Apelin-13 confers Neuropeptide Y–mediated neuroprotection and preserves learning and allocentric memory in D-glutamic acid-induced excitotoxicity in rats

Glutamate-mediated excitotoxicity leads to mitochondrial dysfunction, apoptosis, and neuronal cell death. This study aims to investigate whether NPY2 receptors (NPY2R) and NPY5 receptors (NPY5R) enhance the effects of Apelin-13/APJ signaling pathways as modulatory cofactors in the neuroprotection provided by Apelin-13 against excitotoxic damage and in the prevention of learning-memory disorders. D-Glutamic acid-induced excitotoxicity was established in 42 male Sprague–Dawley rats (6–8 weeks, 200-250 g). Animals were randomly divided into six groups (n = 7); Control (C; 0.9% NaCl, i.p), D-Glutamic Acid (G; 4 mg/kg, i.p), Apelin-13 (A; 300 µg/kg, i.p), D-Glutamic Acid + Apelin-13 (GA), D-Glutamic Acid + Apelin-13 + NPY2R antagonist (GAN2; 1,5 mg/kg, i.p) and D-Glutamic Acid + Apelin-13 + NPY5R antagonist (GAN5; 1,5 mg/kg, i.p). Locomotor activity were evaluated with the Open Field (OFT), short/long-term memory and learning performance, allocentric-egocentric orientation were assesed with novel object recognition (NORT) and Morris water maze (MWM) tests. All parameters were normalized to the C group, and statistical significance between groups was assesed. In group G, a significant decrease (p < 0.001) in Extracellular Signal Regulatory Kinase (ERK1/2) and Protein Kinase B-1 (AKT-1) levels and an increase (p < 0.001) in Caspase-3 were observed. Oxidative parameters increased in the G and GAN2 groups. Antioxidant parameters were also elevated in GA and GAN5, similar to C and A groups. An increase in MWM latency to the target quadrant (p < 0.001) and a decrease in NORT discrimination index (p < 0.001) were found in the G and GAN2 groups compare to the C and A. Histochemical staining scores showed that the protection of Apelin-13 was mediated by NPY2R. In GAN2, blocking NPY2R reduces Apelin-13’s neuroprotection, which is sustained only via NPY5R with limited effect. In GAN5, Apelin-13’s protection was enhanced through NPY2R, as shown with NPY5R blockade. Accordingly, Apelin-13 exerts its neuroprotective effects primarily through NPY2R, its modulatory influence via NPY5R appears to be comparatively limited.

Graphical Abstract

Graphical abstract illustrating the interplay between Apelin-13, NPY2 receptor (NPY2R) antagonist and NPY5 receptor (NPY5R) antagonist in modulating D-Glutamic acid induced excitotoxicity. Left side: This represents the normal glutamate excitotoxicity condition. Excessive glutamate release stimulates NMDA and AMPA receptors found in the postsynaptic membrane, leading to Na ⁺ and Ca ⁺² overload, ROS/RNS production, mitochondrial dysfunction, apoptosis, neuronal death, and cognitive impairment. Middle: Exogenous administration of Apelin-13 along with an NPY2R antagonist results in selective blockade of NPY2R while leaving NPY5R active. NPY2R blockade fails to inhibit presynaptic glutamate release, allowing continued activation of NMDA and AMPA receptors. Therefore, the neuroprotective effects of Apelin-13 are confined to NPY5R-mediated pathways. Right: When Apelin-13 is administered together with the NPY5R antagonist results in selective blockade of NPY5R while leaving NPY2R active. NPY2R activation inhibits presynaptic glutamate release, Apelin-13 also inhibits NMDA and AMPA receptors. Consequently, the neuroprotective effects of Apelin-13 are predominantly dependent on NPY2R.

VGCC: Voltage-gated calcium channel; Glu: Glutamate; APJ: Apelin receptor; NPY2R/NPY5R: Neuropeptide Y receptors 2 and 5; NPY: Neuropeptide Y; ROS/RNS: Reactive oxygen/nitrogen species, AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; NMDA: N-methyl-D-aspartate; Ca ⁺² : Calcium, Na ⁺ : Sodium.