Carola Krüger

The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis

G-CSF is a potent hematopoietic factor that enhances survival and drives differentiation of myeloid lineage cells, resulting in the generation of neutrophilic granulocytes. Here, we show that G-CSF passes the intact blood-brain barrier and reduces infarct volume in 2 different rat models of acute stroke. G-CSF displays strong anti-apoptotic activity in mature neurons and activates multiple cell survival pathways. Both G-CSF and its receptor are widely expressed by neurons in the CNS, and their expression is induced by ischemia, which suggests an autocrine protective signaling mechanism. Surprisingly, the G-CSF receptor was also expressed by adult neural stem cells, and G-CSF induced neuronal differentiation in vitro. G-CSF markedly improved long-term behavioral outcome after cortical ischemia, while stimulating neural progenitor response in vivo, providing a link to functional recovery. Thus, G-CSF is an endogenous ligand in the CNS that has a dual activity beneficial both in counteracting acute neuronal degeneration and contributing to long-term plasticity after cerebral ischemia. We therefore propose G-CSF as a potential new drug for stroke and neurodegenerative diseases.

A Neuroprotective Function for the Hematopoietic Protein Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF)

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine responsible for the proliferation, differentiation, and maturation of cells of the myeloid lineage, which was cloned more than 20 years ago. Here we uncovered a novel function of GM-CSF in the central nervous system (CNS). We identified the GM-CSF α-receptor as an upregulated gene in a screen for ischemia-induced genes in the cortex. This receptor is broadly expressed on neurons throughout the brain together with its ligand and induced by ischemic insults. In primary cortical neurons and human neuroblastoma cells, GM-CSF counteracts programmed cell death and induces BCL-2 and BCL-Xl expression in a dose- and time-dependent manner. Of the signaling pathways studied, GM-CSF most prominently induced the PI3K-Akt pathway, and inhibition of Akt strongly decreased antiapoptotic activity. Intravenously given GM-CSF passes the blood—brain barrier, and decreases infarct damage in two different experimental stroke models (middle cerebral artery occlusion (MCAO), and combined common carotid/distal MCA occlusion) concomitant with induction of BCL-Xl expression. Thus, GM-CSF acts as a neuroprotective protein in the CNS. This finding is remarkably reminiscent of the recently discovered functionality of two other hematopoietic factors, erythropoietin and granulocyte colony-stimulating factor in the CNS. The identification of a third hematopoietic factor acting as a neurotrophic factor in the CNS suggests a common principle in the functional evolution of these factors. Clinically, GM-CSF now broadens the repertoire of hematopoietic factors available as novel drug candidates for stroke and neurodegenerative diseases.

Granulocyte-colony stimulating factor improves outcome in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that results in progressive loss of motoneurons, motor weakness and death within 1–5 years after disease onset. Therapeutic options remain limited despite a substantial number of approaches that have been tested clinically. In particular, various neurotrophic factors have been investigated. Failure in these trials has been largely ascribed to problems of insufficient dosing or inability to cross the blood–brain barrier (BBB). We have recently uncovered the neurotrophic properties of the haematopoietic protein granulocyte-colony stimulating factor (G-CSF). The protein is clinically well tolerated and crosses the intact BBB. This study examined the potential role of G-CSF in motoneuron diseases. We investigated the expression of the G-CSF receptor in motoneurons and studied effects of G-CSF in a motoneuron cell line and in the SOD1(G93A) transgenic mouse model. The neurotrophic growth factor was applied both by continuous subcutaneous delivery and CNS-targeted transgenic overexpression. This study shows that given at the stage of the disease where muscle denervation is already evident, G-CSF leads to significant improvement in motor performance, delays the onset of severe motor impairment and prolongs overall survival of SOD1(G93A)tg mice. The G-CSF receptor is expressed by motoneurons and G-CSF protects cultured motoneuronal cells from apoptosis. In ALS mice, G-CSF increased survival of motoneurons and decreased muscular denervation atrophy. We conclude that G-CSF is a novel neurotrophic factor for motoneurons that is an attractive and feasible drug candidate for the treatment of ALS.

Granulocyte-colony stimulating factor is neuroprotective in a model of Parkinson's disease

We have recently shown that the hematopoietic Granulocyte‐Colony Stimulating Factor (G‐CSF) is neuroprotective in rodent stroke models, and that this action appears to be mediated via a neuronal G‐CSF receptor. Here, we report that the G‐CSF receptor is expressed in rodent dopaminergic substantia nigra neurons, suggesting that G‐CSF might be neuroprotective for dopaminergic neurons and a candidate molecule for the treatment of Parkinsons disease. Thus, we investigated protective effects of G‐CSF in 1‐methyl‐4‐phenylpyridinium (MPP+)‐challenged PC12 cells and primary neuronal midbrain cultures, as well as in the mouse 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) model of Parkinsons disease. Substantial protection was found against MPP+‐induced dopaminergic cell death in vitro. Moreover, subcutaneous application of G‐CSF at a dose of 40 μg/Kg body weight daily over 13 days rescued dopaminergic substantia nigra neurons from MPTP‐induced death in aged mice, as shown by quantification of tyrosine hydroxylase‐positive substantia nigra cells. Using HPLC, a corresponding reduction in striatal dopamine depletion after MPTP application was observed in G‐CSF‐treated mice. Thus our data suggest that G‐CSF is a novel therapeutic opportunity for the treatment of Parkinsons disease, because it is well‐tolerated and already approved for the treatment of neutropenic conditions in humans.

Expression of Hemoglobin in Rodent Neurons

Hemoglobin is the major protein in red blood cells and transports oxygen from the lungs to oxygen-demanding tissues, like the brain. Mechanisms that facilitate the uptake of oxygen in the vertebrate brain are unknown. In invertebrates, neuronal hemoglobin serves as intracellular storage molecule for oxygen. Here, we show by immunohistochemistry that hemoglobin is specifically expressed in neurons of the cortex, hippocampus, and cerebellum of the rodent brain, but not in astrocytes and oligodendrocytes. The neuronal hemoglobin distribution is distinct from the neuroglobin expression pattern on both cellular and subcellular levels. Probing for low oxygen levels in the tissue, we provide evidence that hemoglobin α-positive cells in direct neighborhood with hemoglobin α-negative cells display a better oxygenation than their neighbors and can be sharply distinguished from those. Neuronal hemoglobin expression is upregulated by injection or transgenic overexpression of erythropoietin and is accompanied by enhanced brain oxygenation under physiologic and hypoxic conditions. Thus we provide a novel mechanism for the neuroprotective actions of erythropoietin under ischemic—hypoxic conditions. We propose that neuronal hemoglobin expression is connected to facilitated oxygen uptake in neurons, and hemoglobin might serve as oxygen capacitator molecule.

An extended window of opportunity for G-CSF treatment in cerebral ischemia

BackgroundGranulocyte-colony stimulating factor (G-CSF) is known as a powerful regulator of white blood cell proliferation and differentiation in mammals. We, and others, have shown that G-CSF is effective in treating cerebral ischemia in rodents, both relating to infarct size as well as functional recovery. G-CSF and its receptor are expressed by neurons, and G-CSF regulates apoptosis and neurogenesis, providing a rational basis for its beneficial short- and long-term actions in ischemia. In addition, G-CSF may contribute to re-endothelialisation and arteriogenesis in the vasculature of the ischemic penumbra. In addition to these trophic effects, G-CSF is a potent neuroprotective factor reliably reducing infarct size in different stroke models.ResultsHere, we have further delayed treatment and studied effects of G-CSF on infarct volume in the middle cerebral artery occlusion (MCAO) model and functional outcome in the cortical photothrombotic model. In the MCAO model, we applied a single dose of 60 μg/kg bodyweight G-CSF in rats 4 h after onset of ischemia. Infarct volume was determined 24 h after onset of ischemia. In the rat photothrombotic model, we applied 10 μg/kg bodyweight G-CSF daily for a period of 10 days starting either 24 or 72 h after induction of ischemia. G-CSF both decreased acute infarct volume in the MCAO model, and improved recovery in the photothrombotic model at delayed timepoints.ConclusionThese data further strengthen G-CSFs profile as a unique candidate stroke drug, and provide an experimental basis for application of G-CSF in the post-stroke recovery phase.

Identification of regulated genes during permanent focal cerebral ischaemia: characterization of the protein kinase 9b5/MARKL1/MARK4

Cerebral ischaemia induces transcriptional changes in a number of pathophysiologically important genes. Here we have systematically studied gene expression changes after 90 min and 24 h of permanent focal ischaemia in the mouse by an advanced fragment display technique (restriction‐mediated differential display). We identified 56 transcriptionally altered genes, many of which provide novel hints to ischaemic pathophysiology. Particularly interesting were two pro‐apoptotic genes (Grim19 and Tdag51), whose role in cerebral ischaemia and neuronal cell death has not been recognized so far. Among the unknown sequences, we identified a gene that was rapidly and transiently up‐regulated. The encoded protein displayed high homology to the MARK family of serine–threonine protein kinases and has recently been described as MARKL1/MARK4. Here we demonstrate that this protein is a functional protein kinase with the ability to specifically phosphorylate a cognate peptide substrate for the AMP‐kinase family. Upon overexpression in heterologous cells, the functional wild‐type protein, but not its kinase‐dead mutant, led to decreased cell viability. We conclude that the up‐regulation of this kinase during focal ischaemia may represent an interesting new target for pharmacological intervention.

The functional genome of CA1 and CA3 neurons under native conditions and in response to ischemia

BackgroundThe different physiological repertoire of CA3 and CA1 neurons in the hippocampus, as well as their differing behaviour after noxious stimuli are ultimately based upon differences in the expressed genome. We have compared CA3 and CA1 gene expression in the uninjured brain, and after cerebral ischemia using laser microdissection (LMD), RNA amplification, and array hybridization.ResultsProfiling in CA1 vs. CA3 under normoxic conditions detected more than 1000 differentially expressed genes that belong to different, physiologically relevant gene ontology groups in both cell types. The comparison of each region under normoxic and ischemic conditions revealed more than 5000 ischemia-regulated genes for each individual cell type. Surprisingly, there was a high co-regulation in both regions. In the ischemic state, only about 100 genes were found to be differentially expressed in CA3 and CA1. The majority of these genes were also different in the native state. A minority of interesting genes (e.g. inhibinbetaA) displayed divergent expression preference under native and ischemic conditions with partially opposing directions of regulation in both cell types.ConclusionThe differences found in two morphologically very similar cell types situated next to each other in the CNS are large providing a rational basis for physiological differences. Unexpectedly, the genomic response to ischemia is highly similar in these two neuron types, leading to a substantial attenuation of functional genomic differences in these two cell types. Also, the majority of changes that exist in the ischemic state are not generated de novo by the ischemic stimulus, but are preexistant from the genomic repertoire in the native situation. This unexpected influence of a strong noxious stimulus on cell-specific gene expression differences can be explained by the activation of a cell-type independent conserved gene-expression program. Our data generate both novel insights into the relation of the quiescent and stimulus-induced transcriptome in different cells, and provide a large dataset to the research community, both for mapping purposes, as well as for physiological and pathophysiological research.

The hematopoietic factor granulocyte-colony stimulating factor improves outcome in experimental spinal cord injury.

J. Neurochem. (2010) 113, 930–942.

The hematopoietic factor GM-CSF (Granulocyte-macrophage colony-stimulating factor) promotes neuronal differentiation of adult neural stem cells in vitro

BackgroundGranulocyte-macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factor involved in the generation of granulocytes, macrophages, and dendritic cells from hematopoietic progenitor cells. We have recently demonstrated that GM-CSF has anti-apoptotic functions on neurons, and is neuroprotective in animal stroke models.ResultsThe GM-CSF receptor α is expressed on adult neural stem cells in the rodent brain, and in culture. Addition of GM-CSF to NSCs in vitro increased neuronal differentiation in a dose-dependent manner as determined by quantitative PCR, reporter gene assays, and FACS analysis.ConclusionSimilar to the hematopoietic factor Granulocyte-colony stimulating factor (G-CSF), GM-CSF stimulates neuronal differentiation of adult NSCs. These data highlight the astonishingly similar functions of major hematopoietic factors in the brain, and raise the clinical attractiveness of GM-CSF as a novel drug for neurological disorders.