It is becoming increasingly clear that single cortical neurons encode complex and behaviorally relevant signals, but efficient means to study gene functions in small networks and single neurons in vivo are still lacking. Here, we establish a method for genetic manipulation and subsequent phenotypic analysis of individual cortical neurons in vivo. First, lentiviral vectors are used for neuron-specific gene delivery from ␣-calcium͞calmodulin-dependent protein kinase II or Synapsin I promoters, optionally in combination with gene knockdown by means of U6 promoter-driven expression of short-interfering RNAs. Second, the phenotypic analysis at the level of single cortical cells is carried out by using two-photon microscopy-based techniques: high-resolution two-photon timelapse imaging is used to monitor structural dynamics of dendritic spines and axonal projections, whereas cellular response properties are analyzed electrophysiologically by two-photon microscopydirected whole-cell recordings. This approach is ideally suited for analysis of gene functions in individual neurons in the intact brain.patch-clamp recording ͉ two-photon imaging

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 a-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.

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 a-positive cells in direct neighborhood with hemoglobin a-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.

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.

The efficacy of excitatory transmission in the brain depends to a large extent on synaptic AMPA receptors, hence the importance of understanding the delivery and recycling of the receptors at the synaptic sites. Here we report a novel regulation of the AMPA receptor transport by a PDZ (postsynaptic density-95/Drosophila disc large tumor suppressor zona occludens 1) and LIM (Lin11/rat Isl-1/Mec3) domain-containing protein, RIL (reversion-induced LIM protein). We show that RIL binds to the AMPA glutamate receptor subunit GluR-A C-terminal peptide via its LIM domain and to ␣-actinin via its PDZ domain. RIL is enriched in the postsynaptic density fraction isolated from rat forebrain, strongly localizes to dendritic spines in cultured neurons, and coprecipitates, together with ␣-actinin, in a protein complex isolated by immunoprecipitation of AMPA receptors from forebrain synaptosomes. Functionally, in heterologous cells, RIL links AMPA receptors to the ␣-actinin/actin cytoskeleton, an effect that appears to apply selectively to the endosomal surfaceinternalized population of the receptors. In cultured neurons, an overexpression of recombinant RIL increases the accumulation of AMPA receptors in dendritic spines, both at the total level, as assessed by immunodetection of endogenous GluR-A-containing receptors, and at the synaptic surface, as assessed by recording of miniature EPSCs. Our results thus indicate that RIL directs the transport of GluR-A-containing AMPA receptors to and/or within dendritic spines, in an ␣-actinin/actin-dependent manner, and that such trafficking function promotes the synaptic accumulation of the receptors.

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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motoneurons. We have recently uncovered a new neurotrophic growth factor, granulocyte-colony stimulating factor (G-CSF), which protects α-motoneurons, improves functional outcome, and increases life expectancy of SOD-1 (G93A) mice when delivered subcutaneously. However, chronic systemic delivery of G-CSF is complicated by elevation of neutrophilic granulocytes. Here, we used adeno-associated virus (AAV) to directly target and confine G-CSF expression to the spinal cord. Whereas intramuscular injection of AAV failed to transduce motoneurons retrogradely, and caused a high systemic load of G-CSF, intraspinal delivery led to a highly specific enrichment of G-CSF in the spinal cord with moderate peripheral effects. Intraspinal delivery improved motor functions, delayed disease progression, and increased survival by 10%, longer than after systemic delivery. Mechanistically, we could show that G-CSF in addition to rescuing motoneurons improved neuromuscular junction (NMJ) integrity and enhanced motor axon regeneration after nerve crush injury. Collectively, our results show that intraspinal delivery improves efficacy of G-CSF treatment in an ALS mouse model while minimizing the systemic load of G-CSF, suggesting a new therapeutic option for ALS treatment.

J. Neurochem. (2010) 113, 930–942. Abstract Granulocyte‐colony stimulating factor (G‐CSF) is a potent hematopoietic factor that drives differentiation of neutrophilic granulocytes. We have recently shown that G‐CSF also acts as a neuronal growth factor, protects neurons in vitro and in vivo, and has regenerative potential in various neurological disease models. Spinal cord injury (SCI) following trauma or secondary to skeletal instability is a terrible condition with no effective therapies available at present. In this study, we show that the G‐CSF receptor is up‐regulated upon experimental SCI and that G‐CSF improves functional outcome in a partial dissection model of SCI. G‐CSF significantly decreases apoptosis in an experimental partial spinal transsection model in the mouse and increases expression of the anti‐apoptotic G‐CSF target gene Bcl‐XL. In vitro, G‐CSF enhances neurite outgrowth and branching capacity of hippocampal neurons. In vivo, G‐CSF treatment results in improved functional connectivity of the injured spinal cord as measured by Mn2+‐enhanced MRI. G‐CSF also increased length of the dorsal corticospinal tract and density of serotonergic fibers cranial to the lesion center. Mice treated systemically with G‐CSF as well as transgenic mice over‐expressing G‐CSF in the CNS exhibit a strong improvement in functional outcome as measured by the BBB score and gridwalk analysis. We show that G‐CSF improves outcome after experimental SCI by counteracting apoptosis, and enhancing connectivity in the injured spinal cord. We conclude that G‐CSF constitutes a promising and feasible new therapy option for SCI.