Martin Hasselblatt

Erythropoietin: a candidate compound for neuroprotection in schizophrenia

Erythropoietin (EPO) is a candidate compound for neuroprotection in human brain disease capable of combating a spectrum of pathophysiological processes operational during the progression of schizophrenic psychosis. The purpose of the present study was to prepare the ground for its application in a first neuroprotective add-on strategy in schizophrenia, aiming at improvement of cognitive brain function as well as prevention/slowing of degenerative processes. Using rodent studies, primary hippocampal neurons in culture, immunohistochemical analysis of human post-mortem brain tissue and nuclear imaging technology in man, we demonstrate that: (1) peripherally applied recombinant human (rh) EPO penetrates into the brain efficiently both in rat and humans, (2) rhEPO is enriched intracranially in healthy men and more distinctly in schizophrenic patients, (3) EPO receptors are densely expressed in hippocampus and cortex of schizophrenic subjects but distinctly less in controls, (4) rhEPO attenuates the haloperidol-induced neuronal death in vitro, and (4) peripherally administered rhEPO enhances cognitive functioning in mice in the context of an aversion task involving cortical and subcortical pathways presumably affected in schizophrenia. These observations, together with the known safety of rhEPO, render it an interesting compound for neuroprotective add-on strategies in schizophrenia and other human diseases characterized by a progressive decline in cognitive performance.

The brain erythropoietin system and its potential for therapeutic exploitation in brain disease.

The discovery of the broad neuroprotective potential of erythropoietin (EPO), an endogenous hematopoietic growth factor, has opened new therapeutic avenues in the treatment of brain diseases. EPO expression in the brain is induced by hypoxia. Practically all brain cells are capable of production and release of EPO and expression of its receptor. EPO exerts multifaceted protective effects on brain cells. It protects neuronal cells from noxious stimuli such as hypoxia, excess glutamate, serum deprivation or kainic acid exposure in vitro by targeting a variety of mechanisms and involves neuronal, glial and endothelial cell functions. In rodent models of ischemic stroke, EPO reduces infarct volume and improves functional outcome, but beneficial effects have also been observed in animal models of subarachnoid hemorrhage, intracerebral hemorrhage, traumatic brain injury, and spinal cord injury. EPO has a convenient therapeutic window upon ischemic stroke and favorable pharmacokinetics. Results from first therapeutic trials in humans are promising, but will need to be validated in larger trials. The safety profile and effectiveness of EPO in a wide variety of neurologic disease models make EPO a candidate compound for a potential first-line therapeutic for neurologic emergencies.

Survival of hippocampal neurons in culture upon hypoxia: effect of erythropoietin.

The potential of erythropoietin (EPO) to reduce hypoxia-induced cell death has been investigated in 5-day-old primary cultures of rat postnatal hippocampal neurons. Application of EPO (100 pM) at the start of hypoxia resulted in a significant reduction of neuronal death (33.0 ± 7.5% in cells incubated with EPO vs 56.75 ± 7.3% in non-treated cells; n = 4, p < 0.021). Similar results were obtained upon application of cycloheximide (CHX; 1 μM) simultaneously with hypoxia (34.75 ± 5.6% vs 56.75 ± 7.3% with and without CHX, respectively, n = 4, p < 0.035), indicating that hypoxia-induced neuronal death is an active, protein synthesis-dependent process. Both, EPO and EPO receptor (EPOR) were found to be expressed after hypoxia in hippocampal neurons in vitro and in vivo. These results demonstrate for the first time that EPO can reverse hypoxia-induced neuronal death when applied simultaneously with the hypoxic stimulus.

In vitro Gender Differences in Neuronal Survival on Hypoxia and in 17β-Estradiol-Mediated Neuroprotection

Gender differences in neuropsychiatric disease are recognized but not well understood. Investigating the survival of primary rat hippocampal neurons in culture, we found significant and inverted gender differences on normoxia versus hypoxia. Male cells were more resistant under normoxia but more vulnerable under hypoxia than female cells. Male vulnerability pattern was acquired in cells from neonatally testosterone-primed females. Estrogens, acting via membrane receptors, had a higher neuroprotective power in male neurons, explained at least in part by the pronounced increase in estrogen receptor beta/alpha ratio during hypoxia in male cells only.

Endothelin B receptor deficiency is associated with an increased rate of neuronal apoptosis in the dentate gyrus

The dentate gyrus retains neuronal proliferative potential throughout life. Using immature endothelin B receptor-deficient (sl/sl) rats, a rabbit model of pneumococcal meningitis and autopsy brains from humans who died from pneumococcal meningitis, we explored the role of endothelin B receptors in physiological and pathological neuronal apoptosis in the dentate gyrus. At postnatal days 3-4, the rate of apoptosis in the dentate gyrus was high in all rats, declining to low levels in wild-type rats (+/+) on days 14 and 22, but remaining high in both homozygous (sl/sl) and heterozygous (sl/+) endothelin B receptor-deficient rats. Increased apoptosis was not significantly compensated for by neuronal proliferation. Hippocampal neuronal cultures also exhibited genotype-dependent apoptosis with the highest rate in neurons from homozygous endothelin B receptor-deficient (sl/sl) rats. In rabbit and human pneumococcal meningitis, increased apoptosis in the dentate gyrus was associated with loss of neuronal endothelin B receptor immunoreactivity. In conclusion, endothelin B receptors appear to act as neuronal survival factors in the dentate gyrus in rodents and man, both during postnatal development and under pathological conditions.

ETA and ETB receptor antagonists synergistically increase extracellular endothelin-1 levels in primary rat astrocyte cultures

Astrocytes produce and bind endothelins (ETs), suggesting that these cells have ET autoregulatory and eliminatory functions. To further investigate these functions in primary rat astrocytes, ET-1 levels in the cell culture media (RIA/HPLC) and intracellular content of ET-1 mRNA (RT PCR) were measured under basal and stimulated (thrombin, 2.2 U/ml) conditions in the presence and absence of ETA and ETB selective antagonists (BQ123 or LU135252, and BQ788, respectively). Neither basal nor stimulated ET-1 levels in astrocyte media were influenced by ETA or ETB antagonists alone, but were significantly increased by a combination of both. ir ET-3 levels were not affected by antagonist treatment. Exogenous ET-1, added to the cultures, was rapidly cleared from the supernatant; this clearance was markedly inhibited by a combination of BQ123 and BQ788. ET-1 mRNA levels were not altered by any treatment. To conclude, in primary rat astrocyte cultures, extracellular ET-1 is cleared by binding to ET-receptors, apparently involving both, ETA and ETB sites. Thus, a blockade of the astrocytic ET eliminatory function as a consequence of the in vivo application of non-selective ET receptor antagonists may lead to increased extracellular ET levels in the brain.

Simultaneous monitoring of endothelin-1 and vasopressin plasma levels in migraine.

Vasopressin levels in plasma rise during migraine attacks. Vasopressin also induces endothelin-1 synthesis in endothelial cells, suggesting a role as a mediator of elevated plasma endothelin-1 in migraine. To explore a possible relationship between endothelin-1 and vasopressin in migraine, plasma concentrations of both peptides were monitored simultaneously throughout an attack and during two migraine-free intervals (control) in 20 patients. Endothelin-1 was elevated 6 h after the onset of an attack (3.3 +/- 0.3 pg/ml vs 2.7 +/- 0.2 pg/ml during migraine-free intervals; p = 0.12) whereas vasopressin was increased over control levels (2.8 +/- 0.3 pg/ml) by 3 h (3.6 +/- 0.4 pg/ml; p < 0.05) and remained elevated at 6 h (3.9 +/- 0.5 pg/ml; p < 0.01). These data suggest that vasopressin may act as a peripheral mediator of increased plasma endothelin-1 in migraine.

Endothelin b receptor deficiency augments neuronal damage upon exposure to hypoxia-ischemia in vivo

The role of functional endothelin-B (ETB)-receptors on neuronal survival upon hypoxia-ischemia (HI) has been investigated in 14-day-old ETB-receptor-deficient spotting lethal (sl/sl) and wildtype (+/+) rats. Carotid ligation followed by exposure to 8% oxygen for 2 h produced distinct cortical and hippocampal neuronal damage. Damage severity 24 h after HI was mild to intermediate in +/+ rats whereas large cortical infarcts and profound apoptosis of the hippocampus evolved in sl/sl rats. The number of apoptotic cells in the dentate 24 h after HI amounted to 30 +/- 7 cells/0.1 mm(2) in sl/sl compared to 9 +/- 3 cells/0.1 mm(2) in wildtype rats (mean +/- S.E.M., n=10-11, P=0.0093). In-vitro hypoxia (15 h) resulted in a comparable increase in cell death in primary pure neuronal hippocampal cultures from both groups (49.8 +/- 1.6% in sl/sl, 51.4 +/- 0.9% in +/+, mean +/- S.E.M., n=5, P=0.0560). To conclude, absence of functional ETB receptors is associated with an increased susceptibility to HI in-vivo, which is not intrinsic to neurons. Antagonism of ETB receptors seems not to be desirable in ischemic stroke.

ETA and ETB Specific Ligands Synergistically Antagonize Endothelin-1 Binding to an Atypical Endothelin Receptor in Primary Rat Astrocytes

Abstract: Using a whole‐cell binding procedure with long incubations at low temperature and subsequent acid stripping, we have characterized an atypical endothelin (ET) receptor in primary rat cortical astrocyte cultures. We found the following: (a) no competition for 125I‐ET‐1 binding by the ETA antagonists BQ‐123 and LU 135252 or the ETB agonist IRL 1620; (b) weak competition by the ETB antagonist BQ‐788 and by the predominant ETB ligand ET‐3; (c) potent synergistic competition of ETA and ETB ligands in combination for 125I‐ET‐1 binding; (d) potent competition of ET‐1 with any of the radioligands used, 125I‐ET‐1, 125I‐IRL 1620, and [3H]BQ‐123; (e) lack of competition of IRL 1620 and BQ‐123 with the respective other radioligand; (f) shifting of the amount of acid‐strippable 125I‐ET‐1 binding from 20 to 80% by ETB ligands and to 4% by ETA ligands; and (g) as a control, typical ETA and ETB binding characteristics of the RAT‐1 fibroblast and the U373MG astrocytoma cell line, respectively, under our assay conditions. The unusual binding properties of astrocytic ET receptors described in this study appear to be the result of several binding sites in the receptor for different ET ligands or ligand epitopes.

Effect of Endothelin-1 on Astrocytic Protein Content

The astrocytic endothelin (ET) receptors, ETA and ETB, modulate calcium signaling and the astrocytic gap junctional network. The nonselective ET receptor ligand ET‐1 inhibits gap junction permeability, an effect that can be blocked by tolbutamide. This mechanism may play a role in pathophysiological conditions such as ischemic stroke, characterized by elevated tissue ET‐1 levels and hypertrophic‐appearing reactive astrocytes. Therefore, the effect of ET‐1 on cellular protein content was investigated in confluent once‐passaged rat astrocyte cultures under serum‐free conditions, by the Lowry method. Gap junction permeability was determined by the dye transfer technique. ET‐1 prevented the decrease in astrocytic protein content observed in controls. The effect of ET‐1 on cellular protein content was most pronounced in cultures seeded at high density, but it was attenuated in ETB‐deficient (sl/sl) astrocytes. This effect could be blocked by the nonselective ET antagonist LU 302872 (10 μM), as well as by the protein synthesis inhibitor cycloheximide (10 μM). This increase in astrocytic protein content was inhibited by the ATP‐sensitive K+ channel blocker tolbutamide, which also antagonized the ET‐1‐induced reduction of gap junction permeability and reversed the morphological changes observed in astrocytes upon ET‐1 treatment. Cytosine arabinoside (10 μM), a DNA synthesis blocker, inhibited the ET‐1‐induced BrdU uptake without affecting the ET‐1‐induced increase in astrocytic protein content. To conclude, ET‐1 induces an increase in astrocytic protein content as well as changes in astrocyte morphology in vitro. This hypertrophic response involves uncoupling of the astrocytic gap junctional network and is not dependent on DNA synthesis. GLIA 42:390–397, 2003.