A light-inducible protein clustering system for in vivo analysis of α-synuclein aggregation in Parkinson disease.

Neurodegenerative disorders refer to a group of diseases commonly associated with abnormal protein accumulation and aggregation in the central nervous system. However, the exact role of protein aggregation in the pathophysiology of these disorders remains unclear. This gap in knowledge is due to the lack of experimental models that allow for the spatiotemporal control of protein aggregation, and the investigation of early dynamic events associated with inclusion formation. Here, we report on the development of a light-inducible protein aggregation (LIPA) system that enables spatiotemporal control of α-synuclein (α-syn) aggregation into insoluble deposits called Lewy bodies (LBs), the pathological hallmark of Parkinson disease (PD) and other proteinopathies. We demonstrate that LIPA-α-syn inclusions mimic key biochemical, biophysical, and ultrastructural features of authentic LBs observed in PD-diseased brains. In vivo, LIPA-α-syn aggregates compromise nigrostriatal transmission, induce neurodegeneration and PD-like motor impairments. Collectively, our findings provide a new tool for the generation, visualization, and dissection of the role of α-syn aggregation in PD.

Conditional Deletions of Hdc Confirm Roles of Histamine in Anaphylaxis and Circadian Activity but Not in Autoimmune Encephalomyelitis.

Histamine is best known for its role in allergies, but it could also be involved in autoimmune diseases such as multiple sclerosis. However, studies using experimental autoimmune encephalomyelitis (EAE), the most widely used animal model for multiple sclerosis, have reported conflicting observations and suggest the implication of a nonclassical source of histamine. In this study, we demonstrate that neutrophils are the main producers of histamine in the spinal cord of EAE mice. To assess the role of histamine by taking into account its different cellular sources, we used CRISPR-Cas9 to generate conditional knockout mice for the histamine-synthesizing enzyme histidine decarboxylase. We found that ubiquitous and cell-specific deletions do not affect the course of EAE. However, neutrophil-specific deletion attenuates hypothermia caused by IgE-mediated anaphylaxis, whereas neuron-specific deletion reduces circadian activity. In summary, this study refutes the role of histamine in EAE, unveils a role for neutrophil-derived histamine in IgE-mediated anaphylaxis, and establishes a new mouse model to re-explore the inflammatory and neurologic roles of histamine.

NTPDase8 protects mice from intestinal inflammation by limiting P2Y6 receptor activation: identification of a new pathway of inflammation for the potential treatment of IBD.

OBJECTIVE: Nucleotides are danger signals that activate inflammatory responses via binding P2 receptors. The nucleoside triphosphate diphosphohydrolase-8 (NTPDase8) is an ectonucleotidase that hydrolyses P2 receptor ligands. We investigated the role of NTPDase8 in intestinal inflammation. DESIGN: We generated NTPDase8-deficient (Entpd8-/-) mice to define the role of NTPDase8 in the dextran sodium sulfate (DSS) colitis model. To assess inflammation, colons were collected and analysed by histopathology, reverse transcriptase-quantitative real-time PCR (RT-qPCR) and immunohistochemistry. P2 receptor expression was analysed by RT-qPCR on primary intestinal epithelium and NTPDase8 activity by histochemistry. The role of intestinal P2Y6 receptors was assessed by bone marrow transplantation experiments and with a P2Y6 receptor antagonist. RESULTS: NTPDase8 is the dominant enzyme responsible for the hydrolysis of nucleotides in the lumen of the colon. Compared with wild-type (WT) control mice, the colon of Entpd8-/- mice treated with DSS displayed significantly more histological damage, immune cell infiltration, apoptosis and increased expression of several proinflammatory cytokines. P2Y6 was the dominant P2Y receptor expressed at the mRNA level by the colonic epithelia. Irradiated P2ry6-/- mice transplanted with WT bone marrow were fully protected from DSS-induced intestinal inflammation. In agreement, the daily intrarectal injection of a P2Y6 antagonist protected mice from DSS-induced intestinal inflammation in a dose-dependent manner. Finally, human intestinal epithelial cells express NTPDase8 and P2Y6 similarly as in mice. CONCLUSION: NTPDase8 protects the intestine from inflammation most probably by limiting the activation of P2Y6 receptors in colonic epithelial cells. This may provide a novel therapeutic strategy for the treatment of inflammatory bowel disease.

B cell-dependent EAE induces visual deficits in the mouse with similarities to human autoimmune demyelinating diseases.

BACKGROUND: In the field of autoimmune demyelinating diseases, visual impairments have extensively been studied using the experimental autoimmune encephalomyelitis (EAE) mouse model, which is classically induced by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). However, this model does not involve B cells like its human analogs. New antigens have thus been developed to induce a B cell-dependent form of EAE that better mimics human diseases. METHODS: The present study aimed to characterize the visual symptoms of EAE induced with such an antigen called bMOG. After the induction of EAE with bMOG in C57BL/6J mice, visual function changes were studied by electroretinography and optomotor acuity tests. Motor deficits were assessed in parallel with a standard clinical scoring method. Histological examinations and Western blot analyses allowed to follow retinal neuron survival, gliosis, microglia activation, opsin photopigment expression in photoreceptors and optic nerve demyelination. Disease effects on retinal gene expression were established by RNA sequencing. RESULTS: We observed that bMOG EAE mice exhibited persistent loss of visual acuity, despite partial recovery of electroretinogram and motor functions. This loss was likely due to retinal inflammation, gliosis and synaptic impairments, as evidenced by histological and transcriptomic data. Further analysis suggests that the M-cone photoreceptor pathway was also affected. CONCLUSION: Therefore, by documenting visual changes induced by bMOG and showing similarities to those seen in diseases such as multiple sclerosis and neuromyelitis optica, this study offers a new approach to test protective or restorative ophthalmic treatments.

Single-cell transcriptomics of the ventral posterolateral nucleus-enriched thalamic regions from HSV-1-infected mice reveal a novel microglia/microglia-like transcriptional response.

BACKGROUND: Microglia participate in the immune response upon central nervous system (CNS) infections. However, the role of these cells during herpes simplex encephalitis (HSE) has not been fully characterized. We sought to identify different microglia/microglia-like cells and describe the potential mechanisms and signaling pathways involved during HSE. METHODS: The transcriptional response of CD11b+ immune cells, including microglia/microglia-like cells, was investigated using single-cell RNA sequencing (scRNA-seq) on cells isolated from the ventral posterolateral nucleus (VPL)-enriched thalamic regions of C57BL/6 N mice intranasally infected with herpes simplex virus-1 (HSV-1) (6 × 105 PFUs/20 µl). We further performed scanning electronic microscopy (SEM) analysis in VPL regions on day 6 post-infection (p.i.) to provide insight into microglial functions. RESULTS: We describe a novel microglia-like transcriptional response associated with a rare cell population (7% of all analyzed cells), named "in transition" microglia/microglia-like cells in HSE. This new microglia-like transcriptional signature, found in the highly infected thalamic regions, was enriched in specific genes (Retnlg, Cxcr2, Il1f9) usually associated with neutrophils. Pathway analysis of this cell-type transcriptome showed increased NLRP3-inflammasome-mediated interleukin IL-1ß production, promoting a pro-inflammatory response. These cells' increased expression of viral transcripts suggests that the distinct "in transition" transcriptome corresponds to the intrinsic antiviral immune signaling of HSV-1-infected microglia/microglia-like cells in the thalamus. In accordance with this phenotype, we observed several TMEM119+/IBA-I+ microglia/microglia-like cells immunostained for HSV-1 in highly infected regions. CONCLUSIONS: A new microglia/microglia-like state may potentially shed light on how microglia could react to HSV-1 infection. Our observations suggest that infected microglia/microglia-like cells contribute to an exacerbated CNS inflammation. Further characterization of this transitory state of the microglia/microglia-like cell transcriptome may allow the development of novel immunomodulatory approaches to improve HSE outcomes by regulating the microglial immune response.

Ultrastructural characterization of dark microglia during aging in a mouse model of Alzheimer's disease pathology and in human post-mortem brain samples.

A diverse heterogeneity of microglial cells was previously described in Alzheimer's disease (AD) pathology, including dark microglia, a state characterized by ultrastructural markers of cellular stress. To provide novel insights into the roles of dark microglia during aging in the context of AD pathology, we performed a quantitative density and ultrastructural analysis of these cells using high-throughput scanning electron microscopy in the ventral hippocampus CA1 stratum lacunosum-moleculare of 20-month-old APP-PS1 vs C57BL/6J male mice. The density of dark microglia was significantly higher in APP-PS1 vs C57BL/6J mice, with these cells accounting for nearly half of all microglia observed near amyloid-beta (Aß) plaques. This dark microglial state interacted more with dystrophic neurites compared to other APP-PS1 microglia and possessed glycogen granules, associated with a metabolic shift toward glycolysis, which provides the first ultrastructural evidence of their presence in microglia. Dark microglia were further observed in aging human post-mortem brain samples showing similar ultrastructural features as in mouse. Overall, our results provide a quantitative ultrastructural characterization of a microglial state associated with cellular stress (i.e., dark microglia) that is primarily restricted near Aß plaques and dystrophic neurites. The presence of this microglial state in the aging human post-mortem brain is further revealed.

MicroRNA-223 protects neurons from degeneration in experimental autoimmune encephalomyelitis.

Multiple sclerosis is a chronic inflammatory, demyelinating, and neurodegenerative disease affecting the brain, spinal cord and optic nerves. Neuronal damage is triggered by various harmful factors that engage diverse signalling cascades in neurons; thus, therapeutic approaches to protect neurons will need to focus on agents that can target multiple biological processes. We have therefore focused our attention on microRNAs: small non-coding RNAs that primarily function as post-transcriptional regulators that target messenger RNAs and repress their translation into proteins. A single microRNA can target many functionally related messenger RNAs making microRNAs powerful epigenetic regulators. Dysregulation of microRNAs has been described in many neurodegenerative diseases including multiple sclerosis. Here, we report that two microRNAs, miR-223-3p and miR-27a-3p, are upregulated in neurons in the experimental autoimmune encephalomyelitis mouse model of CNS inflammation and in grey matter-containing multiple sclerosis lesions. Prior work has shown peripheral blood mononuclear cell conditioned media causes sublethal degeneration of neurons in culture. We find overexpression of miR-27a-3p or miR-223-3p protects dissociated cortical neurons from condition media mediated degeneration. Introduction of miR-223-3p in vivo in mouse retinal ganglion cells protects their axons from degeneration in experimental autoimmune encephalomyelitis. In silico analysis revealed that messenger RNAs involved in glutamate receptor signalling are enriched as miR-27a-3p and miR-223-3p targets. We observe that antagonism of NMDA and AMPA type glutamate receptors protects neurons from condition media dependent degeneration. Our results suggest that miR-223-3p and miR-27a-3p are upregulated in response to inflammation to mediate a compensatory neuroprotective gene expression program that desensitizes neurons to glutamate by targeting messenger RNAs involved in glutamate receptor signalling.

Ultrastructural evidence of microglial heterogeneity in Alzheimer's disease amyloid pathology.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disease, characterized by the deposition of extracellular fibrillar amyloid ß (fΑß) and the intracellular accumulation of neurofibrillary tangles. As AD progresses, Aß drives a robust and prolonged inflammatory response via its recognition by microglia, the brain's immune cells. Microglial reactivity to fAß plaques may impair their normal surveillance duties, facilitating synaptic loss and neuronal death, as well as cognitive decline in AD. METHODS: In the current study, we performed correlative light, transmission, and scanning electron microscopy to provide insights into microglial structural and functional heterogeneity. We analyzed microglial cell bodies and processes in areas containing fAß plaques and neuronal dystrophy, dystrophy only, or appearing healthy, among the hippocampus CA1 of 14-month-old APPSwe-PS1Δe9 mice versus wild-type littermates. RESULTS: Our quantitative analysis revealed that microglial cell bodies in the AD model mice were larger and displayed ultrastructural signs of cellular stress, especially nearby plaques. Microglial cell bodies and processes were overall less phagocytic in AD model mice. However, they contained increased fibrillar materials and non-empty inclusions proximal to plaques. Microglial cell bodies and processes in AD model mice also displayed reduced association with extracellular space pockets that contained debris. In addition, microglial processes in healthy subregions of AD model mice encircled synaptic elements more often compared with plaque-associated processes. These observations in mice were qualitatively replicated in post-mortem hippocampal samples from two patients with AD (Braak stage 5). CONCLUSION: Together, our findings identify at the ultrastructural level distinct microglial transformations common to mouse and human in association with amyloid pathology.

CXCL10 triggers early microglial activation in the cuprizone model.

A broad spectrum of diseases is characterized by myelin abnormalities and/or oligodendrocyte pathology. 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 CXCL10-deficient 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.

Dark microglia: A new phenotype predominantly associated with pathological states.

The past decade has witnessed a revolution in our understanding of microglia. These immune cells were shown to actively remodel neuronal circuits, leading to propose new pathogenic mechanisms. To study microglial implication in the loss of synapses, the best pathological correlate of cognitive decline across chronic stress, aging, and diseases, we recently conducted ultrastructural analyses. Our work uncovered the existence of a new microglial phenotype that is rarely present under steady state conditions, in hippocampus, cerebral cortex, amygdala, and hypothalamus, but becomes abundant during chronic stress, aging, fractalkine signaling deficiency (CX3 CR1 knockout mice), and Alzheimer's disease pathology (APP-PS1 mice). Even though these cells display ultrastructural features of microglia, they are strikingly distinct from the other phenotypes described so far at the ultrastructural level. They exhibit several signs of oxidative stress, including a condensed, electron-dense cytoplasm and nucleoplasm making them as "dark" as mitochondria, accompanied by a pronounced remodeling of their nuclear chromatin. Dark microglia appear to be much more active than the normal microglia, reaching for synaptic clefts, while extensively encircling axon terminals and dendritic spines with their highly ramified and thin processes. They stain for the myeloid cell markers IBA1 and GFP (in CX3 CR1-GFP mice), and strongly express CD11b and microglia-specific 4D4 in their processes encircling synaptic elements, and TREM2 when they associate with amyloid plaques. Overall, these findings suggest that dark microglia, a new phenotype that we identified based on their unique properties, could play a significant role in the pathological remodeling of neuronal circuits, especially at synapses.

The inflammasome pyrin contributes to pertussis toxin-induced IL-1ß synthesis, neutrophil intravascular crawling and autoimmune encephalomyelitis.

Microbial agents can aggravate inflammatory diseases, such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). An example is pertussis toxin (PTX), a bacterial virulence factor commonly used as an adjuvant to promote EAE, but whose mechanism of action is unclear. We have reported that PTX triggers an IL-6-mediated signaling cascade that increases the number of leukocytes that patrol the vasculature by crawling on its luminal surface. In the present study, we examined this response in mice lacking either TLR4 or inflammasome components and using enzymatically active and inactive forms of PTX. Our results indicate that PTX, through its ADP-ribosyltransferase activity, induces two series of events upstream of IL-6: 1) the activation of TLR4 signaling in myeloid cells, leading to pro-IL-1ß synthesis; and 2) the formation of a pyrin-dependent inflammasome that cleaves pro-IL-1ß into its active form. In turn, IL-1ß stimulates nearby stromal cells to secrete IL-6, which is known to induce vascular changes required for leukocyte adhesion. Without pyrin, PTX does not induce neutrophil adhesion to cerebral capillaries and is less effective at inducing EAE in transgenic mice with encephalitogenic T lymphocytes. This study identifies the first microbial molecule that activates pyrin, a mechanism by which infections may influence MS and a potential therapeutic target for immune disorders.

Interleukin-36γ is expressed by neutrophils and can activate microglia, but has no role in experimental autoimmune encephalomyelitis.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is a model of inflammatory demyelinating diseases mediated by different types of leukocytes. How these cells communicate with each other to orchestrate autoimmune attacks is not fully understood, especially in the case of neutrophils, whose importance in EAE is newly established. The present study aimed to determine the expression pattern and role of different components of the IL-36 signaling pathway (IL-36α, IL-36ß, IL-36γ, IL-36R) in EAE. METHODS: EAE was induced by either active immunization with myelin peptide, passive transfer of myelin-reactive T cells or injection of pertussis toxin to transgenic 2D2 mice. The molecules of interest were analyzed using a combination of techniques, including quantitative real-time PCR (qRT-PCR), flow cytometry, Western blotting, in situ hybridization, and immunohistochemistry. Microglial cultures were treated with recombinant IL-36γ and analyzed using DNA microarrays. Different mouse strains were subjected to clinical evaluation and flow cytometric analysis in order to compare their susceptibility to EAE. RESULTS: Our observations indicate that both IL-36γ and IL-36R are strongly upregulated in nervous and hematopoietic tissues in different forms of EAE. IL-36γ is specifically expressed by neutrophils, while IL-36R is expressed by different immune cells, including microglia and other myeloid cells. In culture, microglia respond to recombinant IL-36γ by expressing molecules involved in neutrophil recruitment, such as Csf3, IL-1ß, and Cxcl2. However, mice deficient in either IL-36γ or IL-36R develop similar clinical and histopathological signs of EAE compared to wild-type controls. CONCLUSION: This study identifies IL-36γ as a neutrophil-related cytokine that can potentially activate microglia, but that is only correlative and not contributory in EAE.

GPR84 deficiency reduces microgliosis, but accelerates dendritic degeneration and cognitive decline in a mouse model of Alzheimer's disease.

Microglia surrounds the amyloid plaques that form in the brains of patients with Alzheimer's disease (AD), but their role is controversial. Under inflammatory conditions, these cells can express GPR84, an orphan receptor whose pathophysiological role is unknown. Here, we report that GPR84 is upregulated in microglia of APP/PS1 transgenic mice, a model of AD. Without GPR84, these mice display both accelerated cognitive decline and a reduced number of microglia, especially in areas surrounding plaques. The lack of GPR84 affects neither plaque formation nor hippocampal neurogenesis, but promotes dendritic degeneration. Furthermore, GPR84 does not influence the clinical progression of other diseases in which its expression has been reported, i.e., experimental autoimmune encephalomyelitis (EAE) and endotoxic shock. We conclude that GPR84 plays a beneficial role in amyloid pathology by acting as a sensor for a yet unknown ligand that promotes microglia recruitment, a response affecting dendritic degeneration and required to prevent further cognitive decline.

Optimization of a lipid nanoparticle-based protocol for RNA transfection into primary mononuclear phagocytes.

The effective delivery of synthetic RNA into mononuclear phagocytes is a prerequisite for experimental research and therapeutic development. However, traditional methods are highly ineffective and toxic for these cells. Here, we aimed to optimize a transfection protocol for primary bone marrow-derived phagocytes, specifically dendritic cells and macrophages, using lipid nanoparticles generated by microfluidics. Our results show that a lipid mixture similar to that used in Moderna's COVID-19 messenger RNA vaccine outperforms the others tested. Improved messenger RNA transfection can be achieved by replacing uridine with methylpseudouridine but not methoxyuridine, which interferes with transfection. The addition of diphenyleneiodonium or apocynin can enhance transfection in a cell type-dependent manner without adverse effects, while apolipoprotein E provides no added value. These optimized transfection conditions can also be used for microRNA agonists and antagonists. In sum, this study offers a straightforward, highly efficient, reproducible, and nontoxic protocol to deliver RNA into different primary mononuclear phagocytes in culture.

Several classical mouse inbred strains, including DBA/2, NOD/Lt, FVB/N, and SJL/J, carry a putative loss-of-function allele of Gpr84.

G protein-coupled receptor 84 (GPR84) is a 7-transmembrane protein expressed on myeloid cells that can bind to medium-chain free fatty acids in vitro. Here, we report the discovery of a 2-bp frameshift deletion in the second exon of the Gpr84 gene in several classical mouse inbred strains. This deletion generates a premature stop codon predicted to result in a truncated protein lacking the transmembrane domains 4-7. We sequenced Gpr84 exon 2 from 58 strains representing different groups in the mouse family tree and found that 14 strains are homozygous for the deletion. Some of these strains are DBA/1J, DBA/2J, FVB/NJ, LG/J, MRL/MpJ, NOD/LtJ, and SJL/J. However, the deletion was not found in any of the wild-derived inbred strains analyzed. Haplotype analysis suggested that the deletion originates from a unique mutation event that occurred more than 100 years ago, preceding the development of the first inbred strain (DBA), from a Mus musculus domesticus source. As GPR84 ostensibly plays a role in the biology of myeloid cells, it could be relevant 1) to consider the existence of this Gpr84 nonsense mutation in several mouse strains when choosing a mouse model to study immune processes and 2) to consider reevaluating data obtained using such strains.

CXCL1 can be regulated by IL-6 and promotes granulocyte adhesion to brain capillaries during bacterial toxin exposure and encephalomyelitis.

BACKGROUND: Granulocytes generally exert protective roles in the central nervous system (CNS), but recent studies suggest that they can be detrimental in experimental autoimmune encephalomyelitis (EAE), the most common model of multiple sclerosis. While the cytokines and adhesion molecules involved in granulocyte adhesion to the brain vasculature have started to be elucidated, the required chemokines remain undetermined. METHODS: CXCR2 ligand expression was examined in the CNS of mice suffering from EAE or exposed to bacterial toxins by quantitative RT-PCR and in situ hybridization. CXCL1 expression was analyzed in IL-6-treated endothelial cell cultures by quantitative RT-PCR and ELISA. Granulocytes were counted in the brain vasculature after treatment with a neutralizing anti-CXCL1 antibody using stereological techniques. RESULTS: CXCL1 was the most highly expressed ligand of the granulocyte receptor CXCR2 in the CNS of mice subjected to EAE or infused with lipopolysaccharide (LPS) or pertussis toxin (PTX), the latter being commonly used to induce EAE. IL-6 upregulated CXCL1 expression in brain endothelial cells by acting transcriptionally and mediated the stimulatory effect of PTX on CXCL1 expression. The anti-CXCL1 antibody reduced granulocyte adhesion to brain capillaries in the three conditions under study. Importantly, it attenuated EAE severity when given daily for a week during the effector phase of the disease. CONCLUSIONS: This study identifies CXCL1 not only as a key regulator of granulocyte recruitment into the CNS, but also as a new potential target for the treatment of neuroinflammatory diseases such as multiple sclerosis.

Matrix metalloproteinase 2 attenuates brain tumour growth, while promoting macrophage recruitment and vascular repair.

Matrix metalloproteinase 2 (MMP2) is an extracellular protein-degrading enzyme widely believed to be involved in the invasion of brain tumour cells. However, this assumption is mainly based on in vitro studies. By characterizing the transcriptome and in vivo properties of 20 astrocytoma cell lines, we found that the levels of MMP2 were higher in GFAP(-) astrocytoma cells and correlated with their ability to induce vascular changes, a common complication of malignant tumours. To study the relationship between MMP2 expression and vascular alteration, we intracerebrally implanted immunodeficient mice with human astrocytoma cells stably transduced with lentiviral vectors expressing either MMP2 or a short hairpin RNA against MMP2. We found that the tumours depleted of MMP2 were larger, contained more proliferating cells and fewer macrophages, and had a vasculature that was more destabilized and regressed with fewer capillary sprouts. In contrast, the tumours overexpressing MMP2 were smaller and showed no histological difference compared to the controls. We therefore suggest that MMP2 is not the cause of vascular atypia in malignant brain tumours, but is involved in a tissue repair response that tends to limit the growth of these tumours. This study argues against MMP2 inhibition as a therapeutic approach for brain cancer and provides a comprehensive characterization of popular astrocytoma cell lines that should help to identify alternative targets.

Construction of a ganciclovir-sensitive lentiviral vector to assess the influence of angiopoietin-3 and soluble Tie2 on glioma growth.

Malignant brain tumors grow by coopting the existing vasculature, a process involving the release of angiopoietin-2 (Angpt2) from endothelial cells and its binding to the Tie2 receptor. The first goal of this study was to examine the therapeutic potential of two proteins that could interfere with Angpt2, namely Angpt3 and the soluble extracellular domain of Tie2 (sTie2). The second goal was to develop a lentiviral vector capable of delivering such proteins while offering the possibility to identify and destroy the genetically modified cells. To this end, we designed a bicistronic construct expressing the marker enhanced green fluorescent protein fused to the suicide gene herpes simplex virus 1 thymidine kinase. GL261 glioma cells transduced with this vector could be tracked and killed on command by the administration of the prodrug ganciclovir, either in vitro or after implantation into mouse brains. High levels of Angpt3 or sTie2 could be achieved with this vector; however, Angpt3 increased capillary destabilization and glioma growth, whereas sTie2 exerted no effect. Overall, this study helps to understand the importance of the Tie2 signaling pathway in glioma development and the role of Angpt3, but suggests that neither this molecule nor sTie2 are effective agents against malignant gliomas. This study also provides a lentiviral vector design for safer gene therapy.

Rod-Shaped monocytes patrol the brain vasculature and give rise to perivascular macrophages under the influence of proinflammatory cytokines and angiopoietin-2.

The nervous system is constantly infiltrated by blood-derived sentinels known as perivascular macrophages. Their immediate precursors have not yet been identified in situ and the mechanism that governs their recruitment is mostly unknown. Here, we provide evidence that CD68(+)GR1(-) monocytes can give rise to perivascular macrophages in mice suffering from endotoxemia. After adhesion to the endothelium, these monocytes start to crawl, adopt a rod-shaped morphology when passing through capillaries, and can manifest the ability to proliferate and form a long cytoplasmic protuberance. They are attracted in greater numbers during endotoxemia by a combination of vasoregulatory molecules, including TNF (tumor necrosis factor), interleukin-1beta, and angiopoietin-2. After a period of several hours, some of them cross the endothelium to expand the population of perivascular macrophages. Depletion of adherent monocytes and perivascular macrophages can be achieved by injection of anti-angiopoietin-2 peptide-Fc fusion protein. This study extends our understanding of the behavior of monocytes at the blood-brain interface and provides a way to block their infiltration into the nervous tissue under inflammatory conditions.