The brain's immune privilege has been also attributed to the lack of dendritic cells (DC) within ... more The brain's immune privilege has been also attributed to the lack of dendritic cells (DC) within its parenchyma and the adjacent meninges, an assumption, which implies maintenance of antigens rather than their presentation in lymphoid organs. Using mice transcribing the green fluorescent protein under the promoter of the DC marker CD11c (itgax), we identified a juxtavascular population of cells expressing this DC marker and demonstrated their origin from bone marrow and local microglia. We now phenotypically compared this population with CD11c/CD45 double-positive cells from lung, liver, and spleen in healthy mice using seven-color flow cytometry. We identified unique, site-specific expression patterns of F4/80, CD80, CD86, CX3CR1, CCR2, FLT3, CD103, and MHC-II. Furthermore, we observed the two known CD45-positive populations (CD45 high and CD45 int ) in the brain, whereas liver, lung, and spleen exhibited a homogeneous CD45 high population. CD11c-positive microglia lacked MHC-II expression and CD45 high /CD11c-positive cells from the brain have a lower percentage of MHC-IIpositive cells. To test whether phenotypical differences are fixed by origin or specifically develop due to environmental factors, we transplanted brain and spleen mononuclear cells on organotypic slice cultures from brain (OHSC) and spleen (OSSC). We demonstrate that adaption and ramification of MHC-II-positive splenocytes is paralleled by down-regulation of MHC-II, whereas brain-derived mononuclear cells neither ramified nor up-regulated MHC-II in OSSCs. Thus, brain-derived mononuclear cells maintain their MHC-II-negative phenotype within the environment of an immune organ. Intraparenchymal CD11cpositive cells share immunophenotypical characteristics of DCs from other organs but remain unique for their low MHC-II expression.
The chemokine CXCL10 and its receptor CXCR3 are implicated in various CNS pathologies since inter... more The chemokine CXCL10 and its receptor CXCR3 are implicated in various CNS pathologies since interference with CXCL10/CXCR3 signaling alters the onset and progression in various CNS disease models. However, the mechanism and cell-types involved in CXCL10/CXCR3 signaling under pathological conditions are far from understood. Here, we investigated the potential role for CXCL10/CXCR3 signaling in neuronal cell death and glia activation in response to N-methyl-D-aspartic acid (NMDA)-induced excitotoxicity in mouse organotypic hippocampal slice cultures (OHSCs). Our findings demonstrate that astrocytes express CXCL10 in response to excitotoxicity. Experiments in OHSCs derived from CXCL10-deficient (CXCL10 2/2 ) and CXCR3-deficient (CXCR3 2/2 ) revealed that in the absence of CXCL10 or CXCR3, neuronal cell death in the CA1 and CA3 regions was diminished after NMDA-treatment when compared to wild type OHSCs. In contrast, neuronal cell death in the DG region was enhanced in both CXCL10 2/2 and CXCR3 2/2 OHSCs in response to a high (50 lM) NMDAconcentration. Moreover, we show that in the absence of microglia the differential changes in neuronal vulnerability between CXCR3 2/2 and wild type OHSCs are fully abrogated and therefore a prominent role for microglia in this process is suggested. Taken together, our results identify a region-specific role for CXCL10/CXCR3 signaling in neuron-glia and glia-glia interactions under pathological conditions. V V C 2010 Wiley-Liss, Inc.
The immunological response in the brain is crucial to overcome neuropathological events. Some inf... more The immunological response in the brain is crucial to overcome neuropathological events. Some inflammatory mediators, such as the immunoregulatory cytokine interleukin-6 (IL-6) affect neuromodulation and may also play protective roles against various noxious conditions. However, the fundamental mechanisms underlying the long-term effects of IL-6 in the brain remain unclear. We now report that IL-6 increases the expression and function of the neuronal adenosine A 1 receptor, with relevant consequences to synaptic transmission and neuroprotection. IL-6-induced amplification of A 1 receptor function enhances the responses to readily released adenosine during hypoxia, enables neuronal rescue from glutamate-induced death, and protects animals from chemically induced convulsing seizures. Taken together, these results suggest that IL-6 minimizes the consequences of excitotoxic episodes on brain function through the enhancement of endogenous adenosinergic signaling.
Microglia are the resident macrophage population of the CNS and are considered its major immunoco... more 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.
Whenever neurons in the CNS are injured, microglia become activated. In addition to local activat... more Whenever neurons in the CNS are injured, microglia become activated. In addition to local activation, microglia remote from the primary lesion site are stimulated. Because this so-called secondary activation of microglia is instrumental for long-term changes after neuronal injury, it is important to understand how microglia activity is controlled. The remote activation of microglia implies that the activating signals are transported along neuronal projections. However, the identity of these signals has not yet been identified. It is shown here that glutamate-treated neurons rapidly express and release the chemokine CCL21. We also provide evidence that neuronal CCL21 is packed in vesicles and transported throughout neuronal processes to reach presynaptic structures. Chemotaxis assays show that functional CCL21 is released from endangered neurons and activate microglia via the chemokine receptor CXCR3. Based on these findings, we suggest that neuronal CCL21 is important in directed neuron-microglia signaling and that this communication could account for the remote activation of microglia, far distant from a primary lesion.
Neurons are highly polarized cells, and neuron-neuron communication is based on directed transpor... more Neurons are highly polarized cells, and neuron-neuron communication is based on directed transport and release of neurotransmitters, neuropeptides, and neurotrophins. Directed communication may also be attributed to neuron-microglia signaling, since neuronal damage can induce a microglia reaction at specific sites only. However, the mechanism underlying this site-specific microglia reaction is not yet understood. Neuronal CCL21 is a microglia-activating chemokine, which in brain is solely found in endangered neurons and is therefore a candidate for neuronmicroglia signaling. Here we present that neuronal CCL21 is sorted into large dense-core vesicles, the secretory granules of the regulated release pathway of neurons. Live-cell imaging studies show preferential sorting of CCL21-containing vesicles into axons, indicating its directed transport. Thus, mouse neurons express and transport a microglia activating factor very similar to signaling molecules used in neuron-neuron communication. These data show for the first time the directed transport of a microglia activating factor in neurons and corroborate the function of neuronal CCL21 in directed neuron-microglia communication.-Expression, transport, and axonal sorting of neuronal CCL21 in large dense-core vesicles. FASEB J. 22, 4136 -4145 (2008)
Up-regulation of P2X4 receptors in spinal cord microglia is crucial for tactile allodynia, an unt... more Up-regulation of P2X4 receptors in spinal cord microglia is crucial for tactile allodynia, an untreatable pathological pain reaction occurring after peripheral nerve injury. How nerve injury in the periphery leads to this microglia reaction in the dorsal horn of the spinal cord is not yet understood. It is shown here that CCL21 was rapidly expressed in injured small-sized primary sensory neurons and transported to their central terminals in the dorsal horn. Intrathecal administration of a CCL21-blocking antibody diminished tactile allodynia development in wildtype animals. Mice deficient for CCL21 did not develop any signs of tactile allodynia and failed to up-regulate microglial P2X4 receptor expression. Microglia P2X4 expression was enhanced by CCL21 application in vitro and in vivo. A single intrathecal injection of CCL21 to nerve-injured CCL21-deficient mice induced long-lasting allodynia that was undistinguishable from the wild-type response. This effect of CCL21 injection was strictly dependent on P2X4 receptor function. Since neuronal CCL21 is the earliest yet identified factor in the cascade leading to tactile allodynia, these findings may lead to a preventive therapy in neuropathic pain.
Sleep is a complex physiological process and still remains one of the great mysteries of science.... more Sleep is a complex physiological process and still remains one of the great mysteries of science. Over the past 10 y, genetic research has provided a new avenue to address the regulation and function of sleep. Gene loci that contribute quantitatively to sleep characteristics and variability have already been identified. However, up to now, a genetic basis has been established only for a few sleep disorders. Little is yet known about the genetic background of insomnia, one of the most common sleep disorders. According to the conceptualisation of the 3P model of insomnia, predisposing, precipitating and perpetuating factors contribute to the development and maintenance of insomnia. Growing evidence from studies of predisposing factors suggests a certain degree of heritability for insomnia and for a reactivity of sleep patterns to stressful events, explaining the emergence of insomnia in response to stressful life events. While a genetic susceptibility may modulate the impact of stress on the brain, this finding does not provide us with a complete understanding of the capacity of stress to produce long-lasting perturbations of brain and behaviour. Epigenetic geneeenvironment interactions have been identified just recently and may provide a more complex understanding of the genetic control of sleep and its disorders. It was recently hypothesised that stress-response-related brain plasticity might be epigenetically controlled and, moreover, several epigenetic mechanisms have been assumed to be involved in the regulation of sleep. Hence, it might be postulated that insomnia may be influenced by an epigenetic control process of both sleep mechanisms and stress-response-related geneeenvironment interactions having an impact on brain plasticity. This paper reviews the evidence for the genetic basis of insomnia and recent theories about epigenetic mechanisms involved in both sleep regulation and brain-stress response, leading to the hypothesis of an involvement of epigenetic mechanisms in the development and maintenance of insomnia.
It has been established recently that inflammation of the central nervous system (CNS) is accompa... more It has been established recently that inflammation of the central nervous system (CNS) is accompanied by an expression of chemokines within the CNS. Several lines of evidence suggest that chemokines within the CNS initiate and orchestrate the infiltration of the inflamed brain by blood leukocytes. In line with this assumption is the finding that glial cells expressing the chemokine ligands CXCL10 and CXCL9 accompany the presence of CXCR3 positive T-cells in the CNS in demyelinating diseases.
The inhibitory neuromodulator adenosine is released in the brain in high concentrations under con... more The inhibitory neuromodulator adenosine is released in the brain in high concentrations under conditions of exaggerated neuronal activity such as ischemia and seizures, or electroconvulsive treatment. By inhibiting neural overactivity, adenosine counteracts seizure activity and promotes neuronal survival. Since stimulation of adenosine A(2b) receptors on astrocytes induces increased synthesis and release of interleukin-6, which also exerts neuroprotective effects, we hypothesized that the effects of interleukin-6 and of adenosine might be related. We report here that stimulation with interleukin-6 of cultured astrocytes, of cultured organotypic brain slices from newborn rat cortex, and of freshly prepared brain slices from rat cortex induces a concentration- and time-dependent upregulation of adenosine A(1) receptor mRNA. This increased adenosine A(1) receptor mRNA expression is accompanied in astrocytes by an increase in adenosine A(1) receptor-mediated signaling via the phosphoinositide-dependent pathway. Since upregulation of adenosine A(1) receptors leads to increased neuroprotective effects of adenosine, we suggest that the neuroprotective actions of interleukin-6 and adenosine are related and might be mediated at least in part through upregulation of adenosine A(1) receptors. These results may be of relevance for a better understanding of neuroprotection in brain damage but also point to a potential impact of neuroprotection in the mechanisms of the antidepressive effects of chronic carbamazepine, electroconvulsive therapy, and sleep deprivation, which are all accompanied by adenosine A(1) receptor upregulation.
The anticonvulsant and antibipolar drug carbamazepine (CBZ) is known to act as a specific antagon... more The anticonvulsant and antibipolar drug carbamazepine (CBZ) is known to act as a specific antagonist at adenosine A 1 -receptors. After a 3-week application of CBZ, A 1receptors are upregulated in the rat brain. We have investigated the consequences of this upregulation for the A 1 -receptor-mediated signal transduction in primary astrocyte cultures from different regions of the rat brain. CBZ treatment for 10 days had no effect on adenosine A 1receptor mRNA expression in cultures with high basal A 1receptor mRNA levels, but increased A 1 -receptor mRNA in cultures exhibiting low basal A 1 -receptor mRNA levels.
It has been shown that neural stem cells (NSCs) migrate towards areas of brain injury or brain tu... more It has been shown that neural stem cells (NSCs) migrate towards areas of brain injury or brain tumours and that NSCs have the capacity to track infiltrating tumour cells. The possible mechanism behind the migratory behaviour of NSCs is not yet completely understood. As chemokines are involved in the migration of immune cells in the injured brain, they may also be involved in chemoattraction of NSCs towards a brain tumour. Methods: The expression profile of various chemokine receptors in NSCs, harvested from the subventricular zone of adult mice, was investigated by reverse transcriptasepolymerase chain reaction analysis. Furthermore, the functionality of the chemokine receptors was assessed in in vitro chemotaxis assays and calcium signalling experiments. To test the in vivo migration of NSCs, a syngeneic mouse model was developed, whereby a B16F10 mela-noma cell line was grafted into one hemisphere and later NSCs were grafted in the contralateral hemisphere. Furthermore, the expression of chemokines in this melanoma cell line was investigated. Results and conclusions: Adult mouse NSCs functionally express various chemokine receptors of which CXC chemokine receptor (CXCR)4 shows the highest mRNA levels and most pronounced functional responses in vitro. CXC chemokine ligand (CXCL)12, the ligand for CXCR4, is expressed by the melanoma cell line. In this mouse model for metastatic brain tumours, it is shown that NSCs express CXCR4 at their cell membranes while they migrate towards the tumour, which produces CXCL12. It is therefore suggested that the CXCR4/CXCL12 pathway plays a role in the mechanism underlying tumour-mediated attraction of NSCs.
Aging is associated with reduced function, degenerative changes, and increased neuroinflammation ... more Aging is associated with reduced function, degenerative changes, and increased neuroinflammation of the central nervous system (CNS). Increasing evidence suggests that changes in microglia cells contribute to the age-related deterioration of the CNS. The most prominent age-related change of microglia is enhanced sensitivity to inflammatory stimuli, referred to as priming. It is unclear if priming is due to intrinsic microglia ageing or induced by the ageing neural environment. We have studied this in Ercc1 mutant mice, a DNA repair-deficient mouse model that displays features of accelerated aging in multiple tissues including the CNS. In Ercc1 mutant mice, microglia showed hallmark features of priming such as an exaggerated response to peripheral lipopolysaccharide exposure in terms of cytokine expression and phagocytosis. Specific targeting of the Ercc1 deletion to forebrain neurons resulted in a progressive priming response in microglia exemplified by phenotypic alterations. Summarizing, these data show that neuronal genotoxic stress is sufficient to switch microglia from a resting to a primed state.
Microglia are crucial for immune responses in the brain. Although their origin from the yolk sac ... more Microglia are crucial for immune responses in the brain. Although their origin from the yolk sac has been recognized for some time, their precise precursors and the transcription program that is used are not known. We found that mouse microglia were derived from primitive c-kit + erythromyeloid precursors that were detected in the yolk sac as early as 8 d post conception. These precursors developed into CD45 + c-kit lo CX 3 CR1 − immature (A1) cells and matured into CD45 + c-kit − CX 3 CR1 + (A2) cells, as evidenced by the downregulation of CD31 and concomitant upregulation of F4/80 and macrophage colony stimulating factor receptor (MCSF-R). Proliferating A2 cells became microglia and invaded the developing brain using specific matrix metalloproteinases. Notably, microgliogenesis was not only dependent on the transcription factor Pu.1 (also known as Sfpi), but also required Irf8, which was vital for the development of the A2 population, whereas Myb, Id2, Batf3 and Klf4 were not required. Our data provide cellular and molecular insights into the origin and development of microglia.
Whereas chemokines are well known for their ability to induce cell migration, only recently it be... more Whereas chemokines are well known for their ability to induce cell migration, only recently it became evident that chemokines also control a variety of other cell functions and are versatile messengers in the interaction between a diversity of cell types. In the central nervous system (CNS), chemokines are generally found under both physiological and pathological conditions. Whereas many reports describe chemokine expression in astrocytes and microglia and their role in the migration of leukocytes into the CNS, only few studies describe chemokine expression in neurons. Nevertheless, the expression of neuronal chemokines and the corresponding chemokine receptors in CNS cells under physiological and pathological conditions indicates that neuronal chemokines contribute to CNS cell interaction. In this study, we review recent studies describing neuronal chemokine expression and discuss potential roles of neuronal chemokines in neuron-astrocyte, neuronmicroglia, and neuron-neuron interaction.
Background: Most of the known functions of microglia, including neurotoxic and neuroprotective pr... more Background: Most of the known functions of microglia, including neurotoxic and neuroprotective properties, are attributed to morphologically-activated microglia. Resting, ramified microglia are suggested to primarily monitor their environment including synapses. Here, we show an active protective role of ramified microglia in excitotoxicity-induced neurodegeneration.
Background: Microglia are considered a major target for modulating neuroinflammatory and neurodeg... more Background: Microglia are considered a major target for modulating neuroinflammatory and neurodegenerative disease processes. Upon activation, microglia secrete inflammatory mediators that contribute to the resolution or to further enhancement of damage in the central nervous system (CNS). Therefore, it is important to study the intracellular pathways that are involved in the expression of the inflammatory mediators. Particularly, the role of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and glycogen synthase kinase-3 (GSK-3) pathways in activated microglia is unclear. Thus, in the present study we investigated the role of Akt and its downstream pathways, GSK-3 and mTOR, in lipopolysaccharide (LPS)-activated primary rat microglia by pharmacological inhibition of these pathways in regard to the expression of cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1 (mPGES-1) and to the production of prostaglandin (PG) E 2 and PGD 2 . Findings: We show that inhibition of Akt by the Akt inhibitor X enhanced the production of PGE 2 and PGD 2 without affecting the expression of COX-2, mPGES-1, mPGES-2 and cytosolic prostaglandin E synthase (cPGES). Moreover, inhibition of GSK-3 reduced the expression of both COX-2 and mPGES-1. In contrast, the mTOR inhibitor rapamycin enhanced both COX-2 and mPGES-1 immunoreactivity and the release of PGE 2 and PGD 2 . Interestingly, NVP-BEZ235, a dual PI3K/mTOR inhibitor, enhanced COX-2 and reduced mPGES-1 immunoreactivity, albeit PGE 2 and PGD 2 levels were enhanced in LPS-stimulated microglia. However, this compound also increased PGE 2 in nonstimulated microglia.
Our understanding of microglia biology has significantly changed in the last couple of years. Ins... more Our understanding of microglia biology has significantly changed in the last couple of years. Instead of being predominantly detrimental cells showing a stereotypic activation pattern, microglia today are considered highly adaptive elements with many distinct phenotypes. Microglia activity is aimed to protect and to restore and only in case of uncontrolled or impaired microglia function these cells may have detrimental effects. The control of microglia activity is thus an important issue to understand. The family of chemokines are versatile signals specialized to control cell-cell interactions. Neurons express chemokines in a temporarily and spatially regulated manner and microglia respond to these messengers via the appropriate receptors. Due to these features are chemokines ideal messengers for the communication between neurons and microglia.
The brain's immune privilege has been also attributed to the lack of dendritic cells (DC) within ... more The brain's immune privilege has been also attributed to the lack of dendritic cells (DC) within its parenchyma and the adjacent meninges, an assumption, which implies maintenance of antigens rather than their presentation in lymphoid organs. Using mice transcribing the green fluorescent protein under the promoter of the DC marker CD11c (itgax), we identified a juxtavascular population of cells expressing this DC marker and demonstrated their origin from bone marrow and local microglia. We now phenotypically compared this population with CD11c/CD45 double-positive cells from lung, liver, and spleen in healthy mice using seven-color flow cytometry. We identified unique, site-specific expression patterns of F4/80, CD80, CD86, CX3CR1, CCR2, FLT3, CD103, and MHC-II. Furthermore, we observed the two known CD45-positive populations (CD45 high and CD45 int ) in the brain, whereas liver, lung, and spleen exhibited a homogeneous CD45 high population. CD11c-positive microglia lacked MHC-II expression and CD45 high /CD11c-positive cells from the brain have a lower percentage of MHC-IIpositive cells. To test whether phenotypical differences are fixed by origin or specifically develop due to environmental factors, we transplanted brain and spleen mononuclear cells on organotypic slice cultures from brain (OHSC) and spleen (OSSC). We demonstrate that adaption and ramification of MHC-II-positive splenocytes is paralleled by down-regulation of MHC-II, whereas brain-derived mononuclear cells neither ramified nor up-regulated MHC-II in OSSCs. Thus, brain-derived mononuclear cells maintain their MHC-II-negative phenotype within the environment of an immune organ. Intraparenchymal CD11cpositive cells share immunophenotypical characteristics of DCs from other organs but remain unique for their low MHC-II expression.
The chemokine CXCL10 and its receptor CXCR3 are implicated in various CNS pathologies since inter... more The chemokine CXCL10 and its receptor CXCR3 are implicated in various CNS pathologies since interference with CXCL10/CXCR3 signaling alters the onset and progression in various CNS disease models. However, the mechanism and cell-types involved in CXCL10/CXCR3 signaling under pathological conditions are far from understood. Here, we investigated the potential role for CXCL10/CXCR3 signaling in neuronal cell death and glia activation in response to N-methyl-D-aspartic acid (NMDA)-induced excitotoxicity in mouse organotypic hippocampal slice cultures (OHSCs). Our findings demonstrate that astrocytes express CXCL10 in response to excitotoxicity. Experiments in OHSCs derived from CXCL10-deficient (CXCL10 2/2 ) and CXCR3-deficient (CXCR3 2/2 ) revealed that in the absence of CXCL10 or CXCR3, neuronal cell death in the CA1 and CA3 regions was diminished after NMDA-treatment when compared to wild type OHSCs. In contrast, neuronal cell death in the DG region was enhanced in both CXCL10 2/2 and CXCR3 2/2 OHSCs in response to a high (50 lM) NMDAconcentration. Moreover, we show that in the absence of microglia the differential changes in neuronal vulnerability between CXCR3 2/2 and wild type OHSCs are fully abrogated and therefore a prominent role for microglia in this process is suggested. Taken together, our results identify a region-specific role for CXCL10/CXCR3 signaling in neuron-glia and glia-glia interactions under pathological conditions. V V C 2010 Wiley-Liss, Inc.
The immunological response in the brain is crucial to overcome neuropathological events. Some inf... more The immunological response in the brain is crucial to overcome neuropathological events. Some inflammatory mediators, such as the immunoregulatory cytokine interleukin-6 (IL-6) affect neuromodulation and may also play protective roles against various noxious conditions. However, the fundamental mechanisms underlying the long-term effects of IL-6 in the brain remain unclear. We now report that IL-6 increases the expression and function of the neuronal adenosine A 1 receptor, with relevant consequences to synaptic transmission and neuroprotection. IL-6-induced amplification of A 1 receptor function enhances the responses to readily released adenosine during hypoxia, enables neuronal rescue from glutamate-induced death, and protects animals from chemically induced convulsing seizures. Taken together, these results suggest that IL-6 minimizes the consequences of excitotoxic episodes on brain function through the enhancement of endogenous adenosinergic signaling.
Microglia are the resident macrophage population of the CNS and are considered its major immunoco... more 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.
Whenever neurons in the CNS are injured, microglia become activated. In addition to local activat... more Whenever neurons in the CNS are injured, microglia become activated. In addition to local activation, microglia remote from the primary lesion site are stimulated. Because this so-called secondary activation of microglia is instrumental for long-term changes after neuronal injury, it is important to understand how microglia activity is controlled. The remote activation of microglia implies that the activating signals are transported along neuronal projections. However, the identity of these signals has not yet been identified. It is shown here that glutamate-treated neurons rapidly express and release the chemokine CCL21. We also provide evidence that neuronal CCL21 is packed in vesicles and transported throughout neuronal processes to reach presynaptic structures. Chemotaxis assays show that functional CCL21 is released from endangered neurons and activate microglia via the chemokine receptor CXCR3. Based on these findings, we suggest that neuronal CCL21 is important in directed neuron-microglia signaling and that this communication could account for the remote activation of microglia, far distant from a primary lesion.
Neurons are highly polarized cells, and neuron-neuron communication is based on directed transpor... more Neurons are highly polarized cells, and neuron-neuron communication is based on directed transport and release of neurotransmitters, neuropeptides, and neurotrophins. Directed communication may also be attributed to neuron-microglia signaling, since neuronal damage can induce a microglia reaction at specific sites only. However, the mechanism underlying this site-specific microglia reaction is not yet understood. Neuronal CCL21 is a microglia-activating chemokine, which in brain is solely found in endangered neurons and is therefore a candidate for neuronmicroglia signaling. Here we present that neuronal CCL21 is sorted into large dense-core vesicles, the secretory granules of the regulated release pathway of neurons. Live-cell imaging studies show preferential sorting of CCL21-containing vesicles into axons, indicating its directed transport. Thus, mouse neurons express and transport a microglia activating factor very similar to signaling molecules used in neuron-neuron communication. These data show for the first time the directed transport of a microglia activating factor in neurons and corroborate the function of neuronal CCL21 in directed neuron-microglia communication.-Expression, transport, and axonal sorting of neuronal CCL21 in large dense-core vesicles. FASEB J. 22, 4136 -4145 (2008)
Up-regulation of P2X4 receptors in spinal cord microglia is crucial for tactile allodynia, an unt... more Up-regulation of P2X4 receptors in spinal cord microglia is crucial for tactile allodynia, an untreatable pathological pain reaction occurring after peripheral nerve injury. How nerve injury in the periphery leads to this microglia reaction in the dorsal horn of the spinal cord is not yet understood. It is shown here that CCL21 was rapidly expressed in injured small-sized primary sensory neurons and transported to their central terminals in the dorsal horn. Intrathecal administration of a CCL21-blocking antibody diminished tactile allodynia development in wildtype animals. Mice deficient for CCL21 did not develop any signs of tactile allodynia and failed to up-regulate microglial P2X4 receptor expression. Microglia P2X4 expression was enhanced by CCL21 application in vitro and in vivo. A single intrathecal injection of CCL21 to nerve-injured CCL21-deficient mice induced long-lasting allodynia that was undistinguishable from the wild-type response. This effect of CCL21 injection was strictly dependent on P2X4 receptor function. Since neuronal CCL21 is the earliest yet identified factor in the cascade leading to tactile allodynia, these findings may lead to a preventive therapy in neuropathic pain.
Sleep is a complex physiological process and still remains one of the great mysteries of science.... more Sleep is a complex physiological process and still remains one of the great mysteries of science. Over the past 10 y, genetic research has provided a new avenue to address the regulation and function of sleep. Gene loci that contribute quantitatively to sleep characteristics and variability have already been identified. However, up to now, a genetic basis has been established only for a few sleep disorders. Little is yet known about the genetic background of insomnia, one of the most common sleep disorders. According to the conceptualisation of the 3P model of insomnia, predisposing, precipitating and perpetuating factors contribute to the development and maintenance of insomnia. Growing evidence from studies of predisposing factors suggests a certain degree of heritability for insomnia and for a reactivity of sleep patterns to stressful events, explaining the emergence of insomnia in response to stressful life events. While a genetic susceptibility may modulate the impact of stress on the brain, this finding does not provide us with a complete understanding of the capacity of stress to produce long-lasting perturbations of brain and behaviour. Epigenetic geneeenvironment interactions have been identified just recently and may provide a more complex understanding of the genetic control of sleep and its disorders. It was recently hypothesised that stress-response-related brain plasticity might be epigenetically controlled and, moreover, several epigenetic mechanisms have been assumed to be involved in the regulation of sleep. Hence, it might be postulated that insomnia may be influenced by an epigenetic control process of both sleep mechanisms and stress-response-related geneeenvironment interactions having an impact on brain plasticity. This paper reviews the evidence for the genetic basis of insomnia and recent theories about epigenetic mechanisms involved in both sleep regulation and brain-stress response, leading to the hypothesis of an involvement of epigenetic mechanisms in the development and maintenance of insomnia.
It has been established recently that inflammation of the central nervous system (CNS) is accompa... more It has been established recently that inflammation of the central nervous system (CNS) is accompanied by an expression of chemokines within the CNS. Several lines of evidence suggest that chemokines within the CNS initiate and orchestrate the infiltration of the inflamed brain by blood leukocytes. In line with this assumption is the finding that glial cells expressing the chemokine ligands CXCL10 and CXCL9 accompany the presence of CXCR3 positive T-cells in the CNS in demyelinating diseases.
The inhibitory neuromodulator adenosine is released in the brain in high concentrations under con... more The inhibitory neuromodulator adenosine is released in the brain in high concentrations under conditions of exaggerated neuronal activity such as ischemia and seizures, or electroconvulsive treatment. By inhibiting neural overactivity, adenosine counteracts seizure activity and promotes neuronal survival. Since stimulation of adenosine A(2b) receptors on astrocytes induces increased synthesis and release of interleukin-6, which also exerts neuroprotective effects, we hypothesized that the effects of interleukin-6 and of adenosine might be related. We report here that stimulation with interleukin-6 of cultured astrocytes, of cultured organotypic brain slices from newborn rat cortex, and of freshly prepared brain slices from rat cortex induces a concentration- and time-dependent upregulation of adenosine A(1) receptor mRNA. This increased adenosine A(1) receptor mRNA expression is accompanied in astrocytes by an increase in adenosine A(1) receptor-mediated signaling via the phosphoinositide-dependent pathway. Since upregulation of adenosine A(1) receptors leads to increased neuroprotective effects of adenosine, we suggest that the neuroprotective actions of interleukin-6 and adenosine are related and might be mediated at least in part through upregulation of adenosine A(1) receptors. These results may be of relevance for a better understanding of neuroprotection in brain damage but also point to a potential impact of neuroprotection in the mechanisms of the antidepressive effects of chronic carbamazepine, electroconvulsive therapy, and sleep deprivation, which are all accompanied by adenosine A(1) receptor upregulation.
The anticonvulsant and antibipolar drug carbamazepine (CBZ) is known to act as a specific antagon... more The anticonvulsant and antibipolar drug carbamazepine (CBZ) is known to act as a specific antagonist at adenosine A 1 -receptors. After a 3-week application of CBZ, A 1receptors are upregulated in the rat brain. We have investigated the consequences of this upregulation for the A 1 -receptor-mediated signal transduction in primary astrocyte cultures from different regions of the rat brain. CBZ treatment for 10 days had no effect on adenosine A 1receptor mRNA expression in cultures with high basal A 1receptor mRNA levels, but increased A 1 -receptor mRNA in cultures exhibiting low basal A 1 -receptor mRNA levels.
It has been shown that neural stem cells (NSCs) migrate towards areas of brain injury or brain tu... more It has been shown that neural stem cells (NSCs) migrate towards areas of brain injury or brain tumours and that NSCs have the capacity to track infiltrating tumour cells. The possible mechanism behind the migratory behaviour of NSCs is not yet completely understood. As chemokines are involved in the migration of immune cells in the injured brain, they may also be involved in chemoattraction of NSCs towards a brain tumour. Methods: The expression profile of various chemokine receptors in NSCs, harvested from the subventricular zone of adult mice, was investigated by reverse transcriptasepolymerase chain reaction analysis. Furthermore, the functionality of the chemokine receptors was assessed in in vitro chemotaxis assays and calcium signalling experiments. To test the in vivo migration of NSCs, a syngeneic mouse model was developed, whereby a B16F10 mela-noma cell line was grafted into one hemisphere and later NSCs were grafted in the contralateral hemisphere. Furthermore, the expression of chemokines in this melanoma cell line was investigated. Results and conclusions: Adult mouse NSCs functionally express various chemokine receptors of which CXC chemokine receptor (CXCR)4 shows the highest mRNA levels and most pronounced functional responses in vitro. CXC chemokine ligand (CXCL)12, the ligand for CXCR4, is expressed by the melanoma cell line. In this mouse model for metastatic brain tumours, it is shown that NSCs express CXCR4 at their cell membranes while they migrate towards the tumour, which produces CXCL12. It is therefore suggested that the CXCR4/CXCL12 pathway plays a role in the mechanism underlying tumour-mediated attraction of NSCs.
Aging is associated with reduced function, degenerative changes, and increased neuroinflammation ... more Aging is associated with reduced function, degenerative changes, and increased neuroinflammation of the central nervous system (CNS). Increasing evidence suggests that changes in microglia cells contribute to the age-related deterioration of the CNS. The most prominent age-related change of microglia is enhanced sensitivity to inflammatory stimuli, referred to as priming. It is unclear if priming is due to intrinsic microglia ageing or induced by the ageing neural environment. We have studied this in Ercc1 mutant mice, a DNA repair-deficient mouse model that displays features of accelerated aging in multiple tissues including the CNS. In Ercc1 mutant mice, microglia showed hallmark features of priming such as an exaggerated response to peripheral lipopolysaccharide exposure in terms of cytokine expression and phagocytosis. Specific targeting of the Ercc1 deletion to forebrain neurons resulted in a progressive priming response in microglia exemplified by phenotypic alterations. Summarizing, these data show that neuronal genotoxic stress is sufficient to switch microglia from a resting to a primed state.
Microglia are crucial for immune responses in the brain. Although their origin from the yolk sac ... more Microglia are crucial for immune responses in the brain. Although their origin from the yolk sac has been recognized for some time, their precise precursors and the transcription program that is used are not known. We found that mouse microglia were derived from primitive c-kit + erythromyeloid precursors that were detected in the yolk sac as early as 8 d post conception. These precursors developed into CD45 + c-kit lo CX 3 CR1 − immature (A1) cells and matured into CD45 + c-kit − CX 3 CR1 + (A2) cells, as evidenced by the downregulation of CD31 and concomitant upregulation of F4/80 and macrophage colony stimulating factor receptor (MCSF-R). Proliferating A2 cells became microglia and invaded the developing brain using specific matrix metalloproteinases. Notably, microgliogenesis was not only dependent on the transcription factor Pu.1 (also known as Sfpi), but also required Irf8, which was vital for the development of the A2 population, whereas Myb, Id2, Batf3 and Klf4 were not required. Our data provide cellular and molecular insights into the origin and development of microglia.
Whereas chemokines are well known for their ability to induce cell migration, only recently it be... more Whereas chemokines are well known for their ability to induce cell migration, only recently it became evident that chemokines also control a variety of other cell functions and are versatile messengers in the interaction between a diversity of cell types. In the central nervous system (CNS), chemokines are generally found under both physiological and pathological conditions. Whereas many reports describe chemokine expression in astrocytes and microglia and their role in the migration of leukocytes into the CNS, only few studies describe chemokine expression in neurons. Nevertheless, the expression of neuronal chemokines and the corresponding chemokine receptors in CNS cells under physiological and pathological conditions indicates that neuronal chemokines contribute to CNS cell interaction. In this study, we review recent studies describing neuronal chemokine expression and discuss potential roles of neuronal chemokines in neuron-astrocyte, neuronmicroglia, and neuron-neuron interaction.
Background: Most of the known functions of microglia, including neurotoxic and neuroprotective pr... more Background: Most of the known functions of microglia, including neurotoxic and neuroprotective properties, are attributed to morphologically-activated microglia. Resting, ramified microglia are suggested to primarily monitor their environment including synapses. Here, we show an active protective role of ramified microglia in excitotoxicity-induced neurodegeneration.
Background: Microglia are considered a major target for modulating neuroinflammatory and neurodeg... more Background: Microglia are considered a major target for modulating neuroinflammatory and neurodegenerative disease processes. Upon activation, microglia secrete inflammatory mediators that contribute to the resolution or to further enhancement of damage in the central nervous system (CNS). Therefore, it is important to study the intracellular pathways that are involved in the expression of the inflammatory mediators. Particularly, the role of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and glycogen synthase kinase-3 (GSK-3) pathways in activated microglia is unclear. Thus, in the present study we investigated the role of Akt and its downstream pathways, GSK-3 and mTOR, in lipopolysaccharide (LPS)-activated primary rat microglia by pharmacological inhibition of these pathways in regard to the expression of cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1 (mPGES-1) and to the production of prostaglandin (PG) E 2 and PGD 2 . Findings: We show that inhibition of Akt by the Akt inhibitor X enhanced the production of PGE 2 and PGD 2 without affecting the expression of COX-2, mPGES-1, mPGES-2 and cytosolic prostaglandin E synthase (cPGES). Moreover, inhibition of GSK-3 reduced the expression of both COX-2 and mPGES-1. In contrast, the mTOR inhibitor rapamycin enhanced both COX-2 and mPGES-1 immunoreactivity and the release of PGE 2 and PGD 2 . Interestingly, NVP-BEZ235, a dual PI3K/mTOR inhibitor, enhanced COX-2 and reduced mPGES-1 immunoreactivity, albeit PGE 2 and PGD 2 levels were enhanced in LPS-stimulated microglia. However, this compound also increased PGE 2 in nonstimulated microglia.
Our understanding of microglia biology has significantly changed in the last couple of years. Ins... more Our understanding of microglia biology has significantly changed in the last couple of years. Instead of being predominantly detrimental cells showing a stereotypic activation pattern, microglia today are considered highly adaptive elements with many distinct phenotypes. Microglia activity is aimed to protect and to restore and only in case of uncontrolled or impaired microglia function these cells may have detrimental effects. The control of microglia activity is thus an important issue to understand. The family of chemokines are versatile signals specialized to control cell-cell interactions. Neurons express chemokines in a temporarily and spatially regulated manner and microglia respond to these messengers via the appropriate receptors. Due to these features are chemokines ideal messengers for the communication between neurons and microglia.
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Papers by Knut Biber