harald neumann

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Moreover, soluble polySia attenuates LPS-induced production of nitric oxide and proinflammatory cytokines.

Microglia, the immune cells of the CNS, are highly adaptive cells that can acquire different pro- and anti-inflammatory activation states with distinct functions in CNS homeostasis and pathologies.

To study microglial function in vitro, primary microglia or immortalized cell lines are commonly used. An alternative to these cells are embryonic stem cell-derived microglia ESdM.

ESdM have previously been shown to be very similar to primary microglia in terms of expression profiles and surface molecules. In this study, ESdM and primary microglia were treated with different inflammatory stimulants to analyze their ability to adopt different activation states.

Using quantitative real-time PCR, comparative transcriptomics, ELISA, and flow cytometry, we found that different activation states can be induced in ESdM, which are similar to those found in primary microglia.

These states are characterized by specific sets of inflammatory marker molecules and differential transcriptome signatures.

Our results show that ESdM are a valuable alternative cell model to study microglial functions and neuroinflammatory mechanisms.

Loss of the smallest subunit of cytochrome c oxidase, COX8A, causes Leigh-like syndrome and epilepsy. View largeDownload slide COX8A is the smallest subunit of cytochrome c oxidase, the terminal enzyme of the cellular respiration pathway.

Loss of wild-type COX8A reduces the stability of the entire enzyme complex. Anti-inflammatory activity of low molecular weight polysialic acid on human macrophages.

In addition, polySia avDP20 neutralized the LPS-triggered increase in macrophage phagocytosis, but did not affect basal phagocytosis or endocytosis.

Thus, treatment with polySia avDP20 might be a new anti-inflammatory therapeutic strategy that also prevents the oxidative burst of macrophages.

The complement system has been implicated in the removal of dysfunctional synapses and neurites during development and in disease processes in the mouse, but it is unclear how far the mouse data can be transferred to humans.

Here, we co-cultured macrophages derived from human THP1 monocytes and neurons derived from human induced pluripotent stem cells, to study the role of the complement system in a human model.

Components of the complement system were expressed by the human macrophages and human neuronal culture, while receptors of the complement cascade were expressed by human macrophages as shown via gene transcript analysis and flow cytometry.

We mimicked pathological conditions leading to an altered glycocalyx by treatment of human neurons with sialidases. Desialylated human neurites were opsonized by the complement component C1q.

Furthermore, human neurites with an intact sialic acid cap remained untouched, while desialylated human neurites were removed and ingested by human macrophages.

While blockage of the complement receptor 1 CD35 had no effect, blockage of CD11b as part of the complement receptor 3 CR3 reversed the effect on macrophage phagocytosis of desialylated human neurites.

Data demonstrate that in the human system sialylation of the neuronal glycocalyx serves as an inhibitory flag for complement binding and CR3-mediated phagocytosis by macrophages.

Microglia reactivity is a hallmark of neurodegenerative diseases. Microglial proliferation was investigated using flow cytometry, and their phagocytosis was monitored by the uptake of W photoreceptor debris.

This effect was replicated with murine embryonic stem cell-derived microglia ESdM and primary brain microglia.

AMWAP promoted the filopodia formation of microglia and increased the phagocytic uptake of apoptotic W photoreceptor cells.

AMWAP also induces a neuroprotective microglial phenotype with reduced neurotoxicity and increased phagocytosis. We therefore hypothesize that anti-inflammatory whey acidic proteins could have a therapeutic potential in neurodegenerative diseases of the brain and the retina.

In most, if not all, of these diseases, early activation of microglia occurs. Our knowledge regarding the factors triggering early microglia activation is, however, incomplete.

In this study, we used the cuprizone model to investigate the temporal and causal relationship of oligodendrocyte apoptosis and early microglia activation.

Genome-wide gene expression studies revealed the induction of distinct chemokines, among them Cxcl10, Ccl2, and Ccl3 in cuprizone-mediated oligodendrocyte apoptosis.

Early microglia activation was unchanged in CCL2- and CCL3-deficient knockouts, but was significantly reduced in CXCLdeficient mice, resulting in an amelioration of cuprizone toxicity at later time points.

Subsequent in vitro experiments revealed that recombinant CXCL10 induced migration and a proinflammatory phenotype in cultured microglia, without affecting their phagocytic activity or proliferation.

In situ hybridization analyses suggest that Cxcl10 mRNA is mainly expressed by astrocytes, but also oligodendrocytes, in short-term cuprizone-exposed mice.

Our results show that CXCL10 actively participates in the initiation of microglial activation. These findings have implications for the role of CXCL10 as an important mediator during the initiation of neuroinflammatory processes associated with oligodendrocyte pathology.

Polysialic acid and use for treatment of neurodegenerative and neuroinflammatory diseases. Systemic inflammatory reactions have been postulated to exacerbate neurodegenerative diseases via microglial activation.

We now demonstrate in vivo that repeated systemic challenge of mice over four consecutive days with bacterial LPS maintained an elevated microglial inflammatory phenotype and induced loss of dopaminergic neurons in the substantia nigra.

The same total cumulative LPS dose given within a single application did not induce neurodegeneration. Whole-genome transcriptome analysis of the brain demonstrated that repeated systemic LPS application induced an activation pattern involving the classical complement system and its associated phagosome pathway.

Loss of dopaminergic neurons induced by repeated systemic LPS application was rescued in complement C3-deficient mice, confirming the involvement of the complement system in neurodegeneration.

Our data demonstrate that a phagosomal inflammatory response of microglia is leading to complement-mediated loss of dopaminergic neurons.

Sensing the neuronal glycocalyx by glial sialic acid binding immunoglobulin-like lectins. Sialic acid binding immunoglobulin-like lectins Siglecs are cell surface receptors of microglia and oligodendrocytes that recognize the sialic acid cap of healthy neurons and neighboring glial cells.

Upon ligand binding, Siglecs typically signal through an immunoreceptor tyrosine based inhibition motif ITIM to keep the cell in a homeostatic status and support healthy neighboring cells.

Siglecs can be divided into two groups; the first, being conserved among different species. Siglec-4 protects neurons from acute toxicity via interaction of sialic acids bound to neuronal gangliosides.

The second group of Siglecs, named CDrelated Siglecs, is almost exclusively expressed on immune cells and is highly variable among different species.

Microglial Siglec-E is a mouse CDrelated Siglec member that prevents microglial phagocytosis and the associated oxidative burst.

Thus, glial ITIM-signaling Siglecs recognize the intact glycocalyx of neurons and are involved in the modulation of neuron-glial interaction in healthy and diseased brain.

Siglec functions of microglia. Microglia are the resident immune cells of the central nervous system.

They can sense intact or lesioned cells and then respond in an appropriate way. Therefore, microglia need recognition receptors that either lead to the activation or inhibition of the immune response pathways.

Most Siglecs contain an immunoreceptor tyrosine based inhibition motif and its signaling leads to the termination of signals emerging from immunoreceptor tyrosine based activation motif signaling receptors.

Pro-inflammatory immune responses and phagocytosis are turned down in microglia by inhibitory Siglec signaling.

Recently, it was demonstrated that inhibitory Siglecs have neuroprotective effects on cultured neurons by preventing the phagocytosis associated oxidative burst.

Furthermore, microglial mouse Siglec-E and human Siglec have been shown to prevent neurotoxicity via interaction with sialic acid exposed on the neuronal glycocalyx.

Thus, Siglecs sensing the intact glycocalyx of neighboring cells keep microglia in a silent homeostatic status. Translocator protein 18 kDa TSPO is expressed in reactive retinal microglia and modulates microglial inflammation and phagocytosis.

The translocator protein 18 kDa TSPO is a mitochondrial protein expressed on reactive glial cells and a biomarker for gliosis in the brain.

TSPO ligands have been shown to reduce neuroinflammation in several mouse models of neurodegeneration.

TSPO protein analyses were performed in retinoschisin-deficient mouse retinas and human retinas. The migration potential of microglia was determined with wound healing assays and the proliferation was studied with Fluorescence Activated Cell Sorting FACS analysis.

The morphology of microglia was quantified in mouse retinal explants treated with XBD TSPO was strongly up-regulated in microglial cells of the dystrophic mouse retina and also co-localized with microglia in human retinas.

Constitutive TSPO expression was high in the early postnatal Day 3 mouse retina and declined to low levels in the adult tissue. Moreover, treatment with XBD significantly reduced the migratory capacity and proliferation of microglia, their level of NO secretion and their neurotoxic activity on W photoreceptor cells.

Furthermore, XBD treatment of murine and human microglial cells promoted the formation of filopodia and increased their phagocytic capacity to ingest latex beads or photoreceptor debris.

Finally, treatment with XBD reversed the amoeboid alerted phenotype of microglial cells in explanted organotypic mouse retinal cultures after challenge with LPS.

These findings suggest that TSPO is highly expressed in reactive retinal microglia and a promising target to control microglial reactivity during retinal degeneration.

Sialic acid-binding Ig-like lectins Siglecs are members of the Ig superfamily that recognize sialic acid residues of glycoproteins.

Siglec-E is a mouse CDrelated Siglec that preferentially binds to sialic acid residues of the cellular glycocalyx. Here, we demonstrate gene transcription and protein expression of Siglec-E by cultured mouse microglia.

Siglec-E on microglia inhibited phagocytosis of neural debris and prevented the production of superoxide radicals induced by challenge with neural debris.

Soluble extracellular Siglec-E receptor protein bound to the neural glycocalyx. Coculture of mouse microglia and neurons demonstrated a neuroprotective effect of microglial Siglec-E that was dependent on neuronal sialic acid residues.

Increased neurotoxicity of microglia after knockdown of Siglece mRNA was neutralized by the reactive oxygen species scavenger Trolox.

Data suggest that Siglec-E recognizes the intact neuronal glycocalyx and has neuroprotective function by preventing phagocytosis and the associated oxidative burst.

Here, we demonstrate that TREM2 undergoes sequential proteolytic processing by ectodomain shedding and intramembrane proteolysis. Unique transcriptome signature of mouse microglia.

Microglial cells can be derived directly from the dissociated brain tissue by sorting procedures, from postnatal glial cultures by mechanic isolation or from pluripotent stem cells by differentiation.

The detailed molecular phenotype of microglia from different sources is still unclear. Here, we performed a whole transcriptome analysis of flow cytometry-sorted microglia, primary postnatal cultured microglia, embryonic stem cell derived microglia ESdM , and other cell types.

Microglia and ESdM, both cultured in serum-free medium, were closely related to sorted microglia and showed a unique transcriptome profile, clearly distinct to other myeloid cell types, T cells, astrocytes, and neurons.

ESdM and primary cultured microglia showed strong overlap in their transcriptome. Only genes were differentially expressed between both cell types, mainly derived from immune-related genes with a higher activation status of proinflammatory and immune defense genes in primary microglia compared to ESdM.

Flow cytometry analysis of cell surface markers CD54, CD74, and CD selected from the microarray confirmed the close phenotypic relation between ESdM and primary cultured microglia.

Thus, assessment of genome-wide transcriptional regulation demonstrates that microglial cells are unique and clearly distinct from other macrophage cell types.

Siglec-h on activated microglia for recognition and engulfment of glioma cells. Expression of Siglec-h has been observed on plasmacytoid dendritic cells and microglia, but the ligand and the function of Siglec-h remained elusive.

Microglial Siglec-h acted as phagocytosis receptor since targeting of microsphere beads to Siglec-h triggered their uptake into the microglia.

The extracellular domain of Siglec-h protein bound to mouse glioma lines, but not to astrocytes or other normal mouse cells.

Microglial cells stimulated to express Siglec-h engulfed intact glioma cells without prior induction of apoptosis and slightly reduced glioma cell number in culture.

Phagocytosis of glioma cells by activated microglia was dependent on Siglec-h and its adapter molecule DAP Thus, data show that M1-polarized microglial cells can engulf glioma cells via a DAPmediated Siglec-h dependent mechanism.

We propose here that activation of the innate immune system by stimulation of innate immune receptors involved in antiviral and antitumor responses can similarly target different malignant populations of glioma cells.

We used short-term expanded patient-specific primary human GBM cells to study the stimulation of the cytosolic nucleic acid receptors melanoma differentiation-associated gene 5 MDA5 and retinoic acid-inducible gene I RIG-I.

Specifically, we analyzed cells from the tumor core vs. A portfolio of human, non-tumor neural cells was used as a control for these studies.

Receptor stimulation with their respective ligands, p I: C and 3pRNA, led to in vitro evidence for an effective activation of the innate immune system.

Most intriguingly, all investigated cancer cell populations additionally responded with a pronounced induction of apoptotic signaling cascades revealing a second, direct mechanism of antitumor activity.

By contrast, p I: C and 3pRNA induced only little toxicity in human non-malignant neural cells. Granted that the challenge of effective CNS delivery can be overcome, targeting of RIG-I and MDA5 could thus become a quintessential strategy to encounter heterogeneous cancers in the sophisticated environments of the brain.

The genetics of complex disease produce alterations in the molecular interactions of cellular pathways whose collective effect may become clear through the organized structure of molecular networks.

To characterize molecular systems associated with late-onset Alzheimer's disease LOAD , we constructed gene-regulatory networks in 1, postmortem brain tissues from LOAD patients and nondemented subjects, and we demonstrate that LOAD reconfigures specific portions of the molecular interaction structure.

Through an integrative network-based approach, we rank-ordered these network structures for relevance to LOAD pathology, highlighting an immune- and microglia-specific module that is dominated by genes involved in pathogen phagocytosis, contains TYROBP as a key regulator, and is upregulated in LOAD.

Thus the causal network structure is a useful predictor of response to gene perturbations and presents a framework to test models of disease mechanisms underlying LOAD.

Microglia have been regarded as the tissue macrophages of the brain. A study now finds that microglia are quite distinct from blood-borne macrophages and derive from an erythromyeloid precursor cell of the embryonic hematopoiesis.

Beneficial and detrimental consequences of microglial phagocytosis. Microglia are the resident brain macrophages and they have been traditionally studied as orchestrators of the brain inflammatory response during infections and disease.

In addition, microglia has a more benign, less explored role as the brain professional phagocytes. Phagocytosis is a term coined from the Greek to describe the receptor-mediated engulfment and degradation of dead cells and microbes.

Common mechanisms of recognition, engulfment, and degradation of the different types of cargo are assumed, but very little is known about the shared and specific molecules involved in the phagocytosis of each target by microglia.

More importantly, the functional consequences of microglial phagocytosis remain largely unexplored. Overall, phagocytosis is considered a beneficial phenomenon, since it eliminates dead cells and induces an anti-inflammatory response.

However, phagocytosis can also activate the respiratory burst, which produces toxic reactive oxygen species ROS.

Phagocytosis has been traditionally studied in pathological conditions, leading to the assumption that microglia have to be activated in order to become efficient phagocytes.

Recent data, however, has shown that unchallenged microglia phagocytose apoptotic cells during development and in adult neurogenic niches, suggesting an overlooked role in brain remodeling throughout the normal lifespan.

The present review will summarize the current state of the literature regarding the role of microglial phagocytosis in maintaining tissue homeostasis in health as in disease.

Engineered stem cell-derived microglia as therapeutic vehicle for experimental autoimmune encephalomyelitis. Inflammation can be prevented in most inflammatory brain diseases, while tissue repair of the lesioned central nervous system CNS is still a major challenge.

The CNS is difficult to access for protein therapeutics due to the blood-brain barrier. Here, we show that genetically engineered embryonic stem cell-derived microglia ESdM are a suitable therapeutic vehicle for neurotrophin-3 NT3 in experimental autoimmune encephalomyelitis EAE.

The intravenously transplanted ESdM migrated into the inflammatory CNS lesions and engrafted there as microglial cells.

The NT3-transduced ESdM created an anti-inflammatory cytokine milieu in the spinal cord and promoted neuronal sprouting.

Furthermore, mice treated with NT3-transduced ESdM showed less axonal injury and reduced demyelination. Thus, genetically modified ESdM represent a suitable tool to introduce therapeutic neuroprotective and repair-promoting proteins into the CNS in neuroinflammatory diseases.

Gene Therapy advance online publication, 17 January ; doi: Triggering receptor expressed on myeloid cells-2 TREM2 is a microglial surface receptor involved in phagocytosis.

Clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery.

The role of TREM2 following stroke is currently unclear. As an experimental stroke model, the middle cerebral artery of mice was occluded for 30 minutes with a range of reperfusion times duration of reperfusion: Functional consequences of TREM2 knockout were assessed by infarct volumetry.

The acute inflammatory response 12 h reperfusion was very similar between TREM2-KO mice and their littermate controls. No effect on the lesion size was observed.

Although we initially expected an exaggerated pro-inflammatory response following ablation of TREM2, our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in TREM2-KO mice.

We therefore conclude that TREM2 appears to sustain a distinct inflammatory response after stroke. No change in the basal gene transcription of inflammatory mediators in the brain of TREM2 deficient mice.

Microglial activatory immunoreceptor tyrosine-based activation motif - and inhibitory immunoreceptor tyrosine-based inhibition motif -signaling receptors for recognition of the neuronal glycocalyx.

Microglia sense intact or lesioned cells of the central nervous system CNS and respond accordingly. To fulfill this task, microglia express a whole set of recognition receptors.

Those receptors are counter-regulated by immunoreceptor tyrosine-based inhibition motif ITIM -signaling receptors, such as sialic acid-binding immunoglobulin superfamily lectins Siglecs.

Siglecs recognize the sialic acid cap of healthy neurons thus leading to an ITIM signaling that turns down microglial immune responses and phagocytosis.

In contrast, desialylated neuronal processes are phagocytosed by microglial CR3 signaling via an adaptor protein containing an ITAM.

Thus, the aberrant terminal glycosylation of neuronal surface glycoproteins and glycolipids could serve as a flag for microglia, which display a multitude of diverse carbohydrate-binding receptors that monitor the neuronal physical condition and respond via their ITIM- or ITAM-signaling cascade accordingly.

Current knowledge about the pathogenic mechanism of Alzheimer's disease is based mainly on rare, high-penetrance variants in genes encoding amyloid precursor protein, presenilin 1, and presenilin 2, which result in familial early-onset Alzheimer's disease.

However, Alzheimer's disease is predominantly a sporadic late-onset disease with exponentially increasing prevalence starting at the age of 65 years.

Licensing of myeloid cells promotes central nervous system autoimmunity and is controlled by peroxisome proliferator-activated receptor gamma.

During central nervous system autoimmunity, interactions between infiltrating immune cells and brain-resident cells are critical for disease progression and ultimately organ damage.

Here, we demonstrate that local cross-talk between invading autoreactive T cells and auto-antigen-presenting myeloid cells within the central nervous system results in myeloid cell activation, which is crucial for disease progression during experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis.

Together, interference with the disease-promoting cross-talk between central nervous system myeloid cells, autoreactive T cells and brain-resident cells represents a novel therapeutic approach that limits disease progression and lesion development during ongoing central nervous system autoimmunity.

Microglial carbohydrate-binding receptors for neural repair. Microglia are the resident immune cells of the central nervous system CNS and perform typical scavenging and innate immune functions.

Their capacity to eliminate extracellular aggregates and apoptotic neural material without inflammation is crucial for brain tissue homeostasis and repair.

To fulfill these tasks, microglia express a whole set of recognition receptors including toll-like TLRs , carbohydrate-binding, Fc, complement and cytokine receptors.

Receptors recognizing carbohydrate structures are strongly involved in microglial repair function.

Carbohydrate-binding receptors can be divided into two major subgroups: Siglecs bind to the sialic acid cap of the intact glycocalyx. Other lectin family members such as galectins recognize lactosamine units typically exposed after alteration of the glycocalyx.

Dependent on the type of microglial carbohydrate-binding receptors that are stimulated, either a pro-inflammatory cytotoxic or an anti-inflammatory repair-promoting response is evoked.

The carbohydrate-binding receptors are also crucial in regulating microglial function such as phagocytosis during neurodegenerative or neuroinflammatory processes.

A balance between microglial carbohydrate-binding receptor signaling via an immunoreceptor tyrosine-based activation motif or an immunoreceptor tyrosine-based inhibitory motif is required to polarize microglial cells appropriately so that they create a microenvironment permissive for neural regenerative events.

Microglial cells are professional phagocytes of the CNS responsible for clearance of unwanted structures. Neuronal processes are marked by complement C1 before they are removed in development or during disease processes.

Target molecules involved in C1 binding and mechanisms of clearance are still unclear. Here we show that the terminal sugar residue sialic acid of the mouse neuronal glycocalyx determines complement C1 binding and microglial-mediated clearance function.

Several early components of the classical complement cascade including C1q, C1r, C1s, and C3 were produced by cultured mouse microglia.

The opsonin C1q was binding to neurites after enzymatic removal of sialic acid residues from the neuronal glycocalyx.

Desialylated neurites, but not neurites with intact sialic acid caps, were cleared and taken up by cocultured microglial cells. Data demonstrate that mouse microglial cells via CR3 recognize and remove neuronal structures with an altered neuronal glycocalyx lacking terminal sialic acid.

Spatially pathogenic forms of tau detected in Alzheimer's disease brain tissue by fluorescence lifetime-based Forster resonance energy transfer. In tauopathies including Alzheimer's disease AD tau molecules have lost their normal spatial distance to each other and appear in oligomeric or aggregated forms.

Conventional immunostaining methods allow detection of abnormally phosphorylated or conformationally altered aggregated tau proteins, but fail to visualize oligomeric forms of tau.

Here we show that tau molecules that lost their normal spatial localization can be detected on a subcellular level in postmortem central nervous system CNS tissue sections of AD patients by fluorescence lifetime-based Förster resonance energy transfer FRET.

Paraffin sections were co-immunostained with two tau-specific monoclonal antibodies recognizing the same epitope, but labeled with distinct fluorescence dyes suitable for spatial resolution at a nanometer scale by lifetime-based FRET.

A FRET signal was detected in neuritic plaques and neurofibrillary tangles of CNS tissue sections of AD patients, showing associated tau proteins typically reflecting either fibrillary, oligomeric or aggregated tau.

The 'pretangle-like' structures within the neuronal perikarya did not contain spatially pathogenic forms of tau accordingly to this method.

Data demonstrate that fluorescence lifetime-based FRET can be applied to human brain tissue sections to detect pathogenic forms of tau molecules that lost their normal spatial distance.

Protective effects of microGlia in multiple sclerosis. The role of microglia in demyelinating neurodegenerative diseases such as multiple sclerosis MS and its animal model experimental autoimmune encephalomyelitis EAE is still controversial.

Although microglial cells are known as the professional phagocytes and executer of innate immunity in the central nervous system CNS , it is believed that microglia are rather neurotoxic in these diseases.

However, there is recent evidence indicating that microglia could also exert a neuroprotective function in MS and EAE.

First evidence for the protective effect of immune cells in CNS diseases emerged from studies in invertebrates. In the medicinal leech, the process of regeneration begins with rapid activation and accumulation of phagocytic glial cells at the lesion site followed by phagocytosis of damaged tissue by these cells which promoted robust neural regeneration.

In vertebrates, several lines of evidence demonstrate that microglia are also involved in neuroprotection by the secretion of soluble mediators that trigger neural repair and usually contribute to the creation of an environment conductive for regeneration.

The efficient removal of apoptotic cells and clearance of debris at the lesion site and the recruitment of stem cell populations as well as the induction of neurogenesis are directly correlated.

These findings suggest that microglia play a major role in creating a microenvironment for repair and regenerative processes in demyelinating neuroinflammatory diseases.

Siglec-F receptor and neuroprotection in mouse central nervous system. Sep Pharmacological Reports. Generation of microglial cells from mouse embryonic stem cells.

Microglia, the resident immune cells of the brain, are difficult to obtain in high numbers and purity using currently available methods; to date, microglia for experimental research are mainly isolated from the brain or from mixed glial cultures.

In this paper, we describe a basic protocol for the in vitro differentiation of mouse embryonic stem ES cells into microglial precursor cells.

Microglia are obtained by a protocol consisting of five stages: The protocol can be completed in 60 d and results in stably proliferating ESdM lines, which show inducible transcription of inflammatory genes and cell marker expression comparable with primary microglia.

Furthermore, ESdMs are capable of chemokine-directed migration and phagocytosis, which are major functional features of microglia. Functional role of brain-derived neurotrophic factor in neuroprotective autoimmunity: Therapeutic implications in a model of multiple sclerosis.

Brain-derived neurotrophic factor plays a key role in neuronal and axonal survival. Brain-derived neurotrophic factor is expressed in the immune cells in lesions of experimental autoimmune encephalomyelitis and multiple sclerosis, thus potentially mediating neuroprotective effects.

We investigated the functional role of brain-derived neurotrophic factor in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis.

Mice deficient for brain-derived neurotrophic factor in immune cells displayed an attenuated immune response in the acute phase of experimental autoimmune encephalomyelitis, but progressive disability with enhanced axonal loss in the chronic phase of the disease.

In mice deficient for central nervous system-derived brain-derived neurotrophic factor via glial fibrillary acidic protein-crescentin-mediated deletion, a more severe course of experimental autoimmune encephalomyelitis and an overall increased axonal loss was observed.

In a lentiviral approach, injection of brain-derived neurotrophic factor-overexpressing T cells led to a less severe course of experimental autoimmune encephalomyelitis and direct axonal protection.

Our data imply a functional role of brain-derived neurotrophic factor in autoimmune demyelination by mediating axon protection.

Vaccine-based autoimmune anti-amyloid treatments are currently being examined for their therapeutic potential in Alzheimer's disease.

In the present study we examined, in a transgenic model of amyloid pathology, the expression of two molecules previously implicated in decreasing the severity of autoimmune responses: TREM2 triggering receptor expressed on myeloid cells 2 and the intracellular tolerance-associated transcript, Tmemb transmembrane domain protein b.

In situ hybridization analysis revealed that both molecules were highly expressed in plaque-associated microglia, but their expression defined two different zones of plaque-associated activation.

Tmemb expression was highest in the inner zone of amyloid plaques, whereas TREM2 expression was highest in the outer zone. Induced expression of TREM2 occurred co-incident with detection of thioflavine-S-positive amyloid deposits.

TREM2 expression also showed a positive correlation with amyloid phagocytosis in unactivated cells.

Transfection of Tmemb into both microglial and macrophage cell lines increased apoptosis. Elimination of extracellular aggregates and apoptotic neural membranes without inflammation is crucial for brain tissue homeostasis.

In the mammalian central nervous system, essential molecules in this process are the Fc receptors and the DAPassociated receptors which both trigger the microglial immunoreceptor tyrosine-based activation motif- ITAM- Syk-signaling cascade.

Microglial ITAM-signaling receptors are counter-regulated by immunoreceptor tyrosine-based inhibition motif- ITIM- signaling molecules such as sialic acid-binding immunoglobulin superfamily lectins Siglecs.

Siglecs can suppress the proinflammatory and phagocytic activity of microglia via ITIM signaling. Moreover, microglial neurotoxicity is alleviated via interaction of Siglec with sialic acids on the neuronal glycocalyx.

Their dysfunction could lead to impaired phagocytic clearance and neurodegeneration triggered by chronic inflammation.

Alleviation of Neurotoxicity by Microglial Human Siglec Sialic acid-binding Ig superfamily lectins Siglecs are members of the Ig superfamily that recognize sialic acid residues of glycoproteins.

Siglec is a recently identified human-specific CDrelated Siglec that binds to alpha2,8-linked polysialic acids and is expressed on microglia, the brain resident innate immune cells.

We observed gene transcription and protein expression of Siglec splice variant 2 in human brain tissue samples by RT-PCR and Western blot analysis.

Siglec was detected on microglia in human brain tissue by immunohistochemistry. Human Siglec splice variant 2 was ectopically expressed by a lentiviral vector system in cultured murine microglial cells.

Stimulation of Siglec by cross-linking suppressed the lipopolysaccharides LPS -induced gene transcription of the proinflammatory mediators interleukin-1beta and nitric oxide synthase-2 in microglia.

Furthermore, phagocytosis of apoptotic neuronal material was reduced in Siglec transduced microglia. Coculture of microglia transduced with Siglec and neurons demonstrated neuroprotective function of Siglec Thus, data demonstrate that human Siglec ectopically expressed on murine microglia interacts with PSA on neurons, reduces LPS-induced gene transcription of proinflammatory mediators, impairs phagocytosis and alleviates microglial neurotoxicity.

Microglial precursors derived from mouse embryonic stem cells. Microglia are resident immune cells of the central nervous system.

They can be directly isolated from the brain or from mixed postnatal glial cultures. Isolation of primary microglia is inefficient due to low yield.

The cell line BV2 was used as a substitute for primary microglia, but BV2 are oncogenically transformed cells. Here, we established a protocol to generate microglial precursor lines from mouse embryonic stem ES cells.

Microglial precursor cells were obtained from murine ES cells by differentiation of embryoid bodies to microglia within a mixed brain culture.

Several independent ES cell-derived microglial precursor ESdM lines were generated and characterized by flow cytometry, immunocytochemistry, and functional assays.

Stimulation with interferon-gamma or lipopolysaccharide LPS demonstrated upregulation of proinflammatory cytokine gene transcription including nitric oxide synthase-2, interleukin-1beta, and tumor necrosis factor-alpha at levels comparable to primary microglia.

The ESdM showed efficient and rapid phagocytosis of microsphere beads, which was increased after stimulation with LPS.

After in vivo transplantation into postnatal brain tissue, ESdM showed engraftment as cells with a microglial phenotype and morphology.

Thus, ESdM are stable proliferating cells substantially having most characteristics of primary microglia and therefore being a suitable tool to study microglial function in vitro and in vivo.

Signal Regulatory Protein 1: The signal regulatory protein-beta1 SIRPbeta1 is a DAPassociated transmembrane receptor expressed in a subset of hematopoietic cells.

Recently, it was shown that peritoneal macrophages express SIRPbeta1, which positively regulated phagocytosis. Activation of SIRPbeta1 on cultured microglia by cross-linking antibodies induced reorganization of the cytoskeleton protein beta-actin and suppressed lipopolysaccharide-induced gene transcription of tumor necrosis factor-alpha and nitric oxide synthase Furthermore, activation of SIRPbeta1 increased phagocytosis of microsphere beads, neural debris, and fibrillary amyloid-beta Abeta.

Phagocytosis of neural cell debris and Abeta was impaired after lentiviral knockdown of SIRPbeta1 in primary microglial cells. Role of microglia in neuronal degeneration and regeneration.

Microglial cells, the resident macrophage population of the central nervous system CNS , actively scan tissue under both normal and pathologic contexts.

Their resulting engagement can become either neuroprotective or neurotoxic, leading to amelioration or aggravation of disease progression.

In this review, we focus on the molecular signaling molecules involved in microglial responses and discuss observations demonstrating the diverse effects of microglia in animal models of CNS diseases.

Regenerative therapy of experimental autoimmune encephalomyelitis by neurotrophin-3 transduced ES cell derived microglial cells.

Sep 25th Congress of the. Accumulation of tau induced in neurites by microglial proinflammatory mediators. More importantly, the functional consequences of microglial phagocytosis remain largely unexplored.

Overall, phagocytosis is considered a beneficial phenomenon, since it eliminates dead cells and induces an anti-inflammatory response.

However, phagocytosis can also activate the respiratory burst, which produces toxic reactive oxygen species ROS.

Phagocytosis has been traditionally studied in pathological conditions, leading to the assumption that microglia have to be activated in order to become efficient phagocytes.

Recent data, however, has shown that unchallenged microglia phagocytose apoptotic cells during development and in adult neurogenic niches, suggesting an overlooked role in brain remodeling throughout the normal lifespan.

The present review will summarize the current state of the literature regarding the role of microglial phagocytosis in maintaining tissue homeostasis in health as in disease.

Engineered stem cell-derived microglia as therapeutic vehicle for experimental autoimmune encephalomyelitis. Inflammation can be prevented in most inflammatory brain diseases, while tissue repair of the lesioned central nervous system CNS is still a major challenge.

The CNS is difficult to access for protein therapeutics due to the blood-brain barrier. Here, we show that genetically engineered embryonic stem cell-derived microglia ESdM are a suitable therapeutic vehicle for neurotrophin-3 NT3 in experimental autoimmune encephalomyelitis EAE.

The intravenously transplanted ESdM migrated into the inflammatory CNS lesions and engrafted there as microglial cells.

The NT3-transduced ESdM created an anti-inflammatory cytokine milieu in the spinal cord and promoted neuronal sprouting. Furthermore, mice treated with NT3-transduced ESdM showed less axonal injury and reduced demyelination.

Thus, genetically modified ESdM represent a suitable tool to introduce therapeutic neuroprotective and repair-promoting proteins into the CNS in neuroinflammatory diseases.

Gene Therapy advance online publication, 17 January ; doi: Triggering receptor expressed on myeloid cells-2 TREM2 is a microglial surface receptor involved in phagocytosis.

Clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery.

The role of TREM2 following stroke is currently unclear. As an experimental stroke model, the middle cerebral artery of mice was occluded for 30 minutes with a range of reperfusion times duration of reperfusion: Functional consequences of TREM2 knockout were assessed by infarct volumetry.

The acute inflammatory response 12 h reperfusion was very similar between TREM2-KO mice and their littermate controls.

No effect on the lesion size was observed. Although we initially expected an exaggerated pro-inflammatory response following ablation of TREM2, our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in TREM2-KO mice.

We therefore conclude that TREM2 appears to sustain a distinct inflammatory response after stroke. No change in the basal gene transcription of inflammatory mediators in the brain of TREM2 deficient mice.

Microglial activatory immunoreceptor tyrosine-based activation motif - and inhibitory immunoreceptor tyrosine-based inhibition motif -signaling receptors for recognition of the neuronal glycocalyx.

Microglia sense intact or lesioned cells of the central nervous system CNS and respond accordingly. To fulfill this task, microglia express a whole set of recognition receptors.

Those receptors are counter-regulated by immunoreceptor tyrosine-based inhibition motif ITIM -signaling receptors, such as sialic acid-binding immunoglobulin superfamily lectins Siglecs.

Siglecs recognize the sialic acid cap of healthy neurons thus leading to an ITIM signaling that turns down microglial immune responses and phagocytosis.

In contrast, desialylated neuronal processes are phagocytosed by microglial CR3 signaling via an adaptor protein containing an ITAM.

Thus, the aberrant terminal glycosylation of neuronal surface glycoproteins and glycolipids could serve as a flag for microglia, which display a multitude of diverse carbohydrate-binding receptors that monitor the neuronal physical condition and respond via their ITIM- or ITAM-signaling cascade accordingly.

Current knowledge about the pathogenic mechanism of Alzheimer's disease is based mainly on rare, high-penetrance variants in genes encoding amyloid precursor protein, presenilin 1, and presenilin 2, which result in familial early-onset Alzheimer's disease.

However, Alzheimer's disease is predominantly a sporadic late-onset disease with exponentially increasing prevalence starting at the age of 65 years.

Licensing of myeloid cells promotes central nervous system autoimmunity and is controlled by peroxisome proliferator-activated receptor gamma.

During central nervous system autoimmunity, interactions between infiltrating immune cells and brain-resident cells are critical for disease progression and ultimately organ damage.

Here, we demonstrate that local cross-talk between invading autoreactive T cells and auto-antigen-presenting myeloid cells within the central nervous system results in myeloid cell activation, which is crucial for disease progression during experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis.

Together, interference with the disease-promoting cross-talk between central nervous system myeloid cells, autoreactive T cells and brain-resident cells represents a novel therapeutic approach that limits disease progression and lesion development during ongoing central nervous system autoimmunity.

Microglial carbohydrate-binding receptors for neural repair. Microglia are the resident immune cells of the central nervous system CNS and perform typical scavenging and innate immune functions.

Their capacity to eliminate extracellular aggregates and apoptotic neural material without inflammation is crucial for brain tissue homeostasis and repair.

To fulfill these tasks, microglia express a whole set of recognition receptors including toll-like TLRs , carbohydrate-binding, Fc, complement and cytokine receptors.

Receptors recognizing carbohydrate structures are strongly involved in microglial repair function. Carbohydrate-binding receptors can be divided into two major subgroups: Siglecs bind to the sialic acid cap of the intact glycocalyx.

Other lectin family members such as galectins recognize lactosamine units typically exposed after alteration of the glycocalyx.

Dependent on the type of microglial carbohydrate-binding receptors that are stimulated, either a pro-inflammatory cytotoxic or an anti-inflammatory repair-promoting response is evoked.

The carbohydrate-binding receptors are also crucial in regulating microglial function such as phagocytosis during neurodegenerative or neuroinflammatory processes.

A balance between microglial carbohydrate-binding receptor signaling via an immunoreceptor tyrosine-based activation motif or an immunoreceptor tyrosine-based inhibitory motif is required to polarize microglial cells appropriately so that they create a microenvironment permissive for neural regenerative events.

Microglial cells are professional phagocytes of the CNS responsible for clearance of unwanted structures.

Neuronal processes are marked by complement C1 before they are removed in development or during disease processes.

Target molecules involved in C1 binding and mechanisms of clearance are still unclear. Here we show that the terminal sugar residue sialic acid of the mouse neuronal glycocalyx determines complement C1 binding and microglial-mediated clearance function.

Several early components of the classical complement cascade including C1q, C1r, C1s, and C3 were produced by cultured mouse microglia.

The opsonin C1q was binding to neurites after enzymatic removal of sialic acid residues from the neuronal glycocalyx. Desialylated neurites, but not neurites with intact sialic acid caps, were cleared and taken up by cocultured microglial cells.

Data demonstrate that mouse microglial cells via CR3 recognize and remove neuronal structures with an altered neuronal glycocalyx lacking terminal sialic acid.

Spatially pathogenic forms of tau detected in Alzheimer's disease brain tissue by fluorescence lifetime-based Forster resonance energy transfer.

In tauopathies including Alzheimer's disease AD tau molecules have lost their normal spatial distance to each other and appear in oligomeric or aggregated forms.

Conventional immunostaining methods allow detection of abnormally phosphorylated or conformationally altered aggregated tau proteins, but fail to visualize oligomeric forms of tau.

Here we show that tau molecules that lost their normal spatial localization can be detected on a subcellular level in postmortem central nervous system CNS tissue sections of AD patients by fluorescence lifetime-based Förster resonance energy transfer FRET.

Paraffin sections were co-immunostained with two tau-specific monoclonal antibodies recognizing the same epitope, but labeled with distinct fluorescence dyes suitable for spatial resolution at a nanometer scale by lifetime-based FRET.

A FRET signal was detected in neuritic plaques and neurofibrillary tangles of CNS tissue sections of AD patients, showing associated tau proteins typically reflecting either fibrillary, oligomeric or aggregated tau.

The 'pretangle-like' structures within the neuronal perikarya did not contain spatially pathogenic forms of tau accordingly to this method. Data demonstrate that fluorescence lifetime-based FRET can be applied to human brain tissue sections to detect pathogenic forms of tau molecules that lost their normal spatial distance.

Protective effects of microGlia in multiple sclerosis. The role of microglia in demyelinating neurodegenerative diseases such as multiple sclerosis MS and its animal model experimental autoimmune encephalomyelitis EAE is still controversial.

Although microglial cells are known as the professional phagocytes and executer of innate immunity in the central nervous system CNS , it is believed that microglia are rather neurotoxic in these diseases.

However, there is recent evidence indicating that microglia could also exert a neuroprotective function in MS and EAE.

First evidence for the protective effect of immune cells in CNS diseases emerged from studies in invertebrates. In the medicinal leech, the process of regeneration begins with rapid activation and accumulation of phagocytic glial cells at the lesion site followed by phagocytosis of damaged tissue by these cells which promoted robust neural regeneration.

In vertebrates, several lines of evidence demonstrate that microglia are also involved in neuroprotection by the secretion of soluble mediators that trigger neural repair and usually contribute to the creation of an environment conductive for regeneration.

The efficient removal of apoptotic cells and clearance of debris at the lesion site and the recruitment of stem cell populations as well as the induction of neurogenesis are directly correlated.

These findings suggest that microglia play a major role in creating a microenvironment for repair and regenerative processes in demyelinating neuroinflammatory diseases.

Siglec-F receptor and neuroprotection in mouse central nervous system. Sep Pharmacological Reports. Generation of microglial cells from mouse embryonic stem cells.

Microglia, the resident immune cells of the brain, are difficult to obtain in high numbers and purity using currently available methods; to date, microglia for experimental research are mainly isolated from the brain or from mixed glial cultures.

In this paper, we describe a basic protocol for the in vitro differentiation of mouse embryonic stem ES cells into microglial precursor cells. Microglia are obtained by a protocol consisting of five stages: The protocol can be completed in 60 d and results in stably proliferating ESdM lines, which show inducible transcription of inflammatory genes and cell marker expression comparable with primary microglia.

Furthermore, ESdMs are capable of chemokine-directed migration and phagocytosis, which are major functional features of microglia.

Functional role of brain-derived neurotrophic factor in neuroprotective autoimmunity: Therapeutic implications in a model of multiple sclerosis.

Brain-derived neurotrophic factor plays a key role in neuronal and axonal survival. Brain-derived neurotrophic factor is expressed in the immune cells in lesions of experimental autoimmune encephalomyelitis and multiple sclerosis, thus potentially mediating neuroprotective effects.

We investigated the functional role of brain-derived neurotrophic factor in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis.

Mice deficient for brain-derived neurotrophic factor in immune cells displayed an attenuated immune response in the acute phase of experimental autoimmune encephalomyelitis, but progressive disability with enhanced axonal loss in the chronic phase of the disease.

In mice deficient for central nervous system-derived brain-derived neurotrophic factor via glial fibrillary acidic protein-crescentin-mediated deletion, a more severe course of experimental autoimmune encephalomyelitis and an overall increased axonal loss was observed.

In a lentiviral approach, injection of brain-derived neurotrophic factor-overexpressing T cells led to a less severe course of experimental autoimmune encephalomyelitis and direct axonal protection.

Our data imply a functional role of brain-derived neurotrophic factor in autoimmune demyelination by mediating axon protection. Vaccine-based autoimmune anti-amyloid treatments are currently being examined for their therapeutic potential in Alzheimer's disease.

In the present study we examined, in a transgenic model of amyloid pathology, the expression of two molecules previously implicated in decreasing the severity of autoimmune responses: TREM2 triggering receptor expressed on myeloid cells 2 and the intracellular tolerance-associated transcript, Tmemb transmembrane domain protein b.

In situ hybridization analysis revealed that both molecules were highly expressed in plaque-associated microglia, but their expression defined two different zones of plaque-associated activation.

Tmemb expression was highest in the inner zone of amyloid plaques, whereas TREM2 expression was highest in the outer zone.

Induced expression of TREM2 occurred co-incident with detection of thioflavine-S-positive amyloid deposits. TREM2 expression also showed a positive correlation with amyloid phagocytosis in unactivated cells.

Transfection of Tmemb into both microglial and macrophage cell lines increased apoptosis. Elimination of extracellular aggregates and apoptotic neural membranes without inflammation is crucial for brain tissue homeostasis.

In the mammalian central nervous system, essential molecules in this process are the Fc receptors and the DAPassociated receptors which both trigger the microglial immunoreceptor tyrosine-based activation motif- ITAM- Syk-signaling cascade.

Microglial ITAM-signaling receptors are counter-regulated by immunoreceptor tyrosine-based inhibition motif- ITIM- signaling molecules such as sialic acid-binding immunoglobulin superfamily lectins Siglecs.

Siglecs can suppress the proinflammatory and phagocytic activity of microglia via ITIM signaling. Moreover, microglial neurotoxicity is alleviated via interaction of Siglec with sialic acids on the neuronal glycocalyx.

Their dysfunction could lead to impaired phagocytic clearance and neurodegeneration triggered by chronic inflammation. Alleviation of Neurotoxicity by Microglial Human Siglec Sialic acid-binding Ig superfamily lectins Siglecs are members of the Ig superfamily that recognize sialic acid residues of glycoproteins.

Siglec is a recently identified human-specific CDrelated Siglec that binds to alpha2,8-linked polysialic acids and is expressed on microglia, the brain resident innate immune cells.

We observed gene transcription and protein expression of Siglec splice variant 2 in human brain tissue samples by RT-PCR and Western blot analysis.

Siglec was detected on microglia in human brain tissue by immunohistochemistry. Human Siglec splice variant 2 was ectopically expressed by a lentiviral vector system in cultured murine microglial cells.

Stimulation of Siglec by cross-linking suppressed the lipopolysaccharides LPS -induced gene transcription of the proinflammatory mediators interleukin-1beta and nitric oxide synthase-2 in microglia.

Furthermore, phagocytosis of apoptotic neuronal material was reduced in Siglec transduced microglia.

Coculture of microglia transduced with Siglec and neurons demonstrated neuroprotective function of Siglec Thus, data demonstrate that human Siglec ectopically expressed on murine microglia interacts with PSA on neurons, reduces LPS-induced gene transcription of proinflammatory mediators, impairs phagocytosis and alleviates microglial neurotoxicity.

Microglial precursors derived from mouse embryonic stem cells. Microglia are resident immune cells of the central nervous system. They can be directly isolated from the brain or from mixed postnatal glial cultures.

Isolation of primary microglia is inefficient due to low yield. The cell line BV2 was used as a substitute for primary microglia, but BV2 are oncogenically transformed cells.

Here, we established a protocol to generate microglial precursor lines from mouse embryonic stem ES cells. Microglial precursor cells were obtained from murine ES cells by differentiation of embryoid bodies to microglia within a mixed brain culture.

Several independent ES cell-derived microglial precursor ESdM lines were generated and characterized by flow cytometry, immunocytochemistry, and functional assays.

Stimulation with interferon-gamma or lipopolysaccharide LPS demonstrated upregulation of proinflammatory cytokine gene transcription including nitric oxide synthase-2, interleukin-1beta, and tumor necrosis factor-alpha at levels comparable to primary microglia.

The ESdM showed efficient and rapid phagocytosis of microsphere beads, which was increased after stimulation with LPS. After in vivo transplantation into postnatal brain tissue, ESdM showed engraftment as cells with a microglial phenotype and morphology.

Thus, ESdM are stable proliferating cells substantially having most characteristics of primary microglia and therefore being a suitable tool to study microglial function in vitro and in vivo.

Signal Regulatory Protein 1: The signal regulatory protein-beta1 SIRPbeta1 is a DAPassociated transmembrane receptor expressed in a subset of hematopoietic cells.

Recently, it was shown that peritoneal macrophages express SIRPbeta1, which positively regulated phagocytosis. Activation of SIRPbeta1 on cultured microglia by cross-linking antibodies induced reorganization of the cytoskeleton protein beta-actin and suppressed lipopolysaccharide-induced gene transcription of tumor necrosis factor-alpha and nitric oxide synthase Furthermore, activation of SIRPbeta1 increased phagocytosis of microsphere beads, neural debris, and fibrillary amyloid-beta Abeta.

Phagocytosis of neural cell debris and Abeta was impaired after lentiviral knockdown of SIRPbeta1 in primary microglial cells.

Role of microglia in neuronal degeneration and regeneration. Microglial cells, the resident macrophage population of the central nervous system CNS , actively scan tissue under both normal and pathologic contexts.

Their resulting engagement can become either neuroprotective or neurotoxic, leading to amelioration or aggravation of disease progression. In this review, we focus on the molecular signaling molecules involved in microglial responses and discuss observations demonstrating the diverse effects of microglia in animal models of CNS diseases.

Regenerative therapy of experimental autoimmune encephalomyelitis by neurotrophin-3 transduced ES cell derived microglial cells.

Sep 25th Congress of the. Accumulation of tau induced in neurites by microglial proinflammatory mediators. Aggregated fibrillary microtubule-associated protein tau is the major component of neurofibrillary tangles in Alzheimer's disease.

The exact molecular mechanism of tau aggregation is unknown. Microglial cell activation and migration toward amyloid-beta plaques precede the appearance of dysmorphic neurites and formation of neurofibrillary tangles.

Here, we analyzed the accumulation of tau at a distance range of expected spontaneous aggregation by fluorescence lifetime-based Förster resonance energy transfer in cultured primary murine neurons cotransfected with the human tau gene tagged to the green fluorescent protein variants Citrine tau-Citrine and Cerulean tau-Cerulean.

No spontaneous accumulation of cotransfected tau-Citrine and tau-Cerulean was detected in untreated neurons.

Coculture of neurons with activated microglia induced aggregation of tau in neurites. Treatment of neurons with tumor necrosis factor-alpha TNF-alpha stimulated reactive oxygen species generation and resulted in the accumulation of tau-Citrine and tau-Cerulean in neurites, which was inhibited by neutralization of TNF and the free radical inhibitor 6-hydroxy-2,5,7,8-tetramethylchromanecarboxylic acid Trolox.

These data demonstrate that activated microglia and the microglial-derived proinflammatory cytokine TNF can induce accumulation of the aggregation-prone tau molecules in neurites via reactive oxygen species.

Microglial Clearance Function in Health and Disease. Microglial cells are of hematopoietic origin, populate the CNS during early development and form the brain's innate immune cell type.

Besides their well-known role in immune defense, microglia have an active and homeostatic function in the normal CNS based on high motility of their ramified processes and endocytic clearance of apoptotic vesicular material.

During development microglia contribute to the reorganization of neuronal connections, however microglia have also pivotal roles during acute and chronic neurodegeneration.

Microglia become attracted to site of injury by nucleotides released from damaged neurons. Scavenger receptors expressed on microglia bind to debris and microglial phagocytic receptors signal via immunoreceptor tyrosine-based activation motif ITAM --containing adaptor proteins to promote phagocytosis of extracellular material.

Insufficient clearance by microglia appears to be prevalent in neurodegenerative diseases such as Alzheimer's disease. Multiple sclerosis MS is a demyelinating autoimmune disease.

However, the persisting neurological deficits in MS patients result from acute axonal injury and chronic neurodegeneration, which are both triggered by the autoreactive immune response.

Innate immunity, mainly mediated by activated microglial cells and invading macrophages, appears to contribute to chronic neurodegeneration.

Activated microglia produce several reactive oxygen species and proinflammatory cytokines which affect neuronal function, integrity and survival.

Understanding the mechanisms of immune-mediated neuronal damage might help to design novel therapy strategies for MS.

Microglia derived from embryonic stem cells as a tool to study microglia function. Systemic injection of hematopoietic stem cells after ischemic cardiac or neural lesions is one approach to promote tissue repair.

However, mechanisms of possible protective or reparative effects are poorly understood. In this study we analyzed the effect of lineage-negative bone marrow-derived hematopoietic stem and precursor cells Lin - -HSCs on ischemic brain injury in mice.

Lin - -HSCs were injected intravenously at 24 hours after onset of a minute transient cerebral ischemia.

Effects of Lin - -HSCs injection on infarct size, apoptotic cell death, postischemic inflammation and cytokine gene transcription were analyzed.

Green fluorescent protein GFP -marked Lin - -HSCs were detected at 24 hours after injection in the spleen and later in ischemic brain parenchyma, expressing microglial but no neural marker proteins.

Tissue injury assessment showed significantly smaller infarct volumes and less apoptotic neuronal cell death in peri-infarct areas of Lin - -HSC-treated animals.

Analysis of immune cell infiltration in ischemic hemispheres revealed a reduction of invading T cells and macrophages in treated mice.

Moreover, Lin - -HSC therapy counter-regulated proinflammatory cytokine and chemokine receptor gene transcription within the spleen.

Our data demonstrate that systemically applied Lin - -HSCs reduce cerebral postischemic inflammation, attenuate peripheral immune activation and mediate neuroprotection after ischemic stroke.

Von der Entzündung zur Degeneration: Microglia are cells of myeloid origin that populate the CNS during early development and form the brain's innate immune cell type.

They perform homoeostatic activity in the normal CNS, a function associated with high motility of their ramified processes and their constant phagocytic clearance of cell debris.

This debris clearance role is amplified in CNS injury, where there is frank loss of tissue and recruitment of microglia to the injured area.

Recent evidence suggests that this phagocytic clearance following injury is more than simply tidying up, but instead plays a fundamental role in facilitating the reorganization of neuronal circuits and triggering repair.

Insufficient clearance by microglia, prevalent in several neurodegenerative diseases and declining with ageing, is associated with an inadequate regenerative response.

Thus, understanding the mechanism and functional significance of microglial-mediated clearance of tissue debris following injury may open up exciting new therapeutic avenues.

Neuronal 'On' and 'Off' signals control microglia. Recent findings indicate that neurons are not merely passive targets of microglia but rather control microglial activity.

The variety of different signals that neurons use to control microglia can be divided into two categories: They instruct microglia activation under pathological conditions towards a beneficial or detrimental phenotype.

Various neuronal signaling molecules thus actively control microglia function, thereby contribute to the inflammatory milieu of the central nervous system.

Thus, neurons should be envisaged as key immune modulators in the brain. An important step in the pathogenesis of multiple sclerosis is adhesion and transmigration of encephalitogenic T cells across brain endothelial cells EC which strongly relies on interaction with EC-expressed adhesion molecules.

We provide molecular evidence that the transcription factor peroxisome proliferator-activated receptor gamma PPARgamma is a negative regulator of brain EC inflammation.

Adult stem cells are promising cellular vehicles for therapy of malignant gliomas as they have the ability to migrate into these tumors and even track infiltrating tumor cells.

However, their clinical use is limited by a low passaging capacity that impedes large-scale production. In the present study, a bone marrow-derived, highly proliferative subpopulation of mesenchymal stem cells MSCs -here termed bone marrow-derived tumor-infiltrating cells BM-TICs -was genetically modified for the treatment of malignant glioma.

Upon injection into the tumor or the vicinity of the tumor, BM-TICs infiltrated solid parts as well as the border of rat 9L glioma.

Therapeutic efficacy was monitored by PET as well as by magnetic resonance imaging MRI and strongly correlated with histological analysis.

In conclusion, BM-TICs expressing a suicide gene were highly effective in the treatment of malignant glioma in a rat model and therefore hold great potential for the therapy of malignant brain tumors in humans.

A retroviral packaging cell line for pseudotype vectors based on glioma-infiltrating progenitor cells. Early clinical trials for gene therapy of human gliomas with retroviral packaging cells PC have been hampered by low transduction efficacy and lack of dissemination of PC within the tumor.

In the current approach, these issues have been addressed by creating a stable packaging cell line for retroviral vectors pseudotyped with glycoproteins of lymphocytic choriomeningitis virus LCMV based on tumor-infiltrating progenitor cells.

Packaging of a retroviral vector was measured by titration experiments on human fibroblast cells as well as on mouse and human glioma cell lines. Additionally, gene transfer was tested in a rat glioma model in vivo.

This proof-of-principle study shows that primary adult progenitor cells with tumor-infiltrating capacity can be genetically modified to stably produce retroviral LCMV pseudotype vectors.

Cells were analyzed after 2 d of stimulation by flow cytometry. No change in gene transcript levels of inflammatory mediators was detected after stimulation of TREM2 solely.

In multiple sclerosis, inflammation can successfully be prevented, while promoting repair is still a major challenge. Microglial cells, the resident phagocytes of the central nervous system CNS , are hematopoietic-derived myeloid cells and express the triggering receptor expressed on myeloid cells 2 TREM2 , an innate immune receptor.

Myeloid cells are an accessible source for ex vivo gene therapy. We investigated whether myeloid precursor cells genetically modified to express TREM2 affect the disease course of experimental autoimmune encephalomyelitis EAE , an animal model of multiple sclerosis.

EAE was induced in mice by immunization with a myelin autoantigen. Intravenous application of TREM2-transduced bone marrow-derived myeloid precursor cells at the EAE peak led to an amelioration of clinical symptoms, reduction in axonal damage, and prevention of further demyelination.

TREM2-transduced myeloid cells applied intravenously migrated into the inflammatory spinal cord lesions of EAE-diseased mice, showed increased lysosomal and phagocytic activity, cleared degenerated myelin, and created an anti-inflammatory cytokine milieu within the CNS.

Intravenously applied bone marrow-derived and TREM2-tranduced myeloid precursor cells limit tissue destruction and facilitate repair within the murine CNS by clearance of cellular debris during EAE.

TREM2 is a new attractive target for promotion of repair and resolution of inflammation in multiple sclerosis and other neuroinflammatory diseases.

Essential role of the microglial triggering receptor expressed on myeloid cells-2 TREM2 for central nervous tissue immune homeostasis.

While there is a strong evidence for neural tissue destruction mediated by adaptive autoimmune responses, it is still debated how innate immune responses contribute to neuroinflammatory and neurodegenerative diseases such as multiple sclerosis and Alzheimer's disease.

Recently, it was shown that loss-of-function mutations of the innate microglial immune receptor triggering receptor expressed on myeloid cells-2 TREM2 led to a chronic neurodegenerative disease, named Nasu-Hakola disease or polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy PLOSL.

PLOSL is a recessively inherited disease characterized by early onset adult dementia associated with bone cysts. Indeed, TREM2 of microglia fulfils important function of tissue debris clearance and resolution of latent inflammatory reactions.

Absence of TREM2 expression on microglia impairs their capacity to phagocytose cell membrane debris and increases their gene transcription of pro-inflammatory cytokines.

The disease PLOSL and the finding that TREM2 of microglia is required for tissue debris clearance provide prototypic molecular evidence that dysfunctional innate immunity can be disease causative leading to a chronic neurodegenerative process.

Our results provide molecular evidence that the transcription factor peroxisome proliferator-activated receptor gamma PPARg is a negative regulator of brain EC inflammation.

This effect is clearly PPARg mediated, as lentiviral PPARg overexpression in EC results in selective abrogation of inflammation-induced T cell adhesion and subsequent transmigration, but permits adhesion and transmigration of T cells under non-inflammatory conditions.

We therefore propose that PPARg activation in EC selectively curtails inflammation-induced EC-T cell interactions and may therefore be exploited to selectively target detrimental EC-T cell interactions under inflammatory conditions.

Multiple sclerosis MS is pathologically characterized by inflammatory demyelination and neuronal injury. Although phagocytosis of myelin debris by microglia and macrophages in acute MS lesions is well documented, its pathophysiological significance is unclear.

Using real-time quantitative PCR, flow cytometry, ELISA, and reactive oxygen species ROS measurement assays, we demonstrated that phagocytosis of myelin modulates activation of microglial cells prestimulated by interferon-gamma IFN-gamma or a combination of IFN-gamma and lipopolysaccharide with a biphasic temporal pattern, i.

In this second phase, myelin phagocytosis leads to an enhanced release of prostaglandin E2 and ROS in microglia, whereas the production of anti-inflammatory cytokines particularly interleukin remains unchanged.

Suppression of inflammatory microglial activation by myelin phagocytosis was reversed by treatment with superoxide dismutase and catalase, by inhibition of the NADPH-oxidase complex, or by specific knockdown of the NADPH-oxidase-required adaptor pphagocyte oxidase PHOX.

Furthermore, we observed that myelin phagocytosis destabilized tumor necrosis factor-alpha and interferon-induced protein mRNA through an adenine-uridine-rich elements-involved mechanism, which was reversed by blocking the function of NADPH-oxidase complex.

We conclude that phagocytosis of myelin suppresses microglial inflammatory activities via enhancement of pPHOX-mediated ROS generation.

These results suggest that intervention in ROS generation could represent a novel therapeutic strategy to reduce neuroinflammation in MS.

Unloading kinesin transported cargoes from the tubulin track via the inflammatory c-Jun N-terminal kinase pathway. Axonal transport of mitochondria and synaptic vesicle precursors via kinesin motor proteins is essential to keep integrity of axons and synapses.

Disturbance of axonal transport is an early sign of neuroinflammatory and neurodegenerative diseases. Treatment of cultured neurons by the inflammatory cytokine tumor necrosis factor-alpha TNF stimulated phosphorylation of c-Jun N-terminal kinase JNK in neurites.

Furthermore, TNF inhibited axonal transport of mitochondria and synaptophysin by reducing the mobile fraction via JNK.

Thus, TNF produced by activated glial cells in inflammatory or degenerative neurological diseases acts on neurites by acting on the kinesin-tubulin complex and inhibits axonal mitochondria and synaptophysin transport via JNK.

A cellular vehicle for CNS gene therapy. MLD is characterized by progressive demyelination and neurological deficits. Treatment of MLD is still a challenge due to the fact that the blood-brain barrier is a major obstacle for most therapeutic substances.

In this issue of the JCI, Biffi et al. Clearance of tissue debris by TREM2-transduced myeloid cells promotes recovery of experimental autoimmune encephalomyelitis.

Sep 8th International Conference of Neuroimmunology. Bystander mediated therapy of experimental malignant glioma with adult progenitor cells. Sep 51st Annual Meeting of the.

Molecular Therapy 13, S25 [ndash] S25; doi: A receptor for phagocytosis of Alzheimer's amyloid peptide. The amyloid beta peptide 42 Abeta 42 plays a key role in neurotoxicity in Alzheimer's disease.

Recently, the lipopolysaccharide LPS receptor CD14 was shown to mediate phagocytosis of bacterial components and furthermore to contribute to neuroinflammation in Alzheimer's disease.

Here, we investigated whether this key innate immunity receptor can interact with Abeta 42 and mediate phagocytosis of this peptide.

Using flow cytometry, confocal microscopy and two-photon fluorescence lifetime imaging FLIM combined with fluorescence resonance energy transfer FRET , we demonstrated a direct molecular interaction in the range of a few nanometers between Abeta 42 and CD14 in human CDtransfected Chinese hamster ovary cells.

This phagocytosis occurred at Abeta 42 concentration ranges that were considerably lower than the threshold to activate a cellular inflammatory reaction.

In contrast, there was no association of CD14 to microglial internalization of microbeads. In complementary clinical experiments, we detected a pronounced CD14 immunoreactivity on parenchymal microglia spatially correlated to characteristic Alzheimer's disease lesion sites in brain sections of Alzheimer's disease patients but not in brain sections of control subjects.

By showing a close interaction between CD14 and Abeta 42 , demonstrating a direct role of CD14 in Abeta 42 phagocytosis, and detecting CDspecific staining in brains of Alzheimer's disease patients, our results indicate a role of the LPS receptor in the pathophysiology of Alzheimer's disease, which could be of therapeutic relevance.

Malignant gliomas, the most frequent primary brain tumors, still have a dismal prognosis despite advances in neurosurgery, radiation, and chemotherapy.

Gene therapy using viral vectors represents an attractive alternative to conventional cancer therapies. In particular, retroviral vectors have been regarded as potent candidates as they transduce only dividing cells, thereby achieving specificity for tumor cells.

However, clinical trials have failed, because in a given treatment window, only the minority of tumor cells is dividing and therefore susceptible to retroviral infection.

To overcome this problem we used lentiviral vectors, which can transduce dividing as well as quiescent cells, for gene therapy of malignant glioma.

In a previous study we established lentiviral vectors pseudotyped with lymphocytic choriomeningitis LCMV glycoproteins GP and demonstrated efficient transduction of human malignant glioma cells in culture.

In the current investigation, we compared the transduction efficacy of LCMV GP- and vesicular stomatitis virus glycoprotein VSV G -pseudotyped lentiviral vectors for malignant glioma cells and normal brain cells in vitro and in vivo Miletic et al.

LCMV pseudotypes transduced predominantly astrocytes, whereas VSV-G pseudotypes infected neurons as well as astrocytes in vitro and in the hippocampus and striatum of Fischer rats.

LCMV pseudotypes showed an efficient transduction of solid glioma parts of 9L tumors

Thus, genetically modified ESdM represent a suitable tool to introduce therapeutic neuroprotective and repair-promoting proteins into the CNS in neuroinflammatory diseases. Furthermore, TNF inhibited axonal transport of mitochondria and synaptophysin by reducing the mobile fraction via JNK. Thus, data demonstrate that human Siglec ectopically expressed on murine microglia interacts with PSA on neurons, reduces LPS-induced gene transcription of proinflammatory mediators, impairs phagocytosis and alleviates microglial neurotoxicity. Neuronal injury mediated via stimulation of microglial Toll-like receptor 9 TLR9. Control of glial immune function by neurons. To simpsons spiel casino microglial function in vitro, primary microglia or immortalized cell lines are commonly used. A major challenge is to develop methods for their application in effective doses and in Beste Spielothek in Kelz finden precisely timed and localized fashion. The extracellular domain of Siglec-h protein bound to mouse glioma lines, but not to astrocytes or other normal mouse cells. In this study, we report that Fas ligand FasL expression by neurons is involved in protection against perforin-mediated T Beste Spielothek in Großberkenthin finden cytotoxicity. Flow cytometry analysis of cell the club casino luzern markers CD54, CD74, and Bitcoin casino fake selected from the microarray confirmed the close phenotypic relation between ESdM and primary cultured microglia. Polysialylated neuropilin-2 polySia-NRP2 contributes to this pool but further polySia protein carriers have remained elusive. Siglec-4 protects neurons from acute toxicity via interaction of sialic acids bound to neuronal mexiko tipps. Thus, data show that M1-polarized microglial cells can engulf glioma cells via a DAPmediated Siglec-h dependent mechanism.

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Triggering receptor expressed on myeloid cells-2 TREM2 is a microglial surface receptor involved in phagocytosis. Clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery.

The role of TREM2 following stroke is currently unclear. As an experimental stroke model, the middle cerebral artery of mice was occluded for 30 minutes with a range of reperfusion times duration of reperfusion: Functional consequences of TREM2 knockout were assessed by infarct volumetry.

The acute inflammatory response 12 h reperfusion was very similar between TREM2-KO mice and their littermate controls.

No effect on the lesion size was observed. Although we initially expected an exaggerated pro-inflammatory response following ablation of TREM2, our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in TREM2-KO mice.

We therefore conclude that TREM2 appears to sustain a distinct inflammatory response after stroke. No change in the basal gene transcription of inflammatory mediators in the brain of TREM2 deficient mice.

Microglial activatory immunoreceptor tyrosine-based activation motif - and inhibitory immunoreceptor tyrosine-based inhibition motif -signaling receptors for recognition of the neuronal glycocalyx.

Microglia sense intact or lesioned cells of the central nervous system CNS and respond accordingly. To fulfill this task, microglia express a whole set of recognition receptors.

Those receptors are counter-regulated by immunoreceptor tyrosine-based inhibition motif ITIM -signaling receptors, such as sialic acid-binding immunoglobulin superfamily lectins Siglecs.

Siglecs recognize the sialic acid cap of healthy neurons thus leading to an ITIM signaling that turns down microglial immune responses and phagocytosis.

In contrast, desialylated neuronal processes are phagocytosed by microglial CR3 signaling via an adaptor protein containing an ITAM.

Thus, the aberrant terminal glycosylation of neuronal surface glycoproteins and glycolipids could serve as a flag for microglia, which display a multitude of diverse carbohydrate-binding receptors that monitor the neuronal physical condition and respond via their ITIM- or ITAM-signaling cascade accordingly.

Current knowledge about the pathogenic mechanism of Alzheimer's disease is based mainly on rare, high-penetrance variants in genes encoding amyloid precursor protein, presenilin 1, and presenilin 2, which result in familial early-onset Alzheimer's disease.

However, Alzheimer's disease is predominantly a sporadic late-onset disease with exponentially increasing prevalence starting at the age of 65 years.

Licensing of myeloid cells promotes central nervous system autoimmunity and is controlled by peroxisome proliferator-activated receptor gamma.

During central nervous system autoimmunity, interactions between infiltrating immune cells and brain-resident cells are critical for disease progression and ultimately organ damage.

Here, we demonstrate that local cross-talk between invading autoreactive T cells and auto-antigen-presenting myeloid cells within the central nervous system results in myeloid cell activation, which is crucial for disease progression during experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis.

Together, interference with the disease-promoting cross-talk between central nervous system myeloid cells, autoreactive T cells and brain-resident cells represents a novel therapeutic approach that limits disease progression and lesion development during ongoing central nervous system autoimmunity.

Microglial carbohydrate-binding receptors for neural repair. Microglia are the resident immune cells of the central nervous system CNS and perform typical scavenging and innate immune functions.

Their capacity to eliminate extracellular aggregates and apoptotic neural material without inflammation is crucial for brain tissue homeostasis and repair.

To fulfill these tasks, microglia express a whole set of recognition receptors including toll-like TLRs , carbohydrate-binding, Fc, complement and cytokine receptors.

Receptors recognizing carbohydrate structures are strongly involved in microglial repair function.

Carbohydrate-binding receptors can be divided into two major subgroups: Siglecs bind to the sialic acid cap of the intact glycocalyx.

Other lectin family members such as galectins recognize lactosamine units typically exposed after alteration of the glycocalyx.

Dependent on the type of microglial carbohydrate-binding receptors that are stimulated, either a pro-inflammatory cytotoxic or an anti-inflammatory repair-promoting response is evoked.

The carbohydrate-binding receptors are also crucial in regulating microglial function such as phagocytosis during neurodegenerative or neuroinflammatory processes.

A balance between microglial carbohydrate-binding receptor signaling via an immunoreceptor tyrosine-based activation motif or an immunoreceptor tyrosine-based inhibitory motif is required to polarize microglial cells appropriately so that they create a microenvironment permissive for neural regenerative events.

Microglial cells are professional phagocytes of the CNS responsible for clearance of unwanted structures. Neuronal processes are marked by complement C1 before they are removed in development or during disease processes.

Target molecules involved in C1 binding and mechanisms of clearance are still unclear. Here we show that the terminal sugar residue sialic acid of the mouse neuronal glycocalyx determines complement C1 binding and microglial-mediated clearance function.

Several early components of the classical complement cascade including C1q, C1r, C1s, and C3 were produced by cultured mouse microglia.

The opsonin C1q was binding to neurites after enzymatic removal of sialic acid residues from the neuronal glycocalyx.

Desialylated neurites, but not neurites with intact sialic acid caps, were cleared and taken up by cocultured microglial cells.

Data demonstrate that mouse microglial cells via CR3 recognize and remove neuronal structures with an altered neuronal glycocalyx lacking terminal sialic acid.

Spatially pathogenic forms of tau detected in Alzheimer's disease brain tissue by fluorescence lifetime-based Forster resonance energy transfer.

In tauopathies including Alzheimer's disease AD tau molecules have lost their normal spatial distance to each other and appear in oligomeric or aggregated forms.

Conventional immunostaining methods allow detection of abnormally phosphorylated or conformationally altered aggregated tau proteins, but fail to visualize oligomeric forms of tau.

Here we show that tau molecules that lost their normal spatial localization can be detected on a subcellular level in postmortem central nervous system CNS tissue sections of AD patients by fluorescence lifetime-based Förster resonance energy transfer FRET.

Paraffin sections were co-immunostained with two tau-specific monoclonal antibodies recognizing the same epitope, but labeled with distinct fluorescence dyes suitable for spatial resolution at a nanometer scale by lifetime-based FRET.

A FRET signal was detected in neuritic plaques and neurofibrillary tangles of CNS tissue sections of AD patients, showing associated tau proteins typically reflecting either fibrillary, oligomeric or aggregated tau.

The 'pretangle-like' structures within the neuronal perikarya did not contain spatially pathogenic forms of tau accordingly to this method.

Data demonstrate that fluorescence lifetime-based FRET can be applied to human brain tissue sections to detect pathogenic forms of tau molecules that lost their normal spatial distance.

Protective effects of microGlia in multiple sclerosis. The role of microglia in demyelinating neurodegenerative diseases such as multiple sclerosis MS and its animal model experimental autoimmune encephalomyelitis EAE is still controversial.

Although microglial cells are known as the professional phagocytes and executer of innate immunity in the central nervous system CNS , it is believed that microglia are rather neurotoxic in these diseases.

However, there is recent evidence indicating that microglia could also exert a neuroprotective function in MS and EAE.

First evidence for the protective effect of immune cells in CNS diseases emerged from studies in invertebrates. In the medicinal leech, the process of regeneration begins with rapid activation and accumulation of phagocytic glial cells at the lesion site followed by phagocytosis of damaged tissue by these cells which promoted robust neural regeneration.

In vertebrates, several lines of evidence demonstrate that microglia are also involved in neuroprotection by the secretion of soluble mediators that trigger neural repair and usually contribute to the creation of an environment conductive for regeneration.

The efficient removal of apoptotic cells and clearance of debris at the lesion site and the recruitment of stem cell populations as well as the induction of neurogenesis are directly correlated.

These findings suggest that microglia play a major role in creating a microenvironment for repair and regenerative processes in demyelinating neuroinflammatory diseases.

Siglec-F receptor and neuroprotection in mouse central nervous system. Sep Pharmacological Reports. Generation of microglial cells from mouse embryonic stem cells.

Microglia, the resident immune cells of the brain, are difficult to obtain in high numbers and purity using currently available methods; to date, microglia for experimental research are mainly isolated from the brain or from mixed glial cultures.

In this paper, we describe a basic protocol for the in vitro differentiation of mouse embryonic stem ES cells into microglial precursor cells.

Microglia are obtained by a protocol consisting of five stages: The protocol can be completed in 60 d and results in stably proliferating ESdM lines, which show inducible transcription of inflammatory genes and cell marker expression comparable with primary microglia.

Furthermore, ESdMs are capable of chemokine-directed migration and phagocytosis, which are major functional features of microglia.

Functional role of brain-derived neurotrophic factor in neuroprotective autoimmunity: Therapeutic implications in a model of multiple sclerosis. Brain-derived neurotrophic factor plays a key role in neuronal and axonal survival.

Brain-derived neurotrophic factor is expressed in the immune cells in lesions of experimental autoimmune encephalomyelitis and multiple sclerosis, thus potentially mediating neuroprotective effects.

We investigated the functional role of brain-derived neurotrophic factor in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis.

Mice deficient for brain-derived neurotrophic factor in immune cells displayed an attenuated immune response in the acute phase of experimental autoimmune encephalomyelitis, but progressive disability with enhanced axonal loss in the chronic phase of the disease.

In mice deficient for central nervous system-derived brain-derived neurotrophic factor via glial fibrillary acidic protein-crescentin-mediated deletion, a more severe course of experimental autoimmune encephalomyelitis and an overall increased axonal loss was observed.

In a lentiviral approach, injection of brain-derived neurotrophic factor-overexpressing T cells led to a less severe course of experimental autoimmune encephalomyelitis and direct axonal protection.

Our data imply a functional role of brain-derived neurotrophic factor in autoimmune demyelination by mediating axon protection.

Vaccine-based autoimmune anti-amyloid treatments are currently being examined for their therapeutic potential in Alzheimer's disease.

In the present study we examined, in a transgenic model of amyloid pathology, the expression of two molecules previously implicated in decreasing the severity of autoimmune responses: TREM2 triggering receptor expressed on myeloid cells 2 and the intracellular tolerance-associated transcript, Tmemb transmembrane domain protein b.

In situ hybridization analysis revealed that both molecules were highly expressed in plaque-associated microglia, but their expression defined two different zones of plaque-associated activation.

Tmemb expression was highest in the inner zone of amyloid plaques, whereas TREM2 expression was highest in the outer zone. Induced expression of TREM2 occurred co-incident with detection of thioflavine-S-positive amyloid deposits.

TREM2 expression also showed a positive correlation with amyloid phagocytosis in unactivated cells.

Transfection of Tmemb into both microglial and macrophage cell lines increased apoptosis. Elimination of extracellular aggregates and apoptotic neural membranes without inflammation is crucial for brain tissue homeostasis.

In the mammalian central nervous system, essential molecules in this process are the Fc receptors and the DAPassociated receptors which both trigger the microglial immunoreceptor tyrosine-based activation motif- ITAM- Syk-signaling cascade.

Microglial ITAM-signaling receptors are counter-regulated by immunoreceptor tyrosine-based inhibition motif- ITIM- signaling molecules such as sialic acid-binding immunoglobulin superfamily lectins Siglecs.

Siglecs can suppress the proinflammatory and phagocytic activity of microglia via ITIM signaling. Moreover, microglial neurotoxicity is alleviated via interaction of Siglec with sialic acids on the neuronal glycocalyx.

Their dysfunction could lead to impaired phagocytic clearance and neurodegeneration triggered by chronic inflammation. Alleviation of Neurotoxicity by Microglial Human Siglec Sialic acid-binding Ig superfamily lectins Siglecs are members of the Ig superfamily that recognize sialic acid residues of glycoproteins.

Siglec is a recently identified human-specific CDrelated Siglec that binds to alpha2,8-linked polysialic acids and is expressed on microglia, the brain resident innate immune cells.

We observed gene transcription and protein expression of Siglec splice variant 2 in human brain tissue samples by RT-PCR and Western blot analysis.

Siglec was detected on microglia in human brain tissue by immunohistochemistry. Human Siglec splice variant 2 was ectopically expressed by a lentiviral vector system in cultured murine microglial cells.

Stimulation of Siglec by cross-linking suppressed the lipopolysaccharides LPS -induced gene transcription of the proinflammatory mediators interleukin-1beta and nitric oxide synthase-2 in microglia.

Furthermore, phagocytosis of apoptotic neuronal material was reduced in Siglec transduced microglia. Coculture of microglia transduced with Siglec and neurons demonstrated neuroprotective function of Siglec Thus, data demonstrate that human Siglec ectopically expressed on murine microglia interacts with PSA on neurons, reduces LPS-induced gene transcription of proinflammatory mediators, impairs phagocytosis and alleviates microglial neurotoxicity.

Microglial precursors derived from mouse embryonic stem cells. Microglia are resident immune cells of the central nervous system. They can be directly isolated from the brain or from mixed postnatal glial cultures.

Isolation of primary microglia is inefficient due to low yield. The cell line BV2 was used as a substitute for primary microglia, but BV2 are oncogenically transformed cells.

Here, we established a protocol to generate microglial precursor lines from mouse embryonic stem ES cells. Microglial precursor cells were obtained from murine ES cells by differentiation of embryoid bodies to microglia within a mixed brain culture.

Several independent ES cell-derived microglial precursor ESdM lines were generated and characterized by flow cytometry, immunocytochemistry, and functional assays.

Stimulation with interferon-gamma or lipopolysaccharide LPS demonstrated upregulation of proinflammatory cytokine gene transcription including nitric oxide synthase-2, interleukin-1beta, and tumor necrosis factor-alpha at levels comparable to primary microglia.

The ESdM showed efficient and rapid phagocytosis of microsphere beads, which was increased after stimulation with LPS.

After in vivo transplantation into postnatal brain tissue, ESdM showed engraftment as cells with a microglial phenotype and morphology. Thus, ESdM are stable proliferating cells substantially having most characteristics of primary microglia and therefore being a suitable tool to study microglial function in vitro and in vivo.

Signal Regulatory Protein 1: The signal regulatory protein-beta1 SIRPbeta1 is a DAPassociated transmembrane receptor expressed in a subset of hematopoietic cells.

Recently, it was shown that peritoneal macrophages express SIRPbeta1, which positively regulated phagocytosis. Activation of SIRPbeta1 on cultured microglia by cross-linking antibodies induced reorganization of the cytoskeleton protein beta-actin and suppressed lipopolysaccharide-induced gene transcription of tumor necrosis factor-alpha and nitric oxide synthase Furthermore, activation of SIRPbeta1 increased phagocytosis of microsphere beads, neural debris, and fibrillary amyloid-beta Abeta.

Phagocytosis of neural cell debris and Abeta was impaired after lentiviral knockdown of SIRPbeta1 in primary microglial cells. Role of microglia in neuronal degeneration and regeneration.

Microglial cells, the resident macrophage population of the central nervous system CNS , actively scan tissue under both normal and pathologic contexts.

Their resulting engagement can become either neuroprotective or neurotoxic, leading to amelioration or aggravation of disease progression.

In this review, we focus on the molecular signaling molecules involved in microglial responses and discuss observations demonstrating the diverse effects of microglia in animal models of CNS diseases.

Regenerative therapy of experimental autoimmune encephalomyelitis by neurotrophin-3 transduced ES cell derived microglial cells.

Sep 25th Congress of the. Accumulation of tau induced in neurites by microglial proinflammatory mediators. Aggregated fibrillary microtubule-associated protein tau is the major component of neurofibrillary tangles in Alzheimer's disease.

The exact molecular mechanism of tau aggregation is unknown. Microglial cell activation and migration toward amyloid-beta plaques precede the appearance of dysmorphic neurites and formation of neurofibrillary tangles.

Here, we analyzed the accumulation of tau at a distance range of expected spontaneous aggregation by fluorescence lifetime-based Förster resonance energy transfer in cultured primary murine neurons cotransfected with the human tau gene tagged to the green fluorescent protein variants Citrine tau-Citrine and Cerulean tau-Cerulean.

No spontaneous accumulation of cotransfected tau-Citrine and tau-Cerulean was detected in untreated neurons. Coculture of neurons with activated microglia induced aggregation of tau in neurites.

Treatment of neurons with tumor necrosis factor-alpha TNF-alpha stimulated reactive oxygen species generation and resulted in the accumulation of tau-Citrine and tau-Cerulean in neurites, which was inhibited by neutralization of TNF and the free radical inhibitor 6-hydroxy-2,5,7,8-tetramethylchromanecarboxylic acid Trolox.

These data demonstrate that activated microglia and the microglial-derived proinflammatory cytokine TNF can induce accumulation of the aggregation-prone tau molecules in neurites via reactive oxygen species.

Microglial Clearance Function in Health and Disease. Microglial cells are of hematopoietic origin, populate the CNS during early development and form the brain's innate immune cell type.

Besides their well-known role in immune defense, microglia have an active and homeostatic function in the normal CNS based on high motility of their ramified processes and endocytic clearance of apoptotic vesicular material.

During development microglia contribute to the reorganization of neuronal connections, however microglia have also pivotal roles during acute and chronic neurodegeneration.

Microglia become attracted to site of injury by nucleotides released from damaged neurons. Scavenger receptors expressed on microglia bind to debris and microglial phagocytic receptors signal via immunoreceptor tyrosine-based activation motif ITAM --containing adaptor proteins to promote phagocytosis of extracellular material.

Insufficient clearance by microglia appears to be prevalent in neurodegenerative diseases such as Alzheimer's disease.

Multiple sclerosis MS is a demyelinating autoimmune disease. However, the persisting neurological deficits in MS patients result from acute axonal injury and chronic neurodegeneration, which are both triggered by the autoreactive immune response.

Innate immunity, mainly mediated by activated microglial cells and invading macrophages, appears to contribute to chronic neurodegeneration.

Activated microglia produce several reactive oxygen species and proinflammatory cytokines which affect neuronal function, integrity and survival.

Understanding the mechanisms of immune-mediated neuronal damage might help to design novel therapy strategies for MS.

Microglia derived from embryonic stem cells as a tool to study microglia function. Systemic injection of hematopoietic stem cells after ischemic cardiac or neural lesions is one approach to promote tissue repair.

However, mechanisms of possible protective or reparative effects are poorly understood. In this study we analyzed the effect of lineage-negative bone marrow-derived hematopoietic stem and precursor cells Lin - -HSCs on ischemic brain injury in mice.

Lin - -HSCs were injected intravenously at 24 hours after onset of a minute transient cerebral ischemia. Effects of Lin - -HSCs injection on infarct size, apoptotic cell death, postischemic inflammation and cytokine gene transcription were analyzed.

Green fluorescent protein GFP -marked Lin - -HSCs were detected at 24 hours after injection in the spleen and later in ischemic brain parenchyma, expressing microglial but no neural marker proteins.

Tissue injury assessment showed significantly smaller infarct volumes and less apoptotic neuronal cell death in peri-infarct areas of Lin - -HSC-treated animals.

Analysis of immune cell infiltration in ischemic hemispheres revealed a reduction of invading T cells and macrophages in treated mice.

Moreover, Lin - -HSC therapy counter-regulated proinflammatory cytokine and chemokine receptor gene transcription within the spleen.

Our data demonstrate that systemically applied Lin - -HSCs reduce cerebral postischemic inflammation, attenuate peripheral immune activation and mediate neuroprotection after ischemic stroke.

Von der Entzündung zur Degeneration: Microglia are cells of myeloid origin that populate the CNS during early development and form the brain's innate immune cell type.

They perform homoeostatic activity in the normal CNS, a function associated with high motility of their ramified processes and their constant phagocytic clearance of cell debris.

This debris clearance role is amplified in CNS injury, where there is frank loss of tissue and recruitment of microglia to the injured area.

Recent evidence suggests that this phagocytic clearance following injury is more than simply tidying up, but instead plays a fundamental role in facilitating the reorganization of neuronal circuits and triggering repair.

Insufficient clearance by microglia, prevalent in several neurodegenerative diseases and declining with ageing, is associated with an inadequate regenerative response.

Thus, understanding the mechanism and functional significance of microglial-mediated clearance of tissue debris following injury may open up exciting new therapeutic avenues.

Neuronal 'On' and 'Off' signals control microglia. Recent findings indicate that neurons are not merely passive targets of microglia but rather control microglial activity.

The variety of different signals that neurons use to control microglia can be divided into two categories: They instruct microglia activation under pathological conditions towards a beneficial or detrimental phenotype.

Various neuronal signaling molecules thus actively control microglia function, thereby contribute to the inflammatory milieu of the central nervous system.

Thus, neurons should be envisaged as key immune modulators in the brain. An important step in the pathogenesis of multiple sclerosis is adhesion and transmigration of encephalitogenic T cells across brain endothelial cells EC which strongly relies on interaction with EC-expressed adhesion molecules.

We provide molecular evidence that the transcription factor peroxisome proliferator-activated receptor gamma PPARgamma is a negative regulator of brain EC inflammation.

Adult stem cells are promising cellular vehicles for therapy of malignant gliomas as they have the ability to migrate into these tumors and even track infiltrating tumor cells.

However, their clinical use is limited by a low passaging capacity that impedes large-scale production. Siglec was detected on microglia in human brain tissue by immunohistochemistry.

Human Siglec splice variant 2 was ectopically expressed by a lentiviral vector system in cultured murine microglial cells.

Stimulation of Siglec by cross-linking suppressed the lipopolysaccharides LPS -induced gene transcription of the proinflammatory mediators interleukin-1beta and nitric oxide synthase-2 in microglia.

Furthermore, phagocytosis of apoptotic neuronal material was reduced in Siglec transduced microglia. Coculture of microglia transduced with Siglec and neurons demonstrated neuroprotective function of Siglec Thus, data demonstrate that human Siglec ectopically expressed on murine microglia interacts with PSA on neurons, reduces LPS-induced gene transcription of proinflammatory mediators, impairs phagocytosis and alleviates microglial neurotoxicity.

Microglial precursors derived from mouse embryonic stem cells. Microglia are resident immune cells of the central nervous system. They can be directly isolated from the brain or from mixed postnatal glial cultures.

Isolation of primary microglia is inefficient due to low yield. The cell line BV2 was used as a substitute for primary microglia, but BV2 are oncogenically transformed cells.

Here, we established a protocol to generate microglial precursor lines from mouse embryonic stem ES cells. Microglial precursor cells were obtained from murine ES cells by differentiation of embryoid bodies to microglia within a mixed brain culture.

Several independent ES cell-derived microglial precursor ESdM lines were generated and characterized by flow cytometry, immunocytochemistry, and functional assays.

Stimulation with interferon-gamma or lipopolysaccharide LPS demonstrated upregulation of proinflammatory cytokine gene transcription including nitric oxide synthase-2, interleukin-1beta, and tumor necrosis factor-alpha at levels comparable to primary microglia.

The ESdM showed efficient and rapid phagocytosis of microsphere beads, which was increased after stimulation with LPS. After in vivo transplantation into postnatal brain tissue, ESdM showed engraftment as cells with a microglial phenotype and morphology.

Thus, ESdM are stable proliferating cells substantially having most characteristics of primary microglia and therefore being a suitable tool to study microglial function in vitro and in vivo.

Signal Regulatory Protein 1: The signal regulatory protein-beta1 SIRPbeta1 is a DAPassociated transmembrane receptor expressed in a subset of hematopoietic cells.

Recently, it was shown that peritoneal macrophages express SIRPbeta1, which positively regulated phagocytosis.

Activation of SIRPbeta1 on cultured microglia by cross-linking antibodies induced reorganization of the cytoskeleton protein beta-actin and suppressed lipopolysaccharide-induced gene transcription of tumor necrosis factor-alpha and nitric oxide synthase Furthermore, activation of SIRPbeta1 increased phagocytosis of microsphere beads, neural debris, and fibrillary amyloid-beta Abeta.

Phagocytosis of neural cell debris and Abeta was impaired after lentiviral knockdown of SIRPbeta1 in primary microglial cells.

Role of microglia in neuronal degeneration and regeneration. Microglial cells, the resident macrophage population of the central nervous system CNS , actively scan tissue under both normal and pathologic contexts.

Their resulting engagement can become either neuroprotective or neurotoxic, leading to amelioration or aggravation of disease progression.

In this review, we focus on the molecular signaling molecules involved in microglial responses and discuss observations demonstrating the diverse effects of microglia in animal models of CNS diseases.

Regenerative therapy of experimental autoimmune encephalomyelitis by neurotrophin-3 transduced ES cell derived microglial cells.

Sep 25th Congress of the. Accumulation of tau induced in neurites by microglial proinflammatory mediators. Aggregated fibrillary microtubule-associated protein tau is the major component of neurofibrillary tangles in Alzheimer's disease.

The exact molecular mechanism of tau aggregation is unknown. Microglial cell activation and migration toward amyloid-beta plaques precede the appearance of dysmorphic neurites and formation of neurofibrillary tangles.

Here, we analyzed the accumulation of tau at a distance range of expected spontaneous aggregation by fluorescence lifetime-based Förster resonance energy transfer in cultured primary murine neurons cotransfected with the human tau gene tagged to the green fluorescent protein variants Citrine tau-Citrine and Cerulean tau-Cerulean.

No spontaneous accumulation of cotransfected tau-Citrine and tau-Cerulean was detected in untreated neurons.

Coculture of neurons with activated microglia induced aggregation of tau in neurites. Treatment of neurons with tumor necrosis factor-alpha TNF-alpha stimulated reactive oxygen species generation and resulted in the accumulation of tau-Citrine and tau-Cerulean in neurites, which was inhibited by neutralization of TNF and the free radical inhibitor 6-hydroxy-2,5,7,8-tetramethylchromanecarboxylic acid Trolox.

These data demonstrate that activated microglia and the microglial-derived proinflammatory cytokine TNF can induce accumulation of the aggregation-prone tau molecules in neurites via reactive oxygen species.

Microglial Clearance Function in Health and Disease. Microglial cells are of hematopoietic origin, populate the CNS during early development and form the brain's innate immune cell type.

Besides their well-known role in immune defense, microglia have an active and homeostatic function in the normal CNS based on high motility of their ramified processes and endocytic clearance of apoptotic vesicular material.

During development microglia contribute to the reorganization of neuronal connections, however microglia have also pivotal roles during acute and chronic neurodegeneration.

Microglia become attracted to site of injury by nucleotides released from damaged neurons. Scavenger receptors expressed on microglia bind to debris and microglial phagocytic receptors signal via immunoreceptor tyrosine-based activation motif ITAM --containing adaptor proteins to promote phagocytosis of extracellular material.

Insufficient clearance by microglia appears to be prevalent in neurodegenerative diseases such as Alzheimer's disease.

Multiple sclerosis MS is a demyelinating autoimmune disease. However, the persisting neurological deficits in MS patients result from acute axonal injury and chronic neurodegeneration, which are both triggered by the autoreactive immune response.

Innate immunity, mainly mediated by activated microglial cells and invading macrophages, appears to contribute to chronic neurodegeneration.

Activated microglia produce several reactive oxygen species and proinflammatory cytokines which affect neuronal function, integrity and survival.

Understanding the mechanisms of immune-mediated neuronal damage might help to design novel therapy strategies for MS. Microglia derived from embryonic stem cells as a tool to study microglia function.

Systemic injection of hematopoietic stem cells after ischemic cardiac or neural lesions is one approach to promote tissue repair. However, mechanisms of possible protective or reparative effects are poorly understood.

In this study we analyzed the effect of lineage-negative bone marrow-derived hematopoietic stem and precursor cells Lin - -HSCs on ischemic brain injury in mice.

Lin - -HSCs were injected intravenously at 24 hours after onset of a minute transient cerebral ischemia. Effects of Lin - -HSCs injection on infarct size, apoptotic cell death, postischemic inflammation and cytokine gene transcription were analyzed.

Green fluorescent protein GFP -marked Lin - -HSCs were detected at 24 hours after injection in the spleen and later in ischemic brain parenchyma, expressing microglial but no neural marker proteins.

Tissue injury assessment showed significantly smaller infarct volumes and less apoptotic neuronal cell death in peri-infarct areas of Lin - -HSC-treated animals.

Analysis of immune cell infiltration in ischemic hemispheres revealed a reduction of invading T cells and macrophages in treated mice.

Moreover, Lin - -HSC therapy counter-regulated proinflammatory cytokine and chemokine receptor gene transcription within the spleen.

Our data demonstrate that systemically applied Lin - -HSCs reduce cerebral postischemic inflammation, attenuate peripheral immune activation and mediate neuroprotection after ischemic stroke.

Von der Entzündung zur Degeneration: Microglia are cells of myeloid origin that populate the CNS during early development and form the brain's innate immune cell type.

They perform homoeostatic activity in the normal CNS, a function associated with high motility of their ramified processes and their constant phagocytic clearance of cell debris.

This debris clearance role is amplified in CNS injury, where there is frank loss of tissue and recruitment of microglia to the injured area.

Recent evidence suggests that this phagocytic clearance following injury is more than simply tidying up, but instead plays a fundamental role in facilitating the reorganization of neuronal circuits and triggering repair.

Insufficient clearance by microglia, prevalent in several neurodegenerative diseases and declining with ageing, is associated with an inadequate regenerative response.

Thus, understanding the mechanism and functional significance of microglial-mediated clearance of tissue debris following injury may open up exciting new therapeutic avenues.

Neuronal 'On' and 'Off' signals control microglia. Recent findings indicate that neurons are not merely passive targets of microglia but rather control microglial activity.

The variety of different signals that neurons use to control microglia can be divided into two categories: They instruct microglia activation under pathological conditions towards a beneficial or detrimental phenotype.

Various neuronal signaling molecules thus actively control microglia function, thereby contribute to the inflammatory milieu of the central nervous system.

Thus, neurons should be envisaged as key immune modulators in the brain. An important step in the pathogenesis of multiple sclerosis is adhesion and transmigration of encephalitogenic T cells across brain endothelial cells EC which strongly relies on interaction with EC-expressed adhesion molecules.

We provide molecular evidence that the transcription factor peroxisome proliferator-activated receptor gamma PPARgamma is a negative regulator of brain EC inflammation.

Adult stem cells are promising cellular vehicles for therapy of malignant gliomas as they have the ability to migrate into these tumors and even track infiltrating tumor cells.

However, their clinical use is limited by a low passaging capacity that impedes large-scale production. In the present study, a bone marrow-derived, highly proliferative subpopulation of mesenchymal stem cells MSCs -here termed bone marrow-derived tumor-infiltrating cells BM-TICs -was genetically modified for the treatment of malignant glioma.

Upon injection into the tumor or the vicinity of the tumor, BM-TICs infiltrated solid parts as well as the border of rat 9L glioma.

Therapeutic efficacy was monitored by PET as well as by magnetic resonance imaging MRI and strongly correlated with histological analysis.

In conclusion, BM-TICs expressing a suicide gene were highly effective in the treatment of malignant glioma in a rat model and therefore hold great potential for the therapy of malignant brain tumors in humans.

A retroviral packaging cell line for pseudotype vectors based on glioma-infiltrating progenitor cells.

Early clinical trials for gene therapy of human gliomas with retroviral packaging cells PC have been hampered by low transduction efficacy and lack of dissemination of PC within the tumor.

In the current approach, these issues have been addressed by creating a stable packaging cell line for retroviral vectors pseudotyped with glycoproteins of lymphocytic choriomeningitis virus LCMV based on tumor-infiltrating progenitor cells.

Packaging of a retroviral vector was measured by titration experiments on human fibroblast cells as well as on mouse and human glioma cell lines.

Additionally, gene transfer was tested in a rat glioma model in vivo. This proof-of-principle study shows that primary adult progenitor cells with tumor-infiltrating capacity can be genetically modified to stably produce retroviral LCMV pseudotype vectors.

Cells were analyzed after 2 d of stimulation by flow cytometry. No change in gene transcript levels of inflammatory mediators was detected after stimulation of TREM2 solely.

In multiple sclerosis, inflammation can successfully be prevented, while promoting repair is still a major challenge. Microglial cells, the resident phagocytes of the central nervous system CNS , are hematopoietic-derived myeloid cells and express the triggering receptor expressed on myeloid cells 2 TREM2 , an innate immune receptor.

Myeloid cells are an accessible source for ex vivo gene therapy. We investigated whether myeloid precursor cells genetically modified to express TREM2 affect the disease course of experimental autoimmune encephalomyelitis EAE , an animal model of multiple sclerosis.

EAE was induced in mice by immunization with a myelin autoantigen. Intravenous application of TREM2-transduced bone marrow-derived myeloid precursor cells at the EAE peak led to an amelioration of clinical symptoms, reduction in axonal damage, and prevention of further demyelination.

TREM2-transduced myeloid cells applied intravenously migrated into the inflammatory spinal cord lesions of EAE-diseased mice, showed increased lysosomal and phagocytic activity, cleared degenerated myelin, and created an anti-inflammatory cytokine milieu within the CNS.

Intravenously applied bone marrow-derived and TREM2-tranduced myeloid precursor cells limit tissue destruction and facilitate repair within the murine CNS by clearance of cellular debris during EAE.

TREM2 is a new attractive target for promotion of repair and resolution of inflammation in multiple sclerosis and other neuroinflammatory diseases.

Essential role of the microglial triggering receptor expressed on myeloid cells-2 TREM2 for central nervous tissue immune homeostasis.

While there is a strong evidence for neural tissue destruction mediated by adaptive autoimmune responses, it is still debated how innate immune responses contribute to neuroinflammatory and neurodegenerative diseases such as multiple sclerosis and Alzheimer's disease.

Recently, it was shown that loss-of-function mutations of the innate microglial immune receptor triggering receptor expressed on myeloid cells-2 TREM2 led to a chronic neurodegenerative disease, named Nasu-Hakola disease or polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy PLOSL.

PLOSL is a recessively inherited disease characterized by early onset adult dementia associated with bone cysts.

Indeed, TREM2 of microglia fulfils important function of tissue debris clearance and resolution of latent inflammatory reactions. Absence of TREM2 expression on microglia impairs their capacity to phagocytose cell membrane debris and increases their gene transcription of pro-inflammatory cytokines.

The disease PLOSL and the finding that TREM2 of microglia is required for tissue debris clearance provide prototypic molecular evidence that dysfunctional innate immunity can be disease causative leading to a chronic neurodegenerative process.

Our results provide molecular evidence that the transcription factor peroxisome proliferator-activated receptor gamma PPARg is a negative regulator of brain EC inflammation.

This effect is clearly PPARg mediated, as lentiviral PPARg overexpression in EC results in selective abrogation of inflammation-induced T cell adhesion and subsequent transmigration, but permits adhesion and transmigration of T cells under non-inflammatory conditions.

We therefore propose that PPARg activation in EC selectively curtails inflammation-induced EC-T cell interactions and may therefore be exploited to selectively target detrimental EC-T cell interactions under inflammatory conditions.

Multiple sclerosis MS is pathologically characterized by inflammatory demyelination and neuronal injury. Although phagocytosis of myelin debris by microglia and macrophages in acute MS lesions is well documented, its pathophysiological significance is unclear.

Using real-time quantitative PCR, flow cytometry, ELISA, and reactive oxygen species ROS measurement assays, we demonstrated that phagocytosis of myelin modulates activation of microglial cells prestimulated by interferon-gamma IFN-gamma or a combination of IFN-gamma and lipopolysaccharide with a biphasic temporal pattern, i.

In this second phase, myelin phagocytosis leads to an enhanced release of prostaglandin E2 and ROS in microglia, whereas the production of anti-inflammatory cytokines particularly interleukin remains unchanged.

Suppression of inflammatory microglial activation by myelin phagocytosis was reversed by treatment with superoxide dismutase and catalase, by inhibition of the NADPH-oxidase complex, or by specific knockdown of the NADPH-oxidase-required adaptor pphagocyte oxidase PHOX.

Furthermore, we observed that myelin phagocytosis destabilized tumor necrosis factor-alpha and interferon-induced protein mRNA through an adenine-uridine-rich elements-involved mechanism, which was reversed by blocking the function of NADPH-oxidase complex.

We conclude that phagocytosis of myelin suppresses microglial inflammatory activities via enhancement of pPHOX-mediated ROS generation.

These results suggest that intervention in ROS generation could represent a novel therapeutic strategy to reduce neuroinflammation in MS.

Unloading kinesin transported cargoes from the tubulin track via the inflammatory c-Jun N-terminal kinase pathway.

Axonal transport of mitochondria and synaptic vesicle precursors via kinesin motor proteins is essential to keep integrity of axons and synapses.

Disturbance of axonal transport is an early sign of neuroinflammatory and neurodegenerative diseases. Treatment of cultured neurons by the inflammatory cytokine tumor necrosis factor-alpha TNF stimulated phosphorylation of c-Jun N-terminal kinase JNK in neurites.

Furthermore, TNF inhibited axonal transport of mitochondria and synaptophysin by reducing the mobile fraction via JNK. Thus, TNF produced by activated glial cells in inflammatory or degenerative neurological diseases acts on neurites by acting on the kinesin-tubulin complex and inhibits axonal mitochondria and synaptophysin transport via JNK.

A cellular vehicle for CNS gene therapy. MLD is characterized by progressive demyelination and neurological deficits. Treatment of MLD is still a challenge due to the fact that the blood-brain barrier is a major obstacle for most therapeutic substances.

In this issue of the JCI, Biffi et al. Clearance of tissue debris by TREM2-transduced myeloid cells promotes recovery of experimental autoimmune encephalomyelitis.

Sep 8th International Conference of Neuroimmunology. Bystander mediated therapy of experimental malignant glioma with adult progenitor cells.

Sep 51st Annual Meeting of the. Molecular Therapy 13, S25 [ndash] S25; doi: A receptor for phagocytosis of Alzheimer's amyloid peptide. The amyloid beta peptide 42 Abeta 42 plays a key role in neurotoxicity in Alzheimer's disease.

Recently, the lipopolysaccharide LPS receptor CD14 was shown to mediate phagocytosis of bacterial components and furthermore to contribute to neuroinflammation in Alzheimer's disease.

Here, we investigated whether this key innate immunity receptor can interact with Abeta 42 and mediate phagocytosis of this peptide. Using flow cytometry, confocal microscopy and two-photon fluorescence lifetime imaging FLIM combined with fluorescence resonance energy transfer FRET , we demonstrated a direct molecular interaction in the range of a few nanometers between Abeta 42 and CD14 in human CDtransfected Chinese hamster ovary cells.

This phagocytosis occurred at Abeta 42 concentration ranges that were considerably lower than the threshold to activate a cellular inflammatory reaction.

In contrast, there was no association of CD14 to microglial internalization of microbeads. In complementary clinical experiments, we detected a pronounced CD14 immunoreactivity on parenchymal microglia spatially correlated to characteristic Alzheimer's disease lesion sites in brain sections of Alzheimer's disease patients but not in brain sections of control subjects.

By showing a close interaction between CD14 and Abeta 42 , demonstrating a direct role of CD14 in Abeta 42 phagocytosis, and detecting CDspecific staining in brains of Alzheimer's disease patients, our results indicate a role of the LPS receptor in the pathophysiology of Alzheimer's disease, which could be of therapeutic relevance.

Malignant gliomas, the most frequent primary brain tumors, still have a dismal prognosis despite advances in neurosurgery, radiation, and chemotherapy.

Gene therapy using viral vectors represents an attractive alternative to conventional cancer therapies. In particular, retroviral vectors have been regarded as potent candidates as they transduce only dividing cells, thereby achieving specificity for tumor cells.

However, clinical trials have failed, because in a given treatment window, only the minority of tumor cells is dividing and therefore susceptible to retroviral infection.

To overcome this problem we used lentiviral vectors, which can transduce dividing as well as quiescent cells, for gene therapy of malignant glioma.

In a previous study we established lentiviral vectors pseudotyped with lymphocytic choriomeningitis LCMV glycoproteins GP and demonstrated efficient transduction of human malignant glioma cells in culture.

In the current investigation, we compared the transduction efficacy of LCMV GP- and vesicular stomatitis virus glycoprotein VSV G -pseudotyped lentiviral vectors for malignant glioma cells and normal brain cells in vitro and in vivo Miletic et al.

LCMV pseudotypes transduced predominantly astrocytes, whereas VSV-G pseudotypes infected neurons as well as astrocytes in vitro and in the hippocampus and striatum of Fischer rats.

LCMV pseudotypes showed an efficient transduction of solid glioma parts of 9L tumors In contrast, VSV-G pseudotyped lentiviral vectors transduced only a few tumor cells in solid 6.

We could demonstrate a high transduction efficiency of 9L tumors Fig. The Kaplan-Meier survival plot Fig. In conclusion, lentiviral vectors pseudotyped with LCMV glycoproteins represent an attractive option for gene therapy of malignant glioma.

Clearance of apoptotic neurons without inflammation by microglial triggering receptor expressed on myeloid cells Elimination of apoptotic neurons without inflammation is crucial for brain tissue homeostasis, but the molecular mechanism has not been firmly established.

Triggering receptor expressed on myeloid cells-2 TREM2 is a recently identified innate immune receptor. Here, we show expression of TREM2 in microglia.

TREM2 stimulation induced DAP12 phosphorylation, extracellular signal-regulated kinase phosphorylation, and cytoskeleton reorganization and increased phagocytosis.

Knockdown of TREM2 in microglia inhibited phagocytosis of apoptotic neurons and increased gene transcription of tumor necrosis factor alpha and nitric oxide synthase-2, whereas overexpression of TREM2 increased phagocytosis and decreased microglial proinflammatory responses.

Breakdown of axonal synaptic vesicle precursor transport by microglial nitric oxide. The mechanism of axonal injury in inflammatory brain diseases is still unclear.

Increased microglial production of nitric oxide NO is a common early sign in neuroinflammatory diseases. We found by fluorescence correlation spectroscopy that synaptophysin tagged with enhanced green fluorescence protein synaptophysin-EGFP moves anterogradely in axons of cultured neurons.

Thus, overt production of reactive NO by activated microglia blocks the axonal transport of synaptic vesicle precursors via phosphorylation of JNK and could cause axonal and synaptic dysfunction.

Malignant gliomas are the most frequent primary brain tumors and have a dismal prognosis due to their infiltrative growth. In a previous study, we established lentiviral vectors pseudotyped with lymphocytic choriomeningitis virus LCMV glycoproteins GPs and demonstrated transduction of human malignant glioma cells in culture.

In the current approach, we compared the transduction efficacy of LCMV-GP- and vesicular stomatitis virus glycoprotein VSV-G -pseudotyped lentiviral vectors for malignant glioma cells and normal brain cells in vitro and in vivo.

LCMV-GP pseudotypes showed an efficient transduction of solid glioma parts and specific transduction of infiltrating tumor cells.

In contrast, VSV-G-pseudotyped lentiviral vectors transduced only a few tumor cells in solid tumor parts and infected mostly normal brain cells in infiltrating tumor areas.

Specific and efficient transduction of malignant glioma by lentiviral vectors pseudotyped with LCMV glycoproteins.

Oct 49th Annual Meeting of the. Siglecs bind to the sialic acid cap of the intact glycocalyx. Other lectin family members such as galectins recognize lactosamine units typically exposed after alteration of the glycocalyx.

Dependent on the type of microglial carbohydrate-binding receptors that are stimulated, either a pro-inflammatory cytotoxic or an anti-inflammatory repair-promoting response is evoked.

The carbohydrate-binding receptors are also crucial in regulating microglial function such as phagocytosis during neurodegenerative or neuroinflammatory processes.

A balance between microglial carbohydrate-binding receptor signaling via an immunoreceptor tyrosine-based activation motif or an immunoreceptor tyrosine-based inhibitory motif is required to polarize microglial cells appropriately so that they create a microenvironment permissive for neural regenerative events.

Microglial cells are professional phagocytes of the CNS responsible for clearance of unwanted structures.

Neuronal processes are marked by complement C1 before they are removed in development or during disease processes. Target molecules involved in C1 binding and mechanisms of clearance are still unclear.

Here we show that the terminal sugar residue sialic acid of the mouse neuronal glycocalyx determines complement C1 binding and microglial-mediated clearance function.

Several early components of the classical complement cascade including C1q, C1r, C1s, and C3 were produced by cultured mouse microglia.

The opsonin C1q was binding to neurites after enzymatic removal of sialic acid residues from the neuronal glycocalyx.

Desialylated neurites, but not neurites with intact sialic acid caps, were cleared and taken up by cocultured microglial cells.

Data demonstrate that mouse microglial cells via CR3 recognize and remove neuronal structures with an altered neuronal glycocalyx lacking terminal sialic acid.

Spatially pathogenic forms of tau detected in Alzheimer's disease brain tissue by fluorescence lifetime-based Forster resonance energy transfer.

In tauopathies including Alzheimer's disease AD tau molecules have lost their normal spatial distance to each other and appear in oligomeric or aggregated forms.

Conventional immunostaining methods allow detection of abnormally phosphorylated or conformationally altered aggregated tau proteins, but fail to visualize oligomeric forms of tau.

Here we show that tau molecules that lost their normal spatial localization can be detected on a subcellular level in postmortem central nervous system CNS tissue sections of AD patients by fluorescence lifetime-based Förster resonance energy transfer FRET.

Paraffin sections were co-immunostained with two tau-specific monoclonal antibodies recognizing the same epitope, but labeled with distinct fluorescence dyes suitable for spatial resolution at a nanometer scale by lifetime-based FRET.

A FRET signal was detected in neuritic plaques and neurofibrillary tangles of CNS tissue sections of AD patients, showing associated tau proteins typically reflecting either fibrillary, oligomeric or aggregated tau.

The 'pretangle-like' structures within the neuronal perikarya did not contain spatially pathogenic forms of tau accordingly to this method.

Data demonstrate that fluorescence lifetime-based FRET can be applied to human brain tissue sections to detect pathogenic forms of tau molecules that lost their normal spatial distance.

Protective effects of microGlia in multiple sclerosis. The role of microglia in demyelinating neurodegenerative diseases such as multiple sclerosis MS and its animal model experimental autoimmune encephalomyelitis EAE is still controversial.

Although microglial cells are known as the professional phagocytes and executer of innate immunity in the central nervous system CNS , it is believed that microglia are rather neurotoxic in these diseases.

However, there is recent evidence indicating that microglia could also exert a neuroprotective function in MS and EAE. First evidence for the protective effect of immune cells in CNS diseases emerged from studies in invertebrates.

In the medicinal leech, the process of regeneration begins with rapid activation and accumulation of phagocytic glial cells at the lesion site followed by phagocytosis of damaged tissue by these cells which promoted robust neural regeneration.

In vertebrates, several lines of evidence demonstrate that microglia are also involved in neuroprotection by the secretion of soluble mediators that trigger neural repair and usually contribute to the creation of an environment conductive for regeneration.

The efficient removal of apoptotic cells and clearance of debris at the lesion site and the recruitment of stem cell populations as well as the induction of neurogenesis are directly correlated.

These findings suggest that microglia play a major role in creating a microenvironment for repair and regenerative processes in demyelinating neuroinflammatory diseases.

Siglec-F receptor and neuroprotection in mouse central nervous system. Sep Pharmacological Reports. Generation of microglial cells from mouse embryonic stem cells.

Microglia, the resident immune cells of the brain, are difficult to obtain in high numbers and purity using currently available methods; to date, microglia for experimental research are mainly isolated from the brain or from mixed glial cultures.

In this paper, we describe a basic protocol for the in vitro differentiation of mouse embryonic stem ES cells into microglial precursor cells. Microglia are obtained by a protocol consisting of five stages: The protocol can be completed in 60 d and results in stably proliferating ESdM lines, which show inducible transcription of inflammatory genes and cell marker expression comparable with primary microglia.

Furthermore, ESdMs are capable of chemokine-directed migration and phagocytosis, which are major functional features of microglia. Functional role of brain-derived neurotrophic factor in neuroprotective autoimmunity: Therapeutic implications in a model of multiple sclerosis.

Brain-derived neurotrophic factor plays a key role in neuronal and axonal survival. Brain-derived neurotrophic factor is expressed in the immune cells in lesions of experimental autoimmune encephalomyelitis and multiple sclerosis, thus potentially mediating neuroprotective effects.

We investigated the functional role of brain-derived neurotrophic factor in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis.

Mice deficient for brain-derived neurotrophic factor in immune cells displayed an attenuated immune response in the acute phase of experimental autoimmune encephalomyelitis, but progressive disability with enhanced axonal loss in the chronic phase of the disease.

In mice deficient for central nervous system-derived brain-derived neurotrophic factor via glial fibrillary acidic protein-crescentin-mediated deletion, a more severe course of experimental autoimmune encephalomyelitis and an overall increased axonal loss was observed.

In a lentiviral approach, injection of brain-derived neurotrophic factor-overexpressing T cells led to a less severe course of experimental autoimmune encephalomyelitis and direct axonal protection.

Our data imply a functional role of brain-derived neurotrophic factor in autoimmune demyelination by mediating axon protection.

Vaccine-based autoimmune anti-amyloid treatments are currently being examined for their therapeutic potential in Alzheimer's disease.

In the present study we examined, in a transgenic model of amyloid pathology, the expression of two molecules previously implicated in decreasing the severity of autoimmune responses: TREM2 triggering receptor expressed on myeloid cells 2 and the intracellular tolerance-associated transcript, Tmemb transmembrane domain protein b.

In situ hybridization analysis revealed that both molecules were highly expressed in plaque-associated microglia, but their expression defined two different zones of plaque-associated activation.

Tmemb expression was highest in the inner zone of amyloid plaques, whereas TREM2 expression was highest in the outer zone.

Induced expression of TREM2 occurred co-incident with detection of thioflavine-S-positive amyloid deposits. TREM2 expression also showed a positive correlation with amyloid phagocytosis in unactivated cells.

Transfection of Tmemb into both microglial and macrophage cell lines increased apoptosis. Elimination of extracellular aggregates and apoptotic neural membranes without inflammation is crucial for brain tissue homeostasis.

In the mammalian central nervous system, essential molecules in this process are the Fc receptors and the DAPassociated receptors which both trigger the microglial immunoreceptor tyrosine-based activation motif- ITAM- Syk-signaling cascade.

Microglial ITAM-signaling receptors are counter-regulated by immunoreceptor tyrosine-based inhibition motif- ITIM- signaling molecules such as sialic acid-binding immunoglobulin superfamily lectins Siglecs.

Siglecs can suppress the proinflammatory and phagocytic activity of microglia via ITIM signaling. Moreover, microglial neurotoxicity is alleviated via interaction of Siglec with sialic acids on the neuronal glycocalyx.

Their dysfunction could lead to impaired phagocytic clearance and neurodegeneration triggered by chronic inflammation.

Alleviation of Neurotoxicity by Microglial Human Siglec Sialic acid-binding Ig superfamily lectins Siglecs are members of the Ig superfamily that recognize sialic acid residues of glycoproteins.

Siglec is a recently identified human-specific CDrelated Siglec that binds to alpha2,8-linked polysialic acids and is expressed on microglia, the brain resident innate immune cells.

We observed gene transcription and protein expression of Siglec splice variant 2 in human brain tissue samples by RT-PCR and Western blot analysis.

Siglec was detected on microglia in human brain tissue by immunohistochemistry. Human Siglec splice variant 2 was ectopically expressed by a lentiviral vector system in cultured murine microglial cells.

Stimulation of Siglec by cross-linking suppressed the lipopolysaccharides LPS -induced gene transcription of the proinflammatory mediators interleukin-1beta and nitric oxide synthase-2 in microglia.

Furthermore, phagocytosis of apoptotic neuronal material was reduced in Siglec transduced microglia. Coculture of microglia transduced with Siglec and neurons demonstrated neuroprotective function of Siglec Thus, data demonstrate that human Siglec ectopically expressed on murine microglia interacts with PSA on neurons, reduces LPS-induced gene transcription of proinflammatory mediators, impairs phagocytosis and alleviates microglial neurotoxicity.

Microglial precursors derived from mouse embryonic stem cells. Microglia are resident immune cells of the central nervous system. They can be directly isolated from the brain or from mixed postnatal glial cultures.

Isolation of primary microglia is inefficient due to low yield. The cell line BV2 was used as a substitute for primary microglia, but BV2 are oncogenically transformed cells.

Here, we established a protocol to generate microglial precursor lines from mouse embryonic stem ES cells.

Microglial precursor cells were obtained from murine ES cells by differentiation of embryoid bodies to microglia within a mixed brain culture.

Several independent ES cell-derived microglial precursor ESdM lines were generated and characterized by flow cytometry, immunocytochemistry, and functional assays.

Stimulation with interferon-gamma or lipopolysaccharide LPS demonstrated upregulation of proinflammatory cytokine gene transcription including nitric oxide synthase-2, interleukin-1beta, and tumor necrosis factor-alpha at levels comparable to primary microglia.

The ESdM showed efficient and rapid phagocytosis of microsphere beads, which was increased after stimulation with LPS. After in vivo transplantation into postnatal brain tissue, ESdM showed engraftment as cells with a microglial phenotype and morphology.

Thus, ESdM are stable proliferating cells substantially having most characteristics of primary microglia and therefore being a suitable tool to study microglial function in vitro and in vivo.

Signal Regulatory Protein 1: The signal regulatory protein-beta1 SIRPbeta1 is a DAPassociated transmembrane receptor expressed in a subset of hematopoietic cells.

Recently, it was shown that peritoneal macrophages express SIRPbeta1, which positively regulated phagocytosis. Activation of SIRPbeta1 on cultured microglia by cross-linking antibodies induced reorganization of the cytoskeleton protein beta-actin and suppressed lipopolysaccharide-induced gene transcription of tumor necrosis factor-alpha and nitric oxide synthase Furthermore, activation of SIRPbeta1 increased phagocytosis of microsphere beads, neural debris, and fibrillary amyloid-beta Abeta.

Phagocytosis of neural cell debris and Abeta was impaired after lentiviral knockdown of SIRPbeta1 in primary microglial cells. Role of microglia in neuronal degeneration and regeneration.

Microglial cells, the resident macrophage population of the central nervous system CNS , actively scan tissue under both normal and pathologic contexts.

Their resulting engagement can become either neuroprotective or neurotoxic, leading to amelioration or aggravation of disease progression.

In this review, we focus on the molecular signaling molecules involved in microglial responses and discuss observations demonstrating the diverse effects of microglia in animal models of CNS diseases.

Regenerative therapy of experimental autoimmune encephalomyelitis by neurotrophin-3 transduced ES cell derived microglial cells.

Sep 25th Congress of the. Accumulation of tau induced in neurites by microglial proinflammatory mediators. Aggregated fibrillary microtubule-associated protein tau is the major component of neurofibrillary tangles in Alzheimer's disease.

The exact molecular mechanism of tau aggregation is unknown. Microglial cell activation and migration toward amyloid-beta plaques precede the appearance of dysmorphic neurites and formation of neurofibrillary tangles.

Here, we analyzed the accumulation of tau at a distance range of expected spontaneous aggregation by fluorescence lifetime-based Förster resonance energy transfer in cultured primary murine neurons cotransfected with the human tau gene tagged to the green fluorescent protein variants Citrine tau-Citrine and Cerulean tau-Cerulean.

No spontaneous accumulation of cotransfected tau-Citrine and tau-Cerulean was detected in untreated neurons. Coculture of neurons with activated microglia induced aggregation of tau in neurites.

Treatment of neurons with tumor necrosis factor-alpha TNF-alpha stimulated reactive oxygen species generation and resulted in the accumulation of tau-Citrine and tau-Cerulean in neurites, which was inhibited by neutralization of TNF and the free radical inhibitor 6-hydroxy-2,5,7,8-tetramethylchromanecarboxylic acid Trolox.

These data demonstrate that activated microglia and the microglial-derived proinflammatory cytokine TNF can induce accumulation of the aggregation-prone tau molecules in neurites via reactive oxygen species.

Microglial Clearance Function in Health and Disease. Microglial cells are of hematopoietic origin, populate the CNS during early development and form the brain's innate immune cell type.

Besides their well-known role in immune defense, microglia have an active and homeostatic function in the normal CNS based on high motility of their ramified processes and endocytic clearance of apoptotic vesicular material.

During development microglia contribute to the reorganization of neuronal connections, however microglia have also pivotal roles during acute and chronic neurodegeneration.

Microglia become attracted to site of injury by nucleotides released from damaged neurons. Scavenger receptors expressed on microglia bind to debris and microglial phagocytic receptors signal via immunoreceptor tyrosine-based activation motif ITAM --containing adaptor proteins to promote phagocytosis of extracellular material.

Insufficient clearance by microglia appears to be prevalent in neurodegenerative diseases such as Alzheimer's disease. Multiple sclerosis MS is a demyelinating autoimmune disease.

However, the persisting neurological deficits in MS patients result from acute axonal injury and chronic neurodegeneration, which are both triggered by the autoreactive immune response.

Innate immunity, mainly mediated by activated microglial cells and invading macrophages, appears to contribute to chronic neurodegeneration.

Activated microglia produce several reactive oxygen species and proinflammatory cytokines which affect neuronal function, integrity and survival.

Understanding the mechanisms of immune-mediated neuronal damage might help to design novel therapy strategies for MS.

Microglia derived from embryonic stem cells as a tool to study microglia function. Systemic injection of hematopoietic stem cells after ischemic cardiac or neural lesions is one approach to promote tissue repair.

However, mechanisms of possible protective or reparative effects are poorly understood. In this study we analyzed the effect of lineage-negative bone marrow-derived hematopoietic stem and precursor cells Lin - -HSCs on ischemic brain injury in mice.

Lin - -HSCs were injected intravenously at 24 hours after onset of a minute transient cerebral ischemia.

Effects of Lin - -HSCs injection on infarct size, apoptotic cell death, postischemic inflammation and cytokine gene transcription were analyzed.

Green fluorescent protein GFP -marked Lin - -HSCs were detected at 24 hours after injection in the spleen and later in ischemic brain parenchyma, expressing microglial but no neural marker proteins.

Tissue injury assessment showed significantly smaller infarct volumes and less apoptotic neuronal cell death in peri-infarct areas of Lin - -HSC-treated animals.

Analysis of immune cell infiltration in ischemic hemispheres revealed a reduction of invading T cells and macrophages in treated mice.

Moreover, Lin - -HSC therapy counter-regulated proinflammatory cytokine and chemokine receptor gene transcription within the spleen.

Our data demonstrate that systemically applied Lin - -HSCs reduce cerebral postischemic inflammation, attenuate peripheral immune activation and mediate neuroprotection after ischemic stroke.

Von der Entzündung zur Degeneration: Microglia are cells of myeloid origin that populate the CNS during early development and form the brain's innate immune cell type.

They perform homoeostatic activity in the normal CNS, a function associated with high motility of their ramified processes and their constant phagocytic clearance of cell debris.

This debris clearance role is amplified in CNS injury, where there is frank loss of tissue and recruitment of microglia to the injured area.

Recent evidence suggests that this phagocytic clearance following injury is more than simply tidying up, but instead plays a fundamental role in facilitating the reorganization of neuronal circuits and triggering repair.

Insufficient clearance by microglia, prevalent in several neurodegenerative diseases and declining with ageing, is associated with an inadequate regenerative response.

Thus, understanding the mechanism and functional significance of microglial-mediated clearance of tissue debris following injury may open up exciting new therapeutic avenues.

Neuronal 'On' and 'Off' signals control microglia. Recent findings indicate that neurons are not merely passive targets of microglia but rather control microglial activity.

The variety of different signals that neurons use to control microglia can be divided into two categories: They instruct microglia activation under pathological conditions towards a beneficial or detrimental phenotype.

Various neuronal signaling molecules thus actively control microglia function, thereby contribute to the inflammatory milieu of the central nervous system.

Thus, neurons should be envisaged as key immune modulators in the brain. An important step in the pathogenesis of multiple sclerosis is adhesion and transmigration of encephalitogenic T cells across brain endothelial cells EC which strongly relies on interaction with EC-expressed adhesion molecules.

We provide molecular evidence that the transcription factor peroxisome proliferator-activated receptor gamma PPARgamma is a negative regulator of brain EC inflammation.

Adult stem cells are promising cellular vehicles for therapy of malignant gliomas as they have the ability to migrate into these tumors and even track infiltrating tumor cells.

However, their clinical use is limited by a low passaging capacity that impedes large-scale production. In the present study, a bone marrow-derived, highly proliferative subpopulation of mesenchymal stem cells MSCs -here termed bone marrow-derived tumor-infiltrating cells BM-TICs -was genetically modified for the treatment of malignant glioma.

Upon injection into the tumor or the vicinity of the tumor, BM-TICs infiltrated solid parts as well as the border of rat 9L glioma. Therapeutic efficacy was monitored by PET as well as by magnetic resonance imaging MRI and strongly correlated with histological analysis.

In conclusion, BM-TICs expressing a suicide gene were highly effective in the treatment of malignant glioma in a rat model and therefore hold great potential for the therapy of malignant brain tumors in humans.

A retroviral packaging cell line for pseudotype vectors based on glioma-infiltrating progenitor cells. Early clinical trials for gene therapy of human gliomas with retroviral packaging cells PC have been hampered by low transduction efficacy and lack of dissemination of PC within the tumor.

In the current approach, these issues have been addressed by creating a stable packaging cell line for retroviral vectors pseudotyped with glycoproteins of lymphocytic choriomeningitis virus LCMV based on tumor-infiltrating progenitor cells.

Packaging of a retroviral vector was measured by titration experiments on human fibroblast cells as well as on mouse and human glioma cell lines.

Additionally, gene transfer was tested in a rat glioma model in vivo. This proof-of-principle study shows that primary adult progenitor cells with tumor-infiltrating capacity can be genetically modified to stably produce retroviral LCMV pseudotype vectors.

Cells were analyzed after 2 d of stimulation by flow cytometry. No change in gene transcript levels of inflammatory mediators was detected after stimulation of TREM2 solely.

In multiple sclerosis, inflammation can successfully be prevented, while promoting repair is still a major challenge. Microglial cells, the resident phagocytes of the central nervous system CNS , are hematopoietic-derived myeloid cells and express the triggering receptor expressed on myeloid cells 2 TREM2 , an innate immune receptor.

Myeloid cells are an accessible source for ex vivo gene therapy. We investigated whether myeloid precursor cells genetically modified to express TREM2 affect the disease course of experimental autoimmune encephalomyelitis EAE , an animal model of multiple sclerosis.

EAE was induced in mice by immunization with a myelin autoantigen. Intravenous application of TREM2-transduced bone marrow-derived myeloid precursor cells at the EAE peak led to an amelioration of clinical symptoms, reduction in axonal damage, and prevention of further demyelination.

TREM2-transduced myeloid cells applied intravenously migrated into the inflammatory spinal cord lesions of EAE-diseased mice, showed increased lysosomal and phagocytic activity, cleared degenerated myelin, and created an anti-inflammatory cytokine milieu within the CNS.

Intravenously applied bone marrow-derived and TREM2-tranduced myeloid precursor cells limit tissue destruction and facilitate repair within the murine CNS by clearance of cellular debris during EAE.

TREM2 is a new attractive target for promotion of repair and resolution of inflammation in multiple sclerosis and other neuroinflammatory diseases.

Essential role of the microglial triggering receptor expressed on myeloid cells-2 TREM2 for central nervous tissue immune homeostasis.

While there is a strong evidence for neural tissue destruction mediated by adaptive autoimmune responses, it is still debated how innate immune responses contribute to neuroinflammatory and neurodegenerative diseases such as multiple sclerosis and Alzheimer's disease.

Recently, it was shown that loss-of-function mutations of the innate microglial immune receptor triggering receptor expressed on myeloid cells-2 TREM2 led to a chronic neurodegenerative disease, named Nasu-Hakola disease or polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy PLOSL.

PLOSL is a recessively inherited disease characterized by early onset adult dementia associated with bone cysts. Indeed, TREM2 of microglia fulfils important function of tissue debris clearance and resolution of latent inflammatory reactions.

Absence of TREM2 expression on microglia impairs their capacity to phagocytose cell membrane debris and increases their gene transcription of pro-inflammatory cytokines.

The disease PLOSL and the finding that TREM2 of microglia is required for tissue debris clearance provide prototypic molecular evidence that dysfunctional innate immunity can be disease causative leading to a chronic neurodegenerative process.

Our results provide molecular evidence that the transcription factor peroxisome proliferator-activated receptor gamma PPARg is a negative regulator of brain EC inflammation.

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