Abstract
Prolactin (PRL) is a peptidic hormone that displays pleiotropic functions in the organism including different actions in the brain. PRL exerts a neuroprotective effect against excitotoxicity produced by glutamate (Glu) or kainic acid in both in vitro and in vivo models. It is well known that Glu excitotoxicity causes cell death through apoptotic or necrotic pathways due to intracellular calcium ([Ca2+] i) overload. Therefore, the aim of the present study was to assess the molecular mechanisms by which PRL maintains cellular viability of primary cultures of rat hippocampal neurons exposed to Glu excitotoxicity. We determined cell viability by monitoring mitochondrial activity and using fluorescent markers for viable and dead cells. The intracellular calcium level was determined by a fluorometric assay and proteins involved in the apoptotic pathway were determined by immunoblot. Our results demonstrated that PRL afforded neuroprotection against Glu excitotoxicity, as evidenced by a decrease in propidium iodide staining and by the decrease of the LDH activity. In addition, the MTT assay shows that PRL maintains normal mitochondrial activity even in neurons exposed to Glu. Furthermore, the Glu-induced intracellular [Ca2+]i overload was attenuated by PRL. These data correlate with the reduction found in the level of active caspase-3 and the pro-apoptotic ratio (Bax/Bcl-2). Concomitantly, PRL elicited the nuclear translocation of the transcriptional factor NF-κB, which was detected by immunofluorescence and confocal microscopy. To our knowledge, this is the first report demonstrating that PRL prevents Glu excitotoxicity by a mechanism involving the restoration of the intracellular calcium homeostasis and mitochondrial activity, as well as an anti-apoptotic action possibly mediated by the activity of NF-κB. Overall, the current results suggest that PRL could be of potential therapeutic advantage in the treatment of neurodegenerative diseases.
Highlights
It is well known that glutamate (Glu) is the main excitatory neurotransmitter in the central nervous system, and that excitotoxicity is induced by the sustained stimulation of glutamatergic receptors, which include: N-methyl-D-aspartic acid (NMDA), α-amino-3-hydroxy5-methylisoxazole-4-propionate (AMPA) and kainic acid (KA) receptors [1]
PRL prevents cell death and mitochondrial dysfunction caused by Gluinduced excitotoxicity in hippocampal neuronal cultures
The neuroprotective action of PRL observed in the Syto-13/PI-staining and in the LDH assay presented in this study are in accordance with previous reports by our group, in which we demonstrate that PRL stimulates hippocampal neuronal survival, both in lactating rats [13,14] and in ovariectomized PRL-treated animals [16]
Summary
Glu excitotoxicity causes neuronal cell death through the disruption of intracellular calcium ([Ca2+]i) homeostasis, which is followed by mitochondrial uncoupling and activation of the intrinsic mitochondrial apoptosis pathway by triggering caspases activation [2] This condition is one of the main characteristics of many neurodegenerative disorders, including, ischemic injury, epilepsy, traumatic brain injury and neurodegenerative diseases such as Alzheimers and Parkinsons [3,4]. Examples of the former are the induction of maternal behavior [7] and reduction of anxiety [8] Among the latter effects are the stimulation of neurogenesis in the olfactory bulb [9], glia activation [10], remyelination of oligodendrocytes [11], and proliferation of precursor cells in the adult mouse hippocampus [12]. Both in vivo and in vitro studies by our group, have the increased evidence supporting that PRL affords neuroprotection against excitotoxicity induced by kainic acid and Glu [13,14,15,16]
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