represents the percentages of apoptotic cells in each condition, showing an increase in cell death due to ATP treatment that was significantly reduced by Ap4A pre-treatment (mean ± standard deviation, n = 3). ***p < 0.001; **p < 0.01; *p < 0.05; n.s = non significant (Student’s t test)
Fig. 3
Fig. 3
Reduction of intracellular calcium response to ATP by Ap4A treatment in Neuro-2a cells. Representative traces of ATP-induced calcium rise determinations (black) a due to P2 receptor activity and the inhibitory effect of a 24-h pre-treatment with Ap4A (100 μM, 24 h, gray). In b, we summarize the dose response in intracellular calcium by stimulation with ATP with (gray) or without (black) a 24-h pre-treatment with Ap4A
Fig. 4
Fig. 4
Effect of Ap4A on P2 responses and expression. The effect of Ap4A on ATP-induced calcium rise can be due to a partial agonism effect and/or reduction in receptor expression. Representative traces of calcium determinations after addition of ATP (300 μM) and Ap4A (100 μM) (a, b, and c) show a lower effect of Ap4A, maybe due to a partial agonism on P2X receptors. Western blot analysis of P2X and P2Y receptor expression d showed that Ap4A treatment (100 μM, 24 h) reduces the expression of P2X2, P2X7, P2Y1, and P2Y2 receptors in Neuro-2a cells, while the expression of P2X1, P2X4, and P2Y6 receptors remains unchanged. Panel d shows normalized expression versus the housekeeping protein β-tubulin
Fig. 5
Fig. 5
Improvement of mouse locomotor capacities by Ap4A treatment after SCI. We used the open field Basso Mouse Scale (BMS) to determine the locomotor skills of spinal cord injured mice after Ap4A treatment. BMS score (a) revealed that intraperitoneal treatment with Ap4A (20 mg/Kg) ameliorated the locomotor impairment derived from SCI. Improvements become more evident when locomotion parameters are coded according to the BMS subscore (b) (symbols represent mean ± SEM). The specific analysis of interlimb coordination (c) show that Ap4A treatment significantly increases the percentage of animals with coordinative capacities 21 days post-injury (DPI; *p < 0.05 in a chi-square test). In agreement, rotarod analysis of coordination and balance of mice at 14 and 21 DPI (d) reveal an increase of the latency time in Ap4A-treated mice versus vehicle-treated (bars represent the median and dispersion of the sample. *p < 0.05 and ***p < 0.001 vs. vehicle; † p < 0.05, †† p < 0.01, and ††† p < 0.001 vs. sham in Student’s t test; n = 6–12)
Fig. 6
Fig. 6
Reduction of histological damage after SCI by Ap4A treatment. a Representative slices of the spinal cord stained with eriochrome cianine used to measure the spared white matter tissue at 21 DPI. Note that caudal sections in Ap4A-treated animals, but not in vehicle-treated ones, partially preserve the tissue structure of the naïve spinal cord. Scale bar for all images = 0.5 mm. Codes in the y-axis correspond to Cd = caudal; Ep = epicenter, and Rt = rostral. Estimations of the percentage of preserved white matter in transverse sections comprising 3 mm surrounding the injury epicenter of the spinal cord b show that Ap4A causes a significant increase of spared tissue area in caudal segments of the injury zone (symbols represent mean ± SEM; *p < 0.05 in Student’s t test vs. vehicle; n = 4–7). The estimated volume of spared white matter c at the injury epicenter, and the adjacent caudal and rostral regions (300 μm long in the three cases) also reveals the protective effect of Ap4A in the region caudal to the injury zone relative to the vehicle. d This greater tissue conservation in caudal areas significantly correlates with the locomotor improvements observed in BMS scale evaluations (see Fig. 5) (bars represent means ± SEM; † p < 0.05, †† p < 0.01, and ††† p < 0.01 vs. sham; **p < 0.01 in Student’s t test vs. vehicle; n = 4–7)
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