Optimal neuroprotection by erythropoietin requires elevated expression of its receptor in neurons.

Erythropoietin receptor (EpoR) binding mediates neuroprotection by endogenous Epo or by exogenous recombinant human (rh)Epo. The level of EpoR gene expression may determine tissue responsiveness to Epo. Thus, harnessing the neuroprotective power of Epo requires an understanding of the Epo-EpoR system and its regulation. We tested the hypothesis that neuronal expression of EpoR is required to achieve optimal neuroprotection by Epo. The ventral limbic region (VLR) in the rat brain was used because we determined that its neurons express minimal EpoR under basal conditions, and they are highly sensitive to excitotoxic damage, such as occurs with pilocarpine-induced status epilepticus (Pilo-SE). We report that (i) EpoR expression is significantly elevated in nearly all VLR neurons when rats are subjected to 3 moderate hypoxic exposures, with each separated by a 4-day interval; (ii) synergistic induction of EpoR expression is achieved in the dorsal hippocampus and neocortex by the combination of hypoxia and exposure to an enriched environment, with minimal increased expression by either treatment alone; and (iii) rhEpo administered after Pilo-SE cannot rescue neurons in the VLR, unless neuronal induction of EpoR is elicited by hypoxia before Pilo-SE. This study thus demonstrates using environmental manipulations in normal rodents, the strict requirement for induction of EpoR expression in brain neurons to achieve optimal neuroprotection. Our results indicate that regulation of EpoR gene expression may facilitate the neuroprotective potential of rhEpo.

Brain heparanase expression is up-regulated during postnatal development and hypoxia-induced neovascularization in adult rats.

Heparanase is an endo-beta-d-glucuronidase which specifically cleaves extracellular and cell surface heparan sulphates at intra-chain sites. Its enzymatic activity is strongly implicated in cell dissemination associated with tumor metastasis and inflammation. Indeed, heparanase gene is expressed in various tumors and its over-expression is correlated with increased tumor vascularity and metastatic potential of tumor cells. However, heparanase expression in non-invasive and non-immune tissue, including brain, has received less attention. Using RT-qPCR, western blot and histological analysis, we demonstrate in the adult rat that heparanase transcript is differentially expressed according to brain area, and that heparanase protein is mainly detected in neurons. Furthermore, we provide evidence that heparanase transcript and protein reach their greatest levels at early postnatal stages, in particular within the neocortex characterized by intensive structural plasticity. Using the in vitro model of PC12-induced neuronal differentiation, we suggest that developmental regulation of heparanase may coincide with axonal and dendritic pathfinding. At adulthood, we demonstrate that the increased heparanase transcript level correlates in the hippocampus with enhanced angiogenesis following repeated hypoxia exposures. Taken together, our results emphasize the potential importance of heparanase in brain homeostasis, both during development and adaptative responses to severe environmental challenges.

Unexpected expression of orexin-B in basal conditions and increased levels in the adult rat hippocampus during pilocarpine-induced epileptogenesis.

Orexin-A (OX-A) and -B (OX-B) peptides present in the hippocampus are considered to be exclusively contained in fibers arising from hypothalamus neurons, which were established as the only source of orexins (OXs). Because OX-A is known to exert excitatory actions in the hippocampus, we hypothesized that the level of OXs targeted toward the hippocampus may be increased following status-epilepticus (SE)-induced epileptogenesis in the rat pilocarpine model of temporal lobe epilepsy. We found that tissue concentration of prepro-OX mRNA, which encodes for both peptides, rapidly decreased in the hypothalamus of rats having experienced pilocarpine-induced SE (Pilo-SE) followed by a reduced density of OX-A and OX-B immunopositive fibers arising from these neurons. By contrast, it was unexpected to detect within the hippocampus the presence of prepro-OX mRNA in basal conditions and to evidence its up-regulation during the 1- to 3-day period following Pilo-SE. The number of prepro-OX mRNA copies determined by real-time RT-PCR was approximately 50-fold lower in the hippocampus than that in the hypothalamus, precluding the use of in situ hybridization to localize the cells which synthesize the transcript within the hippocampus. The increase in prepro-OX mRNA level within the hippocampus was accompanied by the detection of OX-B-like immunoreactivity 2-3 days post-SE, not only in pyramidal neurons, granule cells and cell bodies resembling interneurons, but also in some astrocytes scattered throughout the hippocampus. The present data suggest that the gene encoding OXs can be activated in the hippocampus, which may play a role in the pathogenesis of epilepsy.

First cellular approach of the effects of global warming on groundwater organisms: a study of the HSP70 gene expression.

Whereas the consequences of global warming at population or community levels are well documented, studies at the cellular level are still scarce. The study of the physiological or metabolic effects of such small increases in temperature (between +2 degrees C and +6 degrees C) is difficult because they are below the amplitude of the daily or seasonal thermal variations occurring in most environments. In contrast, subterranean biotopes are highly thermally buffered (+/-1 degrees C within a year), and underground water organisms could thus be particularly well suited to characterise cellular responses of global warming. To this purpose, we studied genes encoding chaperone proteins of the HSP70 family in amphipod crustaceans belonging to the ubiquitous subterranean genus Niphargus. An HSP70 sequence was identified in eight populations of two complexes of species of the Niphargus genus (Niphargus rhenorhodanensis and Niphargus virei complexes). Expression profiles were determined for one of these by reverse transcription and quantitative polymerase chain reaction, confirming the inducible nature of this gene. An increase in temperature of 2 degrees C seemed to be without effect on N. rhenorhodanensis physiology, whereas a heat shock of +6 degrees C represented an important thermal stress for these individuals. Thus, this study shows that although Niphargus individuals do not undergo any daily or seasonal thermal variations in underground water, they display an inducible HSP70 heat shock response. This controlled laboratory-based physiological experiment constitutes a first step towards field investigations of the cellular consequences of global warming on subterranean organisms.

Erythropoietin receptor expression is concordant with erythropoietin but not with common beta chain expression in the rat brain throughout the life span.

Brain effects of erythropoietin (Epo) are proposed to involve a heteromeric receptor comprising the classical Epo receptor (Epo-R) and the common beta chain (betac). However, data documenting the pattern of betac gene expression in the healthy brain, in comparison with that of the Epo-R gene, are still lacking. The present study is the first to investigate at the same time betac, Epo-R, and Epo gene expression within different rat brain areas throughout the life span, from neonatal to elderly stages, using quantitative RT-PCR for transcripts. Corresponding proteins were localized by using immunohistochemistry. We demonstrate that the betac transcript level does not correlate with that of Epo-R or Epo, whereas the Epo-R transcript level strongly correlates with that of Epo throughout the life span in all brain structures analyzed. Both Epo and Epo-R were detected primarily in neurons. In the hippocampus, the greatest Epo-R mRNA levels were measured during the early postnatal period and in middle-aged rats, associated with an intense neuronal immunolabeling. Conversely, betac protein was barely detectable in the brain at all ages, even in neurons expressing high levels of Epo-R. Finally, betac transcript could not be detected in PC12 cells, even after nerve growth factor-induced neuritogenesis, which is a condition that dramatically enhances Epo-R transcript level. Altogether, our data suggest that most neurons are likely to express high levels of Epo-R but low, if not null, levels of betac. Given that Epo protects extended populations of neurons after injury, a yet-to-be-identified receptor heterocomplex including Epo-R may exist in the large population of brain neurons that does not express betac.

Neuroprotective effects of erythropoietin in the rat hippocampus after pilocarpine-induced status epilepticus.

Neuroprotective functions of erythropoietin (Epo) are thought to involve a heteroreceptor composed of both Epo receptor (Epo-R) and common beta chain (betac). Here, we measured the response of hippocampal Epo system components (Epo, Epo-R and betac) during neurodegenerative processes following pilocarpine-induced status epilepticus (SE), and examined whether recombinant human Epo (rHuEpo) could support neuronal survival. We evidence that Epo is induced in astroglia following SE, in particular within areas displaying delayed neuronal death. In addition, we demonstrate for the first time that rHuEpo reduces considerably hippocampal neurodegeneration following SE. rHuEpo may thus supplement astroglial induction of Epo to promote enhanced hippocampal neuronal survival following SE. We also show that Epo-R is expressed by neurons and astrocytes mainly, while betac is barely detectable in basal conditions and induced in reactive microglia exclusively following SE. Altogether, our results suggest that Epo/rHuEpo exerts neuroprotection, through Epo-R signaling and independently of betac, and, therefore, may be anti-epileptogenic.