Revisiting adult neurogenesis and the role of erythropoietin for neuronal and oligodendroglial differentiation in the hippocampus.

Recombinant human erythropoietin (EPO) improves cognitive performance in neuropsychiatric diseases ranging from schizophrenia and multiple sclerosis to major depression and bipolar disease. This consistent EPO effect on cognition is independent of its role in hematopoiesis. The cellular mechanisms of action in brain, however, have remained unclear. Here we studied healthy young mice and observed that 3-week EPO administration was associated with an increased number of pyramidal neurons and oligodendrocytes in the hippocampus of ~20%. Under constant cognitive challenge, neuron numbers remained elevated until >6 months of age. Surprisingly, this increase occurred in absence of altered cell proliferation or apoptosis. After feeding a 15N-leucine diet, we used nanoscopic secondary ion mass spectrometry, and found that in EPO-treated mice, an equivalent number of neurons was defined by elevated 15N-leucine incorporation. In EPO-treated NG2-Cre-ERT2 mice, we confirmed enhanced differentiation of preexisting oligodendrocyte precursors in the absence of elevated DNA synthesis. A corresponding analysis of the neuronal lineage awaits the identification of suitable neuronal markers. In cultured neurospheres, EPO reduced Sox9 and stimulated miR124, associated with advanced neuronal differentiation. We are discussing a resulting working model in which EPO drives the differentiation of non-dividing precursors in both (NG2+) oligodendroglial and neuronal lineages. As endogenous EPO expression is induced by brain injury, such a mechanism of adult neurogenesis may be relevant for central nervous system regeneration.

Chronophin is a glial tumor modifier involved in the regulation of glioblastoma growth and invasiveness.

Glioblastoma is the most aggressive primary brain tumor in adults. Although the rapid recurrence of glioblastomas after treatment is a major clinical challenge, the relationships between tumor growth and intracerebral spread remain poorly understood. We have identified the cofilin phosphatase chronophin (gene name: pyridoxal phosphatase, PDXP) as a glial tumor modifier. Monoallelic PDXP loss was frequent in four independent human astrocytic tumor cohorts and increased with tumor grade. We found that aberrant PDXP promoter methylation can be a mechanism leading to further chronophin downregulation in glioblastomas, which correlated with shorter glioblastoma patient survival. Moreover, we observed an inverse association between chronophin protein expression and cofilin phosphorylation levels in glioma tissue samples. Chronophin-deficient glioblastoma cells showed elevated cofilin phosphorylation, an increase in polymerized actin, a higher directionality of cell migration, and elevated in vitro invasiveness. Tumor growth of chronophin-depleted glioblastoma cells xenografted into the immunodeficient mouse brain was strongly impaired. Our study suggests a mechanism whereby the genetic and epigenetic alterations of PDXP resulting in altered chronophin expression may regulate the interplay between glioma cell proliferation and invasion.

Neuropsychiatric disease relevance of circulating anti-NMDA receptor autoantibodies depends on blood-brain barrier integrity.

In 2007, a multifaceted syndrome, associated with anti-NMDA receptor autoantibodies (NMDAR-AB) of immunoglobulin-G isotype, has been described, which variably consists of psychosis, epilepsy, cognitive decline and extrapyramidal symptoms. Prevalence and significance of NMDAR-AB in complex neuropsychiatric disease versus health, however, have remained unclear. We tested sera of 2817 subjects (1325 healthy, 1081 schizophrenic, 263 Parkinson and 148 affective-disorder subjects) for presence of NMDAR-AB, conducted a genome-wide genetic association study, comparing AB carriers versus non-carriers, and assessed their influenza AB status. For mechanistic insight and documentation of AB functionality, in vivo experiments involving mice with deficient blood-brain barrier (ApoE(-/-)) and in vitro endocytosis assays in primary cortical neurons were performed. In 10.5% of subjects, NMDAR-AB (NR1 subunit) of any immunoglobulin isotype were detected, with no difference in seroprevalence, titer or in vitro functionality between patients and healthy controls. Administration of extracted human serum to mice influenced basal and MK-801-induced activity in the open field only in ApoE(-/-) mice injected with NMDAR-AB-positive serum but not in respective controls. Seropositive schizophrenic patients with a history of neurotrauma or birth complications, indicating an at least temporarily compromised blood-brain barrier, had more neurological abnormalities than seronegative patients with comparable history. A common genetic variant (rs524991, P=6.15E-08) as well as past influenza A (P=0.024) or B (P=0.006) infection were identified as predisposing factors for NMDAR-AB seropositivity. The >10% overall seroprevalence of NMDAR-AB of both healthy individuals and patients is unexpectedly high. Clinical significance, however, apparently depends on association with past or present perturbations of blood-brain barrier function.

Shared loci for migraine and epilepsy on chromosomes 14q12-q23 and 12q24.2-q24.3.

OBJECTIVES: To describe clinical characteristics and to identify susceptibility loci for epilepsy and migraine in a Finnish family with a complex phenotype. METHODS: Participating family members were interviewed and medical files were reviewed. The seizure classification was made according to International League Against Epilepsy criteria. Migraine diagnosis was made using the validated Finnish Migraine Specific Questionnaire for Family Studies and criteria according to the current International Classification of Headache Disorders-II. DNA samples were obtained from 56 family members and nonparametric genome-wide linkage analyses were performed using 382 polymorphic microsatellite markers. The most promising loci were fine-mapped with additional microsatellite markers. RESULTS: Clinical data were obtained from 60 family members of whom 12 (20%) had idiopathic epileptic seizures. Eight of those 12 (67%) also had migraine. Altogether 33 of the 60 family members (55%) had migraine. Significant evidence of linkage was found between a locus on 14q12-q23 and migraine (p = 0.0001). Suggestive evidence of linkage in this region was also found for epilepsy with generalized tonic-clonic seizures (p = 0.0034). In addition, significant evidence of linkage was found at a locus on 12q24.2-q24.3 (p < 0.001) for migraine alone and for the combined phenotype of migraine and epilepsy. CONCLUSIONS: Our data suggest the occurrence of common susceptibility loci for epilepsy and migraine on chromosomes 14q12-q23 and 12q24.2-q24.3, implicating a shared genetic etiology for these 2 diseases.

Whole-genome linkage scan for epilepsy-related photosensitivity: a mega-analysis.

Photoparoxysmal response (PPR) is considered to be a risk factor for idiopathic generalised epilepsy (IGE) and it has a strong genetic basis. Two genome-wide linkage studies have been published before and they identified loci for PPR at 6p21, 7q32, 13q13, 13q31 and 16p13. Here we combine these studies, augmented with additional families, in a mega-analysis of 100 families. Non-parametric linkage analysis identified three suggestive peaks for photosensitivity, two of which are novel (5q35.3 and 8q21.13) and one has been found before (16p13.3). We found no evidence for linkage at four previously detected loci (6p21, 7q32, 13q13 and 13q31). Our results suggest that the different family data sets are not linked to a shared locus. Detailed analysis showed that the peak at 16p13 was mainly supported by a single subset of families, while the peaks at 5q35 and 8q21 had weak support from multiple subsets. Family studies clearly support the role of PPR as a risk factor for IGE. This mega-analysis shows that distinct loci seem to be linked to subsets of PPR-positive families that may differ in subtle clinical phenotypes or geographic origin. Further linkage studies of PPR should therefore include in-depth phenotyping to make appropriate subsets and increase genetic homogeneity.

Essential role for Stat5 in the neurotrophic but not in the neuroprotective effect of erythropoietin.

The transcription factors signal transducer and activator of transcription 5a and 5b (Stat5) are activated by the neuroprotective and neurotrophic cytokines, erythropoietin (EPO) and growth hormone (GH). Here, we show a dissociation of the intracellular pathway mediating the protective effect of EPO against glutamate toxicity from that needed for its neurotrophic activity using hippocampal neuronal cultures from Stat5a/b-knockout (Stat5(-/-)) mouse fetuses. Both pretreatment and post-treatment with EPO counteracted glutamate-induced cell death in Stat5(+/+) and Stat5(-/-) neurons. Acute pharmacological inhibition of Janus kinase 2 (JAK2)/Stat signalling had no effect on EPO neuroprotection, whereas inhibition of phosphatidylinositol-3' kinase (PI3K)/Akt pathway abolished the protective effect of EPO in both Stat5(+/+) and Stat5(-/-) neurons. GH effectively protected Stat5(+/+) cells against glutamate toxicity but had no effect in Stat5(-/-) neurons or in Stat5(+/+) neurons treated with JAK2/Stat or PI3K inhibitor. EPO and GH stimulated neurite outgrowth and branching of Stat5(+/+) neurons by activating PI3K/Akt signalling but had no trophic effect in Stat5(-/-) cells. We conclude that in hippocampal neurons, Stat5 is not required for neuroprotection by EPO but is together with Akt essential for its neurotrophic activity. Both Stat5 and Akt are needed for neuroprotective and neurotrophic signalling of GH in neurons.

Beneficial effects of antiepileptic medication on absence seizures and cognitive functioning in children.

In this prospective clinical study, the effects on cognitive functioning of absence seizures, epileptiform EEG discharges, and their abolishment by antiepileptic medication were evaluated in patients newly diagnosed with childhood absence epilepsy or juvenile absence epilepsy. Eleven children in the study group and ten age- and gender-matched controls with mild asthma underwent combined video/EEG and neurocognitive assessment (IQ, fine-motor fluency, attention, visual and spatial memory). The neuropsychological assessment was repeated after the introduction of antiepileptic medication. Ten children with absence epilepsy became clinically seizure free. The study group improved in attention, fine-motor fluency, and visual memory. The controls improved only in fine-motor and attention skills. Duration of generalized 3-Hz spike-wave discharges and clinical absence seizures was negatively correlated with performance on the visual memory task. Cessation of seizures induced by antiepileptic medication may support neurocognitive functioning in children.

Improvement of cognitive functions in chronic schizophrenic patients by recombinant human erythropoietin.

Schizophrenia is increasingly recognized as a neurodevelopmental disease with an additional degenerative component, comprising cognitive decline and loss of cortical gray matter. We hypothesized that a neuroprotective/neurotrophic add-on strategy, recombinant human erythropoietin (rhEPO) in addition to stable antipsychotic medication, may be able to improve cognitive function even in chronic schizophrenic patients. Therefore, we designed a double-blind, placebo-controlled, randomized, multicenter, proof-of-principle (phase II) study. This study had a total duration of 2 years and an individual duration of 12 weeks with an additional safety visit at 16 weeks. Chronic schizophrenic men (N=39) with defined cognitive deficit (>or=1 s.d. below normal in the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS)), stable medication and disease state, were treated for 3 months with a weekly short (15 min) intravenous infusion of 40,000 IU rhEPO (N=20) or placebo (N=19). Main outcome measure was schizophrenia-relevant cognitive function at week 12. The neuropsychological test set (RBANS subtests delayed memory, language-semantic fluency, attention and Wisconsin Card Sorting Test (WCST-64) - perseverative errors) was applied over 2 days at baseline, 2 weeks, 4 weeks and 12 weeks of study participation. Both placebo and rhEPO patients improved in all evaluated categories. Patients receiving rhEPO showed a significant improvement over placebo patients in schizophrenia-related cognitive performance (RBANS subtests, WCST-64), but no effects on psychopathology or social functioning. Also, a significant decline in serum levels of S100B, a glial damage marker, occurred upon rhEPO. The fact that rhEPO is the first compound to exert a selective and lasting beneficial effect on cognition should encourage new treatment strategies for schizophrenia.

Hyperekplexia in Kurdish families: a possible GLRA1 founder mutation.

Autosomal recessive hyperekplexia is due to loss-of-function mutations in the GLRA1 gene. The authors describe six patients from two consanguineous families with a homozygous deletion of the first seven GLRA1 exons and provide evidence of a founder effect in Kurds from Turkey. Hyperekplexia may be misdiagnosed as epilepsy.

Endothelins regulate astrocyte gap junctions in rat hippocampal slices.

Gap junctional communication (GJC) is a typical feature of astrocytes proposed to contribute to the role played by these glial cells in brain physiology and pathology. In acutely isolated hippocampal slices from rat (P11-P19), intercellular diffusion of biocytin through gap junction channels was shown to occur between hundreds of cells immuno-positive for astrocytic markers studied in the CA1/CA2 region. Single-cell RT-PCR demonstrated astrocytic mRNA expression of several connexin (Cx) subtypes, the molecular constituent of gap junction channels, whereas immunoblotting confirmed that Cx43 and Cx30 are the main gap junction proteins in hippocampal astrocytes. In the brain, astrocytes represent a major target for endothelins (Ets), a vasoactive family of peptides. Our results demonstrate that Ets decrease the expression of phosphorylated Cx43 forms and are potent inhibitors of GJC. The Et-induced effects were investigated using specific Et receptor agonists and antagonists, including Bosentan (Tracleer trade mark ), an EtA/B receptor antagonist, and using hippocampal slices and cultures from EtB-receptor-deficient rats. Interestingly, the pharmacological profile of Ets effects did not follow the classical profile established in cardiovascular systems. The present study therefore identifies Ets as potent endogenous inhibitory regulators of astrocyte networks. As such, the action of these peptides on astrocyte GJC might be involved in the contribution of astrocytes to neuroprotective processes and have a therapeutic potential in neuropathological situations.

Endothelin B receptor deficient transgenic rescue rats: a rescue phenomenon in the brain.

Homozygous endothelin B receptor deficiency leads to congenital aganglionosis of the gut in rats and mice, equivalent to human Hirschsprung disease. Homozygous endothelin B receptor deficient rats (spotting lethal rats, sl/sl) are characterized not only by this developmental disorder of the enteric nervous system, which limits their life span to 3-4 weeks, but exhibit an increased rate of apoptosis in the dentate gyrus compared to wildtype (+/+) rats. Recently, endothelin B receptor deficient transgenic rescue rats (sl/sl, tg/tg) were created to further investigate the role of the endothelin B receptor in mature animals. Linkage of the human dopamine-beta-hydroxylase promoter to the rat endothelin B receptor gene and expression of this transgenic construct results in normal development of the enteric nervous system. We investigated the expression pattern of this transgenic construct in the brain by using reverse transcriptase polymerase chain reaction. Unexpectedly, transgene mRNA expression was not restricted to the brain stem where adrenergic and noradrenergic nuclei are known to be present but, in addition, was also detectable in hippocampus and cortex. Using in situ tailing technique, cleaved caspase-3 immunohistochemistry and analysis of hematoxylin-eosin-stained serial sections, we found that all studied transgenic animals were rescued from the increased rate of apoptosis in the dentate gyrus characteristic for non-transgenic sl/sl rats. This finding supports our previous observation that the endothelin B receptor might be an important regulatory element supporting cellular survival in the hippocampus during postnatal development. The endothelin B receptor deficient transgenic rescue rats used here are rescued from developmental disorders both in the gut and in the brain.

Expression patterns of erythropoietin and its receptor in the developing midbrain.

The expression patterns of erythropoietin (EPO) and its receptor (EPOR) were investigated in the midbrain and in adjacent parts of the synencephalon and hindbrain of embryonic C57Bl mice. On embryonic (E) day 8 (E8), virtually all neuroepithelial cells expressed EPOR. After neural tube closure, subsets of these cells downregulated EPOR. In contrast, radial glial cells were EPOR-immunolabeled from E11 onwards. Simultaneously, subpopulations of early developing neurons upregulated EPO and expressed HIF-1, known to transcriptionally activate EPO. Three-dimensional reconstructions revealed subpopulations of EPO-expressing neurons: (1) in the trigeminal mesencephalic nucleus (TMN), (2) at the rostral transition of the midbrain and synencephalon, (3) in the basal plate of the midbrain, (4) in the trigeminal motor nucleus, and (5) in the trigeminal principal sensory nucleus. In the rostral midbrain and synencephalon, EPO-immunoreactive neurons were attached to EPOR-expressing radial glial cells. The identity of radial glial cells was proven by their immunoreactivity for antibodies against astrocyte-specific glutamate transporter, brain lipid-binding protein, and nestin. From E12.5 onwards EPOR was downregulated in radial glial cells. Viable neurons of the TMN continued to express EPO and upregulated EPOR. Our findings provide new evidence that components of the EPO system are present in distinct locations of the embryonic brain and, by interactions between neurons and radial glial cells as well as among clustered TMN neurons, may contribute to its morphogenesis. Whether the observed expression patterns of EPO and EPOR may reflect EPO-mediated trophic and/or antiapoptotic effects on neurons is discussed.

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.

Erythropoietin--a novel concept for neuroprotection.

Neuroprotection as a means to prevent or oppose pathological neuronal loss in central nervous system disease of various pathophysiological origins represents a novel therapeutic approach. This approach is supported by extensive experimental evidence on cell culture and animal studies demonstrating beneficial effects of growth factors on neuronal survival and functional recovery. The clinical use of neuroprotective agents has been hampered by the toxicity of many of the compounds that showed promising therapeutic potential in animal studies. The focus of this review is on a novel neuroprotective approach with erythropoietin, a hematopoietic growth factor that: 1) is expressed in the human central nervous system, 2) is hypoxia-inducible, 3) has demonstrated remarkable neuroprotective potential in cell culture and animal models of disease, 4) has multiple protective effects (antiapoptotic, neurotrophic, antioxidant, angiogenic), and 5) is a clinically extremely well tolerated compound.

Proinflammatory cytokine expression contributes to brain injury provoked by chronic monocyte activation.

BACKGROUND: We have proposed that an increased interaction between monocyte/macrophages and blood vessel endothelium predisposes subjects to strokes. The effect of chronic monocyte activation on the development of cerebral infarcts was thus studied in rats after provocation of a modified local Swartzman reaction, in brain vasculature. MATERIALS AND METHODS: Two weeks after an IV bolus of bacillus Calmette-Guérin (BCG), we studied spontaneous superoxide production, integrin expression, endothelial adhesion of monocytes and the neurological symptoms, brain histology, and cytokine immunoreactivity after a provocative dose of LPS (30-300 microg/rat i.c.v.). RESULTS: Monocyte migration into the brain was stimulated by BCG priming. The incidence of paralysis and death in response to LPS was markedly increased in BCG-primed rats. Histological evaluation of the brains of neurologically impaired and moribund animals revealed intravascular thrombosis and pale and hemorrhagic infarcts. Infiltrates of leukocytes expressing immunoreactive IL-1:, IL-6, and TNF-alpha were found around blood vessels, cerebral ventricles, and meninges, and were accompanied by a profound microglial expression of IL1P, endothelial expression of IL-6, and expression of TNF-alpha and TNF-R 1 in glia and neurons of cortex and hippocampus. Treatment (2 x 100 microg/10 ,I, i.c.v.) with recombinant human (rh-)TNF 55kDa receptor completely prevented, and treatment with rh-IL- I receptor antagonist significantly decreased the incidence of paralysis and death in response to BCG + LPS. The improvement of neurological symptoms was accompanied by reduced histological damage and supppression of IL-1P/ expression in the brain tissue. CONCLUSIONS: The data demonstrate that chronic monocyte activation predisposes subjects to thrombosis and hemorrhage via an exaggerated release of proinflammatory cytokines.

Differential cellular expression of tumor necrosis factor-alpha and Type I tumor necrosis factor receptor after transient global forebrain ischemia.

We examined the expression of tumor necrosis factor-alpha (TNF-alpha) and the Type I tumor necrosis factor receptor, (TNFR1), in relation to c-fos, a known regulator gene of immediate cellular responses, after an extended period of global ischemia. The number of TNF-alpha mRNA expressing cells peaked in most brain areas after 8 h of reperfusion. Significant increases in TNFR1 mRNA expression were evident in the cortex at 2 and 8 h of reperfusion and after 8 h of reperfusion in the CA3/CA4 region of the hippocampus. Transient neuronal c-fos mRNA expression preceded these responses. TNF-alpha immunoreactivity was seen in neurons>>>oligodendrocytes=perivascular cells=ependymal cells=vessel wall structures. After ischemia/reperfusion, increased TNF-alpha immunoreactivity was evident only in oligodendrocytes. TNFR1 immunoreactivity in sham brains manifested in bundles of cellular fibers of variable length and thickness. In post-ischemic brains, immunoreactivity in these cellular processes representing mainly astroglial extensions was suppressed at 2 h but recovered partially by 8 and 24 h of reperfusion. In contradiction, transient ischemia-induced TNFR1 immunoreactivity was observed in somas of large cortical neurons, in activated microglia/macrophages, perivascular and endothelial cells.Taken together, the increase in neuronal TNF-alpha mRNA appeared not to be followed by substantial translation to protein in the cerebral tissue after an extended period of global ischemia. However, there was increased neuronal TNFR1 immunostaining in conjunction with increased immunostaining for TNF-alpha in oligoglial elements, which suggests signaling to neurons by enhanced oligoglial TNF-alpha.

Erythropoietin and erythropoietin receptor in human ischemic/hypoxic brain.

Using immunohistochemistry, expression of erythropoietin (EPO), a hypoxia-inducible neuroprotective factor, and its receptor (EPOR) were investigated in human brain tissue after ischemia/hypoxia. Autopsy brains of neuropathologically normal subjects were compared to those with ischemic infarcts or hypoxic damage. In normal brain, weak EPO/EPOR immunoreactivity was mainly neuronal. In fresh infarcts, EPO immunoreactivity appeared in vascular endothelium, EPOR in microvessels and neuronal fibers. In older infarcts reactive astrocytes exhibited EPO/EPOR immunoreactivity. Acute hypoxic brain damage was associated with vascular EPO expression, older hypoxic damage with EPO/EPOR immunoreactivity in reactive astrocytes. The pronounced up-regulation of EPO/EPOR in human ischemic/hypoxic brains underlines their role as an endogenous neuroprotective system and suggests a novel therapeutic potential in cerebrovascular disease for EPO, a clinically well-characterized and safe compound.

Role of the astrocytic ET(B) receptor in the regulation of extracellular endothelin-1 during hypoxia.

Astrocytes are known to possess an effective endothelin (ET) eliminatory system which involves astrocytic ET(A) and ET(B) receptors and may become particularly relevant under pathophysiological conditions. The present study has therefore been designed to explore the effect of standardized hypoxia on extracellular concentrations of endothelin-1 (ET-1) and on endothelin-converting enzyme (ECE) activity in primary rat astrocytes genetically (sl/sl) or experimentally (dexamethasone) deficient in ET(B) receptors. The results revealed (1) a hypoxia-mediated decrease of extracellular ET-1 in wildtype astrocytes (+/+) that was not observed in ET(B)-deficient (sl/sl) cultures; (2) an ET receptor antagonist-induced increase in ET-1 in the media of both genotypes with further elevation upon hypoxia in +/+ cultures only; (3) augmentation of the dexamethasone-induced increase in extracellular ET-1 by hypoxia in +/+, but not in sl/sl cultures; (4) synergistic reduction of ET(B) gene transcription by hypoxia and dexamethasone; and (5) significant increases in endothelin-converting enzyme activity in the presence of hypoxia. To conclude, hypoxia stimulates astrocytic release of mature ET-1. This stimulation is (over)compensated for by increased ET-1 binding to functional ET(B) receptors. ET(B) deficiency, whether genetic or experimentally induced, impairs elimination of extracellular ET-1.