Epo In Brain Research Articles

Tracking the ancestral functions of erythropoietin: neuroprotection & mitochondria

It has long been thought that erythropoietin (Epo) is exclusively involved in erythropoiesis; now, it is known that EPO in mammal's brain plays key roles in the development, maintenance, protection, and repair of the nervous system. Also, EPO in mammals contributes to the efficient use of oxygen through the regulation of mitochondrial bioenergetic. Remarkably, a similar neuroprotective impact of recombinant human EPO (rhEPO) has been found in the brain of grasshoppers, raising questions about the evolutive origin of the EPO and its generic molecular function. The objective of this study is to show that the neuroprotective effect of rhEPO in insects involves the regulation of mitochondrial functions. The experiments were performed in crickets (Acheta domesticus). These insects were exposed under normoxia and hypoxia (5 days; 6% O2) conditions. Before experimentation, the animals were treated with EPO (30 IU/ml ‐ intra‐lymphatic injection) or PBS, as a control. The brains of the crickets were removed, and then we determined the mitochondrial respiration and production of mitochondrial ROS using our system oxygraphy ‐ 2K (ORORBOROS). Our results show that compares to normoxia; hypoxia significantly reduces mitochondrial respiration of complexes 1 and 1&2. On the other hand, the treatment of EPO in hypoxia, despite significantly increasing these parameters, does not recover the levels of mitochondrial respiration under normoxic conditions. In addition, the activity of complex IV (an indicator of the number of mitochondria) does not vary significantly between any of the treatments. Furthermore, we observed that while hypoxia did not significantly affect H2O2 production, the treatment with EPO increased ROS production under normoxic but not hypoxic conditions. Our data suggest that rhEPO regulates in some way the mitochondrial respiration and ROS production in the brain of crickets. Considering that insects appeared during a geological period (Cambrian explosion) in which the atmospheric O2 was increasing, which could cause great oxidative stress due to the change in the metabolism of these animals, this molecule would have appeared as a regulator of mitochondrial functions.

Hypercapnic ventilatory response is decreased in a mouse model of excessive erythrocytosis.

The impact of cerebral erythropoietin (Epo) in the regulation of the hypercapnic ventilatory response (HcVR) is controversial. While we reported that cerebral Epo does not affect the central chemosensitivity in C57Bl6 mice receiving an intracisternal injection of sEpoR (the endogenous antagonist of Epo), a recent study in transgenic mice with constitutive high levels of human Epo in brain and circulation (Tg6) and in brain only (Tg21), showed that Epo blunts the HcVR, maybe by interacting with central and peripheral chemoreceptors. High Epo serum levels in Tg6 mice lead to excessive erythrocytosis (hematocrit ~80-90%), the main symptom of chronic mountain sickness (CMS). These latter results support the hypothesis that reduced central chemosensitivity accounts for the hypoventilation observed in CMS patients. To solve this intriguing divergence, we reevaluate HcVR in Tg6 and Tg21 mouse lines, by assessing the metabolic rate [O consumption (V̇) and CO production (V̇)], a key factor modulating ventilation, the effect of which was not considered in the previous study. Our results showed that the decreased HcVR observed in Tg6 mice (~70% reduction; < 0.01) was due to a significant decrease in the metabolism (~40%; < 0.0001) rather than Epo's effect on CO chemosensitivity. Additional analysis in Tg21 mice did not reveal differences of HcVR or metabolism. We concluded that cerebral Epo does not modulate the central chemosensitivity system, and that a metabolic effect upon CO inhalation is responsible for decreased HcVR observed in Tg6 animals. As CMS patients also show decreased HcVR, our findings might help to better understand respiratory disorders at high altitude.

Polycythemia and high levels of erythropoietin in blood and brain blunt the hypercapnic ventilatory response in adult mice.

Changes in arterial Po2, Pco2, and pH are the strongest stimuli sensed by peripheral and central chemoreceptors to adjust ventilation to the metabolic demand. Erythropoietin (Epo), the main regulator of red blood cell production, increases the hypoxic ventilatory response, an effect attributed to the presence of Epo receptors in both carotid bodies and key brainstem structures involved in integration of peripheral inputs and control of breathing. However, it is not known whether Epo also has an effect on the hypercapnic chemoreflex. In a first attempt to answer this question, we tested the hypothesis that Epo alters the ventilatory response to increased CO2 levels. Basal ventilation and hypercapnic ventilatory response (HCVR) were recorded from control mice and from two transgenic mouse lines constitutively expressing high levels of human Epo in brain only (Tg21) or in brain and plasma (Tg6), the latter leading to polycythemia. To tease apart the potential effects of polycythemia and levels of plasma Epo in the HCVR, control animals were injected with an Epo analog (Aranesp), and Tg6 mice were treated with the hemolytic agent phenylhydrazine after splenectomy. Ventilatory parameters measured by plethysmography in conscious mice were consistent with data from electrophysiological recordings in anesthetized animals and revealed a blunted HCVR in Tg6 mice. Polycythemia alone and increased levels of plasma Epo blunt the HCVR. In addition, Tg21 mice with an augmented level of cerebral Epo also had a decreased HCVR. We discuss the potential implications of these findings in several physiopathological conditions.

Cerebral erythropoietin regulates the respiratory control system during early postnatal life

Apart from its role in neuroprotection, erythropoietin (Epo) synthesized by neurons and astrocytes modulates the respiratory control system in adult mice. However, the impact of Epo in the regulation of the neural control of breathing during the postnatal development remains unknown. Here we use a transgenic mouse line (Tg21) constitutively overexpressing human Epo in brain to investigate whether Epo interacts with the brainstem centers associated with respiration during postnatal life. Our immunohistochemical analysis revealed that Epo and Epo receptor (EpoR) are expressed in the main central respiratory nuclei at 1, 4, 7, 10, 15 and 21 days of age. Furthermore, by using antibodies to choline acetyltranferase (ChAT), somatostatin (SST) and neurokinin receptor 1 (NK1), EpoR was localized to the pre‐Bötzinger complex nucleus at all studied postnatal days. Whole body phtethysmography technique was used to evaluate basal ventilation and ventilatory response to hypoxia at 10% and 6% O2. Compared to wild type animals, Tg21 mice showed improved hypoxic ventilation (both at 10% and 6% O2) at postnatal days 15 and 21, but not 7 and 10. These results are in line with data obtained by radioimmunoassay, showing that overexpression of Epo in transgenic mice brains dramatically increases after 10 days of postnatal life. These results imply that cerebral Epo plays a key‐regulating role in the neural control of breathing in a period in which the central respiratory network undergoes full development and maturation.Source of research support is Molly Towell Perinatal Research Foundation (MTPRF).

Acute and chronic exposure to hypoxia alters ventilatory pattern but not minute ventilation of mice overexpressing erythropoietin

Apart from enhancing red blood cell production, erythropoietin (Epo) has been shown to modulate the ventilatory response to reduced oxygen supply. Both functions are crucial for the organism to cope with increased oxygen demand. In the present work, we analyzed the impact of Epo and the resulting excessive erythrocytosis in the neural control of normoxic and hypoxic ventilation. To this end, we used our transgenic mouse line (Tg6) that shows high levels of human Epo in brain and plasma, the latter leading to a hematocrit of approximately 80%. Interestingly, while normoxic and hypoxic ventilation in Tg6 mice was similar to WT mice, Tg6 mice showed an increased respiratory frequency but a decreased tidal volume. Knowing that Epo modulates catecholaminergic activity, the altered catecholaminergic metabolism measured in brain stem suggested that the increased respiratory frequency in Tg6 mice was related to the overexpression of Epo in brain. In the periphery, higher response to hyperoxia (Dejours test), as well as reduced tyrosine hydroxylase activity in carotid bodies, revealed a higher chemosensitivity to oxygen in transgenic mice. Moreover, in line with the decreased activity of the rate-limiting enzyme for dopamine synthesis, the intraperitoneal injection of a highly specific peripheral ventilatory stimulant, domperidone, did not stimulate hypoxic ventilatory response in Tg6 mice. These results suggest that high Epo plasma levels modulate the carotid body's chemotransduction. All together, these findings are relevant for understanding the cross-talk between the ventilatory and erythropoietic systems exposed to hypoxia.

Brain‐derived erythropoietin protects from focal cerebral ischemia by dual activation of ERK‐1/‐2 and Akt pathways

Apart from its hematopoietic function, erythropoietin (Epo) exerts neuroprotective functions in brain hypoxia and ischemia. To examine the mechanisms mediating Epo's neuroprotective activity in vivo, we made use of our transgenic mouse line tg21 that constitutively expresses human Epo in brain without inducing excessive erythrocytosis. We show that human Epo is expressed in tg21 brains and that cortical and striatal neurons carry the Epo receptor. After middle cerebral artery occlusion, human Epo potently protected brains of tg21 mice against ischemic injury, both when severe (90 min) and mild (30 min) ischemia was imposed. Histochemical studies revealed that Epo induced an activation of JAK-2, ERK-1/-2, and Akt pathways in the ischemic brain. This activation was associated with elevated Bcl-XL and decreased NO synthase-1 and -2 levels in neurons. Intracerebroventricular injections of selective inhibitors of ERK-1/-2 (PD98059) or Akt (wortmannin) pathways revealed that both ERK-1/-2 and Akt were required for Epo's neuroprotective function, antagonization of either pathway completely abolishing tissue protection. On the other hand, ERK-1/-2 and Akt blockade did not reverse the neuronal NO synthase-1/-2 inhibition, indicating that Epo down-regulates these NO synthases in an ERK-1/-2 and Akt independent manner. On the basis of our data, the dual activation of ERK-1/-2 and Akt is crucial for Epo's neuroprotective activity.

Erythropoietin regulates hypoxic ventilation in mice by interacting with brainstem and carotid bodies

Apart from its role in elevating red blood cell number, erythropoietin (Epo) exerts protective functions in brain, retina and heart upon ischaemic injury. However, the physiological non-erythroid functions of Epo remain unclear. Here we use a transgenic mouse line (Tg21) constitutively overexpressing human Epo in brain to investigate Epo's impact on ventilation upon hypoxic exposure. Tg21 mice showed improved ventilatory response to severe acute hypoxia and moreover improved ventilatory acclimatization to chronic hypoxic exposure. Furthermore, following bilateral transection of carotid sinus nerves that uncouples the brain from the carotid body, Tg21 mice adapted their ventilation to acute severe hypoxia while chemodenervated wild-type (WT) animals developed a life-threatening apnoea. These results imply that Epo in brain modulates ventilation. Additional analysis revealed that the Epo receptor (EpoR) is expressed in the main brainstem respiratory centres and suggested that Epo stimulates breathing control by alteration of catecholaminergic metabolism in brainstem. The modulation of hypoxic pattern of ventilation after i.v. injection of recombinant human Epo in WT mice and the dense EpoR immunosignal observed in carotid bodies showed that these chemoreceptors are sensitive to plasma levels of Epo. In summary, our results suggest that Epo controls ventilation at the central (brainstem) and peripheral (carotid body) levels. These novel findings are relevant to understanding better respiratory disorders including those occurring at high altitude.