Anna-Leena Sirén

Erythropoietin and erythropoietin receptor in human ischemic/hypoxic brain

Abstract. 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.

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 (⩾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.

Reduced oxidative damage in ALS by high‐dose enteral melatonin treatment

Abstract:  Amyotrophic lateral sclerosis (ALS) is the collective term for a fatal motoneuron disease of different etiologies, with oxidative stress as a common molecular denominator of disease progression. Melatonin is an amphiphilic molecule with a unique spectrum of antioxidative effects not conveyed by classical antioxidants. In preparation of a possible future clinical trial, we explored the potential of melatonin as neuroprotective compound and antioxidant in: (1) cultured motoneuronal cells (NSC‐34), (2) a genetic mouse model of ALS (SOD1G93A‐transgenic mice), and (3) a group of 31 patients with sporadic ALS. We found that melatonin attenuates glutamate‐induced cell death of cultured motoneurons. In SOD1G93A‐transgenic mice, high‐dose oral melatonin delayed disease progression and extended survival. In a clinical safety study, chronic high‐dose (300 mg/day) rectal melatonin was well tolerated during an observation period of up to 2 yr. Importantly, circulating serum protein carbonyls, which provide a surrogate marker for oxidative stress, were elevated in ALS patients, but were normalized to control values by melatonin treatment. This combination of preclinical effectiveness and proven safety in humans suggests that high‐dose melatonin is suitable for clinical trials aimed at neuroprotection through antioxidation in ALS.

Therapeutic potential of erythropoietin and its structural or functional variants in the nervous system

SummaryThe growth factor erythropoietin (EPO) and erythropoietin receptors (EPOR) are expressed in the nervous system. Neuronal expression of EPO and EPOR peaks during brain development and is upregulated in the adult brain after injury. Peripherally administered EPO, and at least some of its variants, cross the blood-brain barrier, stimulate neurogenesis, neuronal differentiation, and activate brain neurotrophic, anti-apoptotic, anti-oxidant and anti-inflammatory signaling. These mechanisms underlie their tissue protective effects in nervous system disorders. As the tissue protective functions of EPO can be separated from its stimulatory action on hematopoiesis, novel EPO derivatives and mimetics, such as asialo-EPO and carbamoylated EPO have been developed. While the therapeutic potential of the novel EPO derivatives continues to be characterized in preclinical studies, the experimental findings in support for the use of recombinant human (rh)EPO in human brain disease have already been translated to clinical studies in acute ischemic stroke, chronic schizophrenia, and chronic progressive multiple sclerosis. In this review article, we assess the studies on EPO and, in particular, on its structural or functional variants in experimental models of nervous system disorders, and we provide a short overview of the completed and ongoing clinical studies testing EPO as neuroprotective/neuroregenerative treatment option in neuropsychiatric disease.

Erythropoietin--a novel concept for neuroprotection.

Abstract 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.

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.

Upregulation of intercellular adhesion molecule 1 (ICAM-1) on brain microvascular endothelial cells in rat ischemic cortex.

The expression of intercellular adhesion molecule 1 (ICAM-1) was studied in rat focal ischemic cortex. A significant increase in ICAM-1 mRNA expression in the ischemic cortex over levels in contralateral (nonischemic) site was observed by means of Northern blot analysis following either permanent or temporary occlusion with reperfusion of the middle cerebral artery (PMCAO or MCAO with reperfusion) in spontaneously hypertensive rats. In the ischemic cortex, levels of ICAM-1 mRNA increased significantly at 3 h (2.6-fold, n = 3, P < 0.05), peaked at 6 to 12 h (6.0-fold, P < 0.01) and remained elevated up to 5 days (2.5-fold, P < 0.05) after PMCAO. The profile of ICAM-1 mRNA expression in the ischemic cortex following MCAO with reperfusion was similar to that following PMCAO, except that ICAM-1 mRNA was significantly increased as early as 1 h (6.3-fold, n = 3, P < 0.05) and then gradually reached a peak at 12 h (12-fold, P < 0.01) after reperfusion. ICAM-1 mRNA expression in ischemic cortex following PMCAO was significantly greater in hypertensive rats than in two normotensive rat strains. Immunostaining using anti-ICAM-1 antibodies indicated that upregulated ICAM-1 expression was localized to endothelial cells of intraparenchymal blood vessels in the ischemic but not contralateral cortex. The data suggest that an upregulation of ICAM-1 mRNA and protein on brain capillary endothelium may play an important role in leukocyte migration into ischemic brain tissue.

Stiff person syndrome-associated autoantibodies to amphiphysin mediate reduced GABAergic inhibition

Synaptic inhibition is a central factor in the fine tuning of neuronal activity in the central nervous system. Symptoms consistent with reduced inhibition such as stiffness, spasms and anxiety occur in paraneoplastic stiff person syndrome with autoantibodies against the intracellular synaptic protein amphiphysin. Here we show that intrathecal application of purified anti-amphiphysin immunoglobulin G antibodies induces stiff person syndrome-like symptoms in rats, including stiffness and muscle spasms. Using in vivo recordings of Hoffmann reflexes and dorsal root potentials, we identified reduced presynaptic GABAergic inhibition as an underlying mechanism. Anti-amphiphysin immunoglobulin G was internalized into neurons by an epitope-specific mechanism and colocalized in vivo with presynaptic vesicular proteins, as shown by stimulation emission depletion microscopy. Neurons from amphiphysin deficient mice that did not internalize the immunoglobulin provided additional evidence of the specificity in antibody uptake. GABAergic synapses appeared more vulnerable than glutamatergic synapses to defective endocytosis induced by anti-amphiphysin immunoglobulin G, as shown by increased clustering of the endocytic protein AP180 and by defective loading of FM 1-43, a styryl dye used to label cell membranes. Incubation of cultured neurons with anti-amphiphysin immunoglobulin G reduced basal and stimulated release of γ-aminobutyric acid substantially more than that of glutamate. By whole-cell patch-clamp analysis of GABAergic inhibitory transmission in hippocampus granule cells we showed a faster, activity-dependent decrease of the amplitude of evoked inhibitory postsynaptic currents in brain slices treated with antibodies against amphiphysin. We suggest that these findings may explain the pathophysiology of the core signs of stiff person syndrome at the molecular level and show that autoantibodies can alter the function of inhibitory synapses in vivo upon binding to an intraneuronal key protein by disturbing vesicular endocytosis.

Global forebrain ischemia results in differential cellular expression of interleukin-1β (IL-1β) and its receptor at mRNA and protein level

The mRNA expression of the proinflammatory cytokine interleukin-1β (IL-1β) has been shown to be induced in neural elements during ischemia. It is not clear which cells generate the IL-1β mRNA and eventually synthesize IL-1 protein and which cells respond to this signaling by producing IL-1 receptors during ischemia. To clarify this question, rats were subjected to global ischemia by bilateral carotid occlusion and hypotension for 20 minutes, followed by reperfusion for 2 hours (n = 7), 8 hours (n = 7), or 24 hours (n = 7). Cryostat sections were hybridized using antisense oligonucleotide probes (30 dimer). Multiple cell markers were used in immunohistochemical staining to identify the cells expressing IL-1β and IL-1R protein. The sham animals (n = 5) showed no or only a weak expression of IL-1R or IL-1β mRNA. The number of IL-1β mRNA-expressing cells was significantly increased by 2 hours of reperfusion in several brain areas including cortex (12-fold compared with sham) and caudate-putamen (14-fold), and was maximally increased in most hippocampal regions by 8 hours of reperfusion (mean ± SD of positive cells/field versus sham equivalent being 37.9 ± 12.3 versus 4.0 ± 3.3; 30.6 ± 9.0 versus 3.1 ± 2.3; 41.3 ± 17.5 versus 2.9 ± 1.9; in CA1; CA2; CA3/CA4 regions of the hippocampus, respectively). IL-1β mRNA signal was also intensified in the white matter areas. Changes in IL-1R mRNA were seen in the hippocampus (after 2 hours CA1: 16-fold; CA2: 17-fold; DG: 24-fold increase; and CA3/CA4: 10-fold increase after 8 hours), and the expression was prolonged especially in CA1 and CA2 regions up to 24 hours of reperfusion. The major cellular source of IL-1β protein was glia (astrocytes, oligodendrocytes, microglia, and scattered perivascular macrophages/monocytes), while neurons and sporadic microvascular endothelia showed IL-1R immunoreactivity. The data suggest that neurons in discrete areas vulnerable for selective neuronal death, and possibly the vascular endothelium, are target cells for ischemia-induced glial IL-1β production.