Prolactin-induced neuroprotection against glutamate excitotoxicity is mediated by the reduction of [Ca2+]i overload and NF-κB activation.

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.

Caspases and their role in inflammation and ischemic neuronal death. Focus on caspase-12.

Caspases are cysteine proteases, which play important roles in different processes including, apoptosis and inflammation. Caspase-12, expressed in mouse and human, is classified as an inflammatory caspase. However, in humans caspase-12 gene has acquired different mutations that result in the expression of different variants. Caspase-12 is generally recognized as a negative regulator of the inflammatory response induced by infections, because it inhibits the activation of caspase-1 in inflammasome complexes, the production of the pro-inflammatory cytokines IL-1ß and IL-18 and the overall response to sepsis. In contrast, caspase-4, the human paralog of caspase-12, exerts a positive modulatory action of the inflammatory response to infectious agents. The role of caspase-12 and caspase-4 in inflammation associated with cerebral ischemia, a condition that results from a transient or permanent reduction of cerebral blood flow, is still unknown. Among the mechanisms involved in ischemic brain injury, apoptosis and inflammation have important roles. Under these conditions, disturbances in the homeostasis of the endoplasmic reticulum (ER) take place, leading to ER stress, caspase activation and apoptosis. Caspase-12 up-regulation and processing has been observed after the ischemic episode but its role in apoptosis is controversial. Cleavage of caspase-4 also occurs during ER stress but its role in ischemic brain injury is unknown. Throughout this review evidence supporting a role of caspase-12 and caspase-4 on the modulation of the inflammatory response to infection and their potential contribution to ER stress-induced apoptosis, is discussed. Understanding the actions of rodent caspase-12 and human caspase-4 will help us to elucidate their role in different pathological conditions, which to date is not well understood.

Neuroprotective role of estradiol against neuronal death induced by glucose deprivation in cultured rat hippocampal neurons.

Studies have reported the protective effect of estradiol (E(2)) against neuronal death induced by several insults including oxygen deprivation, mitochondrial toxins and activation of glutamate receptors. Glucose deprivation (GD) is associated with ischemia and hypoglycemia, and to date there is no effective therapeutic agent able to prevent neuronal damage induced by these conditions. In this study, we have investigated the effects of 17ß-E(2) and the selective agonists of the alpha (ERα) and beta (ERß) estrogen receptors, propyl pyrazole triol (PPT) and diarylpropionitrile (DPN), respectively, on neuronal death induced by GD in cultured rat hippocampal neurons. We have also analyzed the expression of both ER isoforms after GD. Results show that GD for 2 and 4 h reduces cell survival by 42 and 55%, respectively. Treatment with 17ß-E(2) (10 nM to 10 µM) induces a dose-dependent protective effect that is blocked by ICI 182,780, an ER antagonist, and by 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(-piperidinylethoxy)phenol]-1H'pyrazole dihydrochloride (MPP) and 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP), selective ERα and ERß antagonists, respectively. The ERα and ERß agonists PPT and DPN show a similar neuroprotective effect to that of 17ß-E(2), but DPN is more efficient. In addition, hippocampal neurons under normal conditions show a higher expression of the ERß isoform. When exposed to GD during 4 h, the expression of both ER isoforms is increased, while only that of the ERß isoform significantly increases after 2 h of GD. Results demonstrate that E(2) prevents neuronal death induced by GD through its interaction with ER, although the ERß isoform might have a predominant role. Results also suggest that GD differentially alters the expression of ERα and ERß in hippocampal neurons.