Neuropeptide Y receptors activation protects rat retinal neural cells against necrotic and apoptotic cell death induced by glutamate

Abstract

It has been claimed that glutamate excitotoxicity might have a role in the pathogenesis of several retinal degenerative diseases, including glaucoma and diabetic retinopathy. Neuropeptide Y (NPY) has neuroprotective properties against excitotoxicity in the hippocampus, through the activation of Y1, Y2 and/or Y5 receptors. The principal objective of this study is to investigate the potential protective role of NPY against glutamate-induced toxicity in rat retinal cells (in vitro and in an animal model), unraveling the NPY receptors and intracellular mechanisms involved. Rat retinal neural cell cultures were prepared from newborn Wistar rats (P3-P5) and exposed to glutamate (500 μM) for 24 h. Necrotic cell death was evaluated by propidium iodide (PI) assay and apoptotic cell death using TUNEL and caspase-3 assays. The cell types present in culture were identified by immunocytochemistry. The involvement of NPY receptors was assessed using selective agonists and antagonists. Pre-treatment of cells with NPY (100 nM) inhibited both necrotic cell death (PI-positive cells) and apoptotic cell death (TUNEL-positive cells and caspase 3-positive cells) triggered by glutamate, with the neurons being the cells most strongly affected. The activation of NPY Y2, Y4 and Y5 receptors inhibited necrotic cell death, while apoptotic cell death was only prevented by the activation of NPY Y5 receptor. Moreover, NPY neuroprotective effect was mediated by the activation of PKA and p38K. In the animal model, NPY (2.35 nmol) was intravitreally injected 2 h before glutamate (500 nmol) injection into the vitreous. The protective role of NPY was assessed 24 h after glutamate (or saline) injection by TUNEL assay and Brn3a (marker of ganglion cells) immunohistochemistry. NPY inhibited the increase in the number of TUNEL-positive cells and the decrease in the number of Brn3a-positive cells induced by glutamate. In conclusion, NPY and NPY receptors can be considered potential targets to treat retinal degenerative diseases, such as glaucoma and diabetic retinopathy.

Neuropeptide Y system in the retina: From localization to function

“…More recently, we showed that microglial cells present NPY Y 1 , Y 2 and Y 5 receptors-IR (Ambrosio et al, 2013;Santos-Carvalho et al, 2013a). Additionally, when these rat retinal cell cultures are exposed to an excitotoxic insult (glutamate), there is an increase of microglia proliferation and activation (Santos-Carvalho et al, 2013b). We also showed that the activation of NPY Y 1 receptor has an anti-inflammatory effect in retinal microglia (Ambrosio et al, 2013), suggesting that NPY system can be targeted as a strategy to delay the progression of retinal degenerative diseases with a neuroinflammatory component.…”

“…The neuroprotective effects of NPY against excitotoxicity have been investigated in different CNS regions, and also in the retina (Alvaro et al, 2008b(Alvaro et al, , 2009Santos-Carvalho et al, 2013b;Silva et al, 2003Silva et al, , 2005Smialowska et al, 2009;Xapelli et al, 2006Xapelli et al, , 2007. We have shown that NPY protects retinal neurons against 3,4-methylenedioxymethamphetamine (MDMA)-induced toxicity in rat retinal neural cell cultures (Alvaro et al, 2008b).…”

“…More recently, we also showed, in rat retinal cell cultures, that the activation of NPY Y 2 , Y 4 or Y 5 receptors protects retinal cells against necrotic cell death (Santos-Carvalho et al, 2013b). In addition, the NPY Y 5 receptor activation protects retinal cells against glutamateinduced apoptotic cell death (Santos-Carvalho et al, 2013b). Moreover, the neuroprotective effect of NPY is mediated by the activation of protein kinase A (PKA) and protein-38-kinase (p38K).…”

New strategies for neuroprotection in glaucoma, a disease that affects the central nervous system

“…Y2 and Y4 receptors are involved in the protective effect of neuropeptide Y against necrotic, while Y5 against necrotic and apoptotic cell death induced by glutamate (Santos-Carvalho et al, 2013).…”

Adenosine A 3 receptor activation is neuroprotective against retinal neurodegeneration

“…In order to assess the potential neuroprotective effects mediated by A 3 R activation, TUNEL assay ( Figure 1A and B) was performed in a primary retinal neural cell culture, well characterized in our lab (Costa et al, 2009;Santiago et al, 2007;Santos-Carvalho et al, 2013b). Retinal cultures were incubated with the selective A 3 R agonist ( …”