Biomedical subjects

Robert Nitsch

Publications and source records attributed to Robert Nitsch.

At least 19 recordsLinked to original sources

PubMed2005

Circulating monocytic cells infiltrate layers of anterograde axonal degeneration where they transform into microglia.

In this study, we demonstrate the infiltration of blood-derived monocytic cells and their morphologic transformation into microglia in zones of acute, anterograde (Wallerian) axonal degeneration induced by entorhinal cortex lesion (ECL). ECL was performed in mice which had received green fluorescent protein (GFP)-transduced bone marrow grafts allowing identification of blood-derived elements within the brain. While in the unlesioned hemisphere GFP+ cells were restricted to perivascular and leptomeningeal sites, many round fluorescent cells appeared in hippocampal zones of axonal degeneration at 24 h post lesion (hpl). Within 72 hpl, these GFP+ cells acquired ramified, microglia-like morphologies, which persisted for at least 7 days post ECL. Differentiation of GFP+ cells into glial fibrillary acidic protein (GFAP)+ astrocytes was never observed. To exclude that this recruitment is an artifact of irradiation or bone marrow transplantation, the fluorescent cell tracker 6-carboxylfluorescein diacetate (CFDA) was injected into spleens of normal mice 1 day before ECL. Again, fluorescent cells appeared at the lesion site and along the layers of axonal degeneration at 48 hpl and CFDA+/MAC-1+, cells exhibited amoeboid and ramified morphologies. Thus, blood-derived cells infiltrate not only the site of mechanical lesion, but also the layers of anterograde axonal degeneration, where they readily transform into microglia-like elements. A role for infiltrating leukocytes in facilitating or modulating postlesional plasticity, e.g., by phagocytosis of growth-inhibiting myelin should now be considered. Moreover, monocytic cells may serve as vehicles to transport therapeutic substances such as neurotrophic factors or caspase inhibitors to zones of axonal degeneration.

Animals↗

PubMed2005

Regulation of miRNA expression during neural cell specification.

MicroRNA (miRNA) are a newly recognized class of small, noncoding RNA molecules that participate in the developmental control of gene expression. We have studied the regulation of a set of highly expressed neural miRNA during mouse brain development. Temporal control is a characteristic of miRNA regulation in C. elegans and Drosophila, and is also prominent in the embryonic brain. We observed significant differences in the onset and magnitude of induction for individual miRNAs. Comparing expression in cultures of embryonic neurons and astrocytes we found marked lineage specificity for each of the miRNA in our study. Two of the most highly expressed miRNA in adult brain were preferentially expressed in neurons (mir-124, mir-128). In contrast, mir-23, a miRNA previously implicated in neural specification, was restricted to astrocytes. mir-26 and mir-29 were more strongly expressed in astrocytes than neurons, others were more evenly distributed (mir-9, mir-125). Lineage specificity was further explored using reporter constructs for two miRNA of particular interest (mir-125 and mir-128). miRNA-mediated suppression of both reporters was observed after transfection of the reporters into neurons but not astrocytes. miRNA were strongly induced during neural differentiation of embryonic stem cells, suggesting the validity of the stem cell model for studying miRNA regulation in neural development.

Animals↗

PubMed2004

CXCR3-dependent microglial recruitment is essential for dendrite loss after brain lesion.

Microglia are the resident macrophage population of the CNS and are considered its major immunocompetent elements. They are activated by any type of brain pathology and can migrate to the lesion site. The chemokine CXCL10 is expressed in neurons in response to brain injury and is a signaling candidate for activating microglia and directing them to the lesion site. We recently identified CXCR3, the corresponding receptor for CXCL10, in microglia and demonstrated that this receptor system controls microglial migration. We have now tested the impact of CXCR3 signaling on cellular responses after entorhinal cortex lesion. In wild-type mice, microglia migrate within the first 3 d after lesion into the zone of axonal degeneration, where 8 d after lesion denervated dendrites of interneurons are subsequently lost. In contrast, the recruitment of microglia was impaired in CXCR3 knock-out mice, and, strikingly, denervated distal dendrites were maintained in zones of axonal degeneration. No differences between wild-type and knock-out mice were observed after facial nerve axotomy, as a lesion model for assessing microglial proliferation. This shows that CXCR3 signaling is crucial in microglia recruitment but not proliferation, and this recruitment is an essential element for neuronal reorganization.

Animals↗

PubMed2004

Identification of macrophage/microglia activation factor (MAF) associated with late endosomes/lysosomes in microglial cells.

Damage to the central nervous system triggers rapid activation and specific migration of glial cells towards the lesion site. There, glial cells contribute heavily to secondary neuronal changes that take place after lesion. In an attempt to identify the molecular cues of glial activation following brain trauma we performed differential display reverse transcription-polymerase chain reaction screenings from lesioned and control hippocampus. Here we report on the identification of the macrophage/microglia activation factor (MAF), a new membrane protein with seven putative transmembrane domains. Expression analysis revealed that MAF is predominantly expressed in microglial cells in the brain, and is upregulated following brain lesion. Overexpression of MAF in non-glial cells shows an intracellular codistribution with the lysosomal marker endosome/lysosome-associated membrane protein-1 (lamp-1). Furthermore, MAF-transfected cells show that MAF is primarily associated with late endosomes/lysosomes, and that this association can be disrupted by activation of protein kinase C-dependent pathways. In conclusion, these results imply that MAF is involved in the dynamics of lysosomal membranes associated with microglial activation following brain lesion.

Amino Acid Sequence↗

PubMed2004

Direct impact of T cells on neurons revealed by two-photon microscopy in living brain tissue.

Encephalitogenic T cells invade the brain during neuroinflammation such as multiple sclerosis (MS), inducing damage to myelin sheaths and oligodendrocytes. Only recently, neuronal structures were reported to be a crucial target in the disease. Here, two-photon microscopy using ion-sensitive dyes revealed that within the complex cellular network of living brain tissue, proteolipid protein (PLP)-specific T cells and T cells recognizing the nonmurine antigen ovalbumin (OVA) directly and independently of the major histocompatibility complex (MHC) contact neurons in which they induce calcium oscillations. T cell contact finally resulted in a lethal increase in neuronal calcium levels. This could be prevented by blocking both perforin and glutamate receptors. For the first time, our data provide direct insight into the activity of T cells in the living brain and their detrimental impact on neurons.

Animals↗

PubMed2004

Astrocytes protect the CNS: antigen-specific T helper cell responses are inhibited by astrocyte-induced upregulation of CTLA-4 (CD152).

Astrocytes are the first cells that are encountered by T cells invading the central nervous system (CNS) by crossing the blood-brain barrier. We show that primary astrocytes contribute to the immune privilege of the CNS by suppressing Th1 and Th2 cell activation, proliferation and effector function. Moreover, this astrocyte-mediated inhibition of Th effector cells was effective on already activated, proliferating cells. Transforming growth factor (TGF)-beta secreted by astrocytes or T cells was not the major factor in the inhibition. The inhibition of T-cell proliferation induced by astrocytes was mainly mediated by upregulation of CTLA-4 on already activated T cells, which occurred both with and without cell-cell contact. Upregulation of the inhibitory molecule CTLA-4 on autoreactive Th cells, as mediated by astrocytes, thus represents a novel mechanism for securing the immune privilege of the CNS.

Antigens, CD↗

PubMed2004

Selenium and brain function: a poorly recognized liaison.

Molecular biology has recently contributed significantly to the recognition of selenium (Se)2 and Se-dependent enzymes as modulators of brain function. Increased oxidative stress has been proposed as a pathomechanism in neurodegenerative diseases including, among others, Parkinson's disease, stroke, and epilepsy. Glutathione peroxidases (GPx), thioredoxin reductases, and one methionine-sulfoxide-reductase are selenium-dependent enzymes involved in antioxidant defense and intracellular redox regulation and modulation. Selenium depletion in animals is associated with decreased activities of Se-dependent enzymes and leads to enhanced cell loss in models of neurodegenerative disease. Genetic inactivation of cellular GPx increases the sensitivity towards neurotoxins and brain ischemia. Conversely, increased GPx activity as a result of increased Se supply or overexpression ameliorates the outcome in the same models of disease. Genetic inactivation of selenoprotein P leads to a marked reduction of brain Se content, which has not been achieved by dietary Se depletion, and to a movement disorder and spontaneous seizures. Here we review the role of Se for the brain under physiological as well as pathophysiological conditions and highlight recent findings which open new vistas on an old essential trace element.

Animals↗

PubMed2004

Production of neuroprotective NGF in astrocyte-T helper cell cocultures is upregulated following antigen recognition.

Astrocytic production of nerve growth factor (NGF) is increased during inflammation of the central nervous system (CNS). Here we show that cell-cell interaction between primary murine astrocytes and myelin basic protein (MBP)-specific T cell receptor (TCR) transgenic Th1 and Th2 cells significantly increased production of NGF. This upregulation was found to be dependent on antigen recognition. Neutralization of cytokines produced in cocultures did not affect NGF production. This novel finding suggests a neuroprotective role of astrocytes during T cell-mediated inflammation in the CNS.

Animals↗

PubMed2004

Molecular cloning and expression regulation of PRG-3, a new member of the plasticity-related gene family.

Phospholipid-mediated signalling on neurons provokes diverse responses such as neurogenesis, pattern formation and neurite remodelling. We have recently uncovered a novel set of molecules in the mammalian brain, named plasticity-related genes (PRGs), which mediate lipid phosphate phosphatase activity and provide evidence for their involvement in mechanisms of neuronal plasticity. Here, we report on a new member of the vertebrate-specific PRG family, which we have named plasticity-related gene-3 (PRG-3). PRG-3 is heavily expressed in the brain and shows a specific expression pattern during brain development where PRG-3 expression is found predominantly in neuronal cell layers and is already expressed at embryonic day 16. In the mature brain, strongest PRG-3 expression occurs in the hippocampus and cerebellum. Overexcitation of neurons induced by kainic acid leads to a transient down-regulation of PRG-3. Furthermore, PRG-3 is expressed on neurite extensions and promotes neurite growth and a spreading-like cell body in neuronal cells and COS-7 cells. In contrast to previously described members of the PRG family, PRG-3 does not perform its function through enzymatic phospholipid degradation. In summary, our findings feature a new member of the PRG family which shows dynamic expression regulation during brain development and neuronal excitation.

Amino Acid Sequence↗

PubMed2004

An impaired neocortical Ih is associated with enhanced excitability and absence epilepsy.

Neuronal subthreshold excitability and firing behaviour are markedly influenced by the activation and deactivation of the somato-dendritic hyperpolarization-activated cation current (Ih). Here, we evaluated possible contributions of Ih to hyperexcitability in an animal model of absence seizures (WAG/Rij rats). We investigated pyramidal neurons of the somatosensory neocortex, the site of generation of spike-wave discharges. Ih-mediated functions in neurons from WAG/Rij rats, Wistar rats (sharing the same genetic background with WAG/Rij, but less epilepsy-prone) and ACI rats (an inbred strain, virtually free of seizures) were compared. We complemented whole-cell recordings from layer 2-3 pyramidal neurons with immunohistochemistry, Western blot and RT-PCR analysis of the h-channel subunits HCN1-4. The fast component of Ih activation in WAG/Rij neurons was significantly reduced (50% reduction in the h-current density) and four times slower than in neurons from nonepileptic Wistar or ACI rats. The results showing decreases in currents corresponded to a 34% reduction in HCN1 protein in the WAG/Rij compared to the Wistar neocortex, but HCN1 mRNA showed stable expression. The other three Ih subunit mRNAs and proteins (HCN2-4) were not affected. The alterations in Ih magnitude and kinetics of gating in WAG/Rij neurons may contribute to augmented excitatory postsynaptic potentials, the increase in their temporal summation and the facilitation of burst firing of these neurons because each of these effects could be mimicked by the selective Ih antagonist ZD 7288. We suggest that the deficit in Ih-mediated functions may contribute to the development and onset of spontaneously occurring hyperexcitability in a rat model of absence seizures.

2-Amino-5-phosphonovalerate↗

PubMed2004

Entorhinal cortex lesion in the mouse induces transsynaptic death of perforant path target neurons.

Entorhinal cortex lesion (ECL) is a well described model of anterograde axonal degeneration, subsequent sprouting and reactive synaptogenesis in the hippocampus. Here, we show that such lesions induce transsynaptic degeneration of the target cells of the lesions pathway in the dentate gyrus. Peaking between 24 and 36 hours post-lesion, dying neurons were labeled with DeOlmos silver-staining and antisera against activated caspase 3 (CCP32), a downstream inductor of programmed cell death. Within caspase 3-positive neurons, fragmented nuclei were co-localized using Hoechst 33342 staining. Chromatin condensation and nuclear fragmentation were also evident in semithin sections and at the ultrastructural level, where virtually all caspase 3-positive neurons showed these hallmarks of apoptosis. There is a well-described upregulation of the apoptosis-inducing CD95/L system within the CNS after trauma, yet a comparison of caspase 3-staining patterns between CD95 (Ipr)- and CD95L (gld)-deficient with non-deficient mice (C57/bl6) provided no evidence for CD95L-mediated neuronal cell death in this setting. However, inhibition of NMDA receptors with MK-801 completely suppressed caspase 3 activation, pointing to glutamate neurotoxicity as the upstream inducer of the observed cell death. Thus, these data show that axonal injury in the CNS does not only damage the axotomized neurons themselves, but can also lethally affect their target cells, apparently by activating glutamate-mediated intracellular pathways of programmed cell death.

Animals↗

PubMed2003

Activation of microglial poly(ADP-ribose)-polymerase-1 by cholesterol breakdown products during neuroinflammation: a link between demyelination and neuronal damage.

Multiple sclerosis (MS) is a chronic demyelinating disease in which it has only recently been suggested that damage to neuronal structures plays a key role. Here, we uncovered a link between the release of lipid breakdown products, found in the brain and cerebrospinal fluid (CSF) of MS patients as well as in experimental autoimmune encephalomyelitis, and neuronal damage mediated by microglial activation. The concentrations of the breakdown product 7-ketocholesterol detected in the CSF of MS patients were capable of inducing neuronal damage via the activation and migration of microglial cells in living brain tissue. 7-ketocholesterol rapidly entered the nucleus and activated poly(ADP-ribose)-polymerase (PARP)-1, followed by the expression of migration-regulating integrins CD11a and intercellular adhesion molecule 1. These findings reveal a novel mechanism linking demyelination and progressive neuronal damage, which might represent an underlying insidious process driving disease beyond a primary white matter phenomenon and rendering the microglial PARP-1 a possible antiinflammatory therapeutic target.

Animals↗

PubMed2003

Bone marrow-derived cells expressing green fluorescent protein under the control of the glial fibrillary acidic protein promoter do not differentiate into astrocytes in vitro and in vivo.

Differentiation of bone marrow (BM) cells into astroglia expressing the glial fibrillary acidic protein (GFAP) has been reported in vitro and after intracerebral or systemic BM transplantation. In contrast, recent data suggest that astrocytic differentiation does not occur from BM-derived cells in vivo. Using transgenic mice that express the enhanced green fluorescent protein (GFP) under the control of the human glial fibrillary acidic protein (GFAP) promoter, we investigated the potential of adult murine BM-derived cells to differentiate into macroglia. In the brains of GFAP-GFP transgenic mice, astrocytes were brightly fluorescent from the expression of GFP. When BM from these animals was transplanted into lethally irradiated wild-type animals, the transgene was detected in the reconstituted hematopoietic system, but no GFP expression was found in the nervous system. In contrast, GFAP-GFP neuroectodermal anlage grafted into adult wild-type striatum gave rise to GFP-expressing astrocytes. Because cerebral ischemia has been suggested to promote the differentiation of BM-derived cells into astrocytes, BM chimeric mice were subjected to focal cerebral ischemia. No GFP-positive cells were found in the ischemic or contralateral hemispheres of these brains. Even after direct injection of GFAP-GFP transgenic BM cells into wild-type striatum, no GFP-expressing astroglia were detected. To test the hypothesis that the in vitro environment might be more permissible for astroglial differentiation, we cultured BM from mice that constitutively express GFP, BM cells expressing GFP from a retroviral vector, and BM from GFAP-GFP transgenic mice on astrocytes and on organotypic hippocampal slices. In all experimental paradigms, BM-derived cells were found to differentiate into ramified microglia but not into GFAP-expressing astrocytes.

Animals↗

PubMed2003

Molecular analysis of Nogo expression in the hippocampus during development and following lesion and seizure.

The Nogo gene encodes an integral membrane protein mainly responsible for the neurite inhibition properties of myelin. Here, we analyzed the expression pattern of Nogo-A, Nogo-B, and Nogo-C and Nogo-66 receptor (Ng66R) mRNA during hippocampal development and lesion-induced axonal sprouting. Nogo-A and Nogo-B and Ng66R transcripts preceded the progress of myelination and were highly expressed at postnatal day zero (P0) in all principal hippocampal cell layers, with the exception of dentate granule cells. Only a slight Nogo-C expression was found at P0 in the principal cell layers of the hippocampus. During adulthood, all Nogo splice variants and their receptor were expressed in the neuronal cell layers of the hippocampus, in contrast to the myelin basic protein mRNA expression pattern, which revealed a neuronal source of Nogo gene expression in addition to oligodendrocytes. After hippocampal denervation, the Nogo genes showed an isoform-specific temporal regulation. All Nogo genes were strongly regulated in the hippocampal cell layers, whereas the Ng66R transcripts showed a significant increase in the contralateral cortex. These data could be confirmed on protein levels. Furthermore, Nogo-A expression was up-regulated after kainate-induced seizures. Our data show that neurons express Nogo genes with a clearly distinguishable pattern during development. This expression is further dynamically and isoform-specifically altered after lesioning during the early phase of structural rearrengements. Thus, our results indicate a role for Nogo-A, -B, and -C during development and during the stabilization phase of hippocampal reorganization. Taken together with these data, the finding that neurons in a highly plastic brain region express Nogo genes supports the hypothesis that Nogo may function beyond its known neuronal growth inhibition activity in shaping neuronal circuits.

Animals↗

PubMed2003

Modification of microglia function protects from lesion-induced neuronal alterations and promotes sprouting in the hippocampus.

Primary neuronal destruction in the central nervous system triggers rapid changes in glial morphology and function, after which activated glial cells contribute to secondary neuronal changes. Here we show that, after entorhinal cortex lesion, activation of microglia, but not other glial cells, leads to massive secondary dendritic changes of deafferentiated hippocampal neurons. Blocking of microglial activation in vivo reduced this secondary neuronal damage and enhanced regenerative axonal sprouting. In contrast, abolishing astrocytes or oligodendroglia did not result in specific neuronal changes. Furthermore, primary damage leads to an interleukin 1beta up-regulation, which is attenuated by the immuno-modulator transforming growth factor beta1, whereas tumor necrosis factor alpha is not affected. Modification of microglial activity following denervation of the hippocampus protects neurons from secondary dendritic alterations and therefore enables their reinnervation. These data render activated microglia a putative therapeutic target during the course of axonal degeneration.

Animals↗

PubMed2003

Treatment of relapsing paralysis in experimental encephalomyelitis by targeting Th1 cells through atorvastatin.

Statins, known as inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, exhibit numerous functions related to inflammation, such as MHC class II down-regulation, interference with T cell adhesion, and induction of apoptosis. Here we demonstrate that both subcutaneous and oral administration of atorvastatin inhibit the development of actively induced chronic experimental autoimmune encephalomyelitis in SJL/J mice and significantly reduce the inflammatory infiltration into the central nervous system (CNS). When treatment was started after disease onset, atorvastatin reduced the incidence of relapses and protected from the development of further disability. Both the reduced autoreactive T cell response measured by proliferation toward the encephalitogenic peptide PLP139-151 and the cytokine profile indicate a potent blockade of T helper cell type 1 immune response. In in vitro assays atorvastatin not only inhibited antigen-specific responses, but also decreased T cell proliferation mediated by direct TCR engagement independently of MHC class II and LFA-1. Inhibition of proliferation was not due to apoptosis induction, but linked to a negative regulation on cell cycle progression. However, early T cell activation was unaffected, as reflected by unaltered calcium fluxes. Thus, our results provide evidence for a beneficial role of statins in the treatment of autoimmune attack on the CNS.

Animals↗

PubMed2003

IDO (indolamine 2,3-dioxygenase) expression and function in the CNS.

From an immunological perspective the placenta is an allograft and therefore requires a special immune suppressive status termed immune privilege. Other organs of the body, which possess poor regenerative capacity share this special status, e.g. the brain, the eye and the gonads. The biological function of immune privilege in all these tissues is to protect them from inflammation-mediated injury. The mechanism maintaining immune privilege are poorly understood and are apparently site-specific. In the placenta, inhibition of IDO leads to spontaneous abortion, showing the crucial role of this enzyme for the maintenance of immune privilege. By catabolizing extracellular tryptophan IDO inhibits local T cell proliferation thereby preventing placental rejection. Here, we show that this mechanism can also be active in suppressing inflammatory responses in the CNS, where inflammations must be tightly regulated to prevent the loss of irreplaceable neurons. Employing RT-PCR and Western blot analysis we could show that, upon activation with the pro-inflammatory cytokine interferon-gamma, astrocytes and microglia are capable of expressing IDO in vitro and in vivo. To test the functional capacity of IDO in the CNS, we performed blockade experiments using actively induced experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease which correlates to the human disease multiple sclerosis (MS). Inhibition of IDO activity by daily subcutaneous administration of the specific IDO inhibitor 1-methyl-DL-tryptophan during EAE significantly exacerbates EAE, shown by comparing clinical disease scores. Thus, local expression of IDO during inflammation is apparently a self-protection mechanism which limits antigen-specific immune responses in the CNS.

Animals↗

PubMed2003

Identification of neuronal cell death in a model of degeneration in the hippocampus.

Neuronal cell death and microglial changes are both hallmarks of neurodegenerative disorders. Therefore, analysis of degenerating neurons related to microglial changes are addressed in many studies of neurosciences. Here we compared different lesion models and two markers for neurodegeneration (Fluoro-Jade and propidium iodide) in an in vivo as well as an in vitro approach. Fluoro-Jade is a specific and selective marker to identify neurons undergoing degeneration. We also tested this marker to analyze neurodegeneration in organotypic hippocampal slice cultures. We could show that activation of microglia is followed by neuronal cell death. Most degeneration markers, such as propidium iodide, only stain the neuronal cell body excluding the axonal and dendritic processes. Fluoro-Jade is able to stain the distal portion as well as the proximal portion of the dissected axon including the axotomized neuron, as so called anterograde and retrograde degeneration after axotomy. To analyze the specificity of Fluoro-Jade, we used primary microglial and BV-2 cells, a well-described murine microglial cell line. Treatment of microglial and BV-2 cells with an excess of L-glutamate induces cell death which could be detected by propidium iodide staining, but not by Fluoro-Jade, demonstrating its specificity to monitor neuronal cell death.

Animals↗