Early amyloid-induced changes in microglia gene expression in male APP/PS1 mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia, characterized by deposition of extracellular amyloid-beta (Aß) aggregates and intraneuronal hyperphosphorylated Tau. Many AD risk genes, identified in genome-wide association studies (GWAS), are expressed in microglia, the innate immune cells of the central nervous system. Specific subtypes of microglia emerged in relation to AD pathology, such as disease-associated microglia (DAMs), which increased in number with age in amyloid mouse models and in human AD cases. However, the initial transcriptional changes in these microglia in response to amyloid are still unknown. Here, to determine early changes in microglia gene expression, hippocampal microglia from male APPswe/PS1dE9 (APP/PS1) mice and wild-type littermates were isolated and analyzed by RNA sequencing (RNA-seq). By bulk RNA-seq, transcriptomic changes were detected in hippocampal microglia from 6-months-old APP/PS1 mice. By performing single-cell RNA-seq of CD11c-positive and negative microglia from 6-months-old APP/PS1 mice and analysis of the transcriptional trajectory from homeostatic to CD11c-positive microglia, we identified a set of genes that potentially reflect the initial response of microglia to Aß.

Epigenetic regulation of innate immune memory in microglia.

BACKGROUND: Microglia are the tissue-resident macrophages of the CNS. They originate in the yolk sac, colonize the CNS during embryonic development and form a self-sustaining population with limited turnover. A consequence of their relative slow turnover is that microglia can serve as a long-term memory for inflammatory or neurodegenerative events. METHODS: Using ATAC-, ChIP- and RNA-sequencing, we characterized the epigenomes and transcriptomes of FACS-purified microglia from mice exposed to different stimuli. A repeated endotoxin challenge (LPS) was used to induce tolerance in microglia, while genotoxic stress (DNA repair deficiency-induced accelerated aging through Ercc1 deficiency) resulted in primed (hypersensitive) microglia. RESULTS: Whereas the enrichment of permissive epigenetic marks at enhancer regions could explain training (hyper-responsiveness) of primed microglia to an LPS challenge, the tolerized response of microglia seems to be regulated by loss of permissive epigenetic marks. We identify that inflammatory stimuli and accelerated aging as a result of genotoxic stress activate distinct gene networks. These gene networks and associated biological processes are partially overlapping, which is likely driven by specific transcription factor networks, resulting in altered epigenetic signatures and distinct functional (desensitized vs. primed) microglia phenotypes. CONCLUSION: This study provides insight into epigenetic profiles and transcription factor networks associated with transcriptional signatures of tolerized and trained microglia in vivo, leading to a better understanding of innate immune memory of microglia.

Brain macrophages acquire distinct transcriptomes in multiple sclerosis lesions and normal appearing white matter.

Multiple sclerosis (MS) is a disease of the central nervous system that is characterized by inflammation and focal areas of demyelination, ultimately resulting in axonal degradation and neuronal loss. Several lines of evidence point towards a role for microglia and other brain macrophages in disease initiation and progression, but exactly how lesion formation is triggered is currently unknown. Here, we characterized early changes in MS brain tissue through transcriptomic analysis of normal appearing white matter (NAWM). We found that NAWM was characterized by enriched expression of genes associated with inflammation and cellular stress derived from brain macrophages. Single cell RNA sequencing confirmed a stress response in brain macrophages in NAWM and identified specific microglia and macrophage subsets at different stages of demyelinating lesions. We identified both phagocytic/activated microglia and CAM clusters that were associated with various MS lesion types. These overall changes in microglia and macrophages associated with lesion development in MS brain tissue may provide therapeutic targets to limit lesion progression and demyelination.

VISTA regulates microglia homeostasis and myelin phagocytosis, and is associated with MS lesion pathology.

V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA) is a negative checkpoint regulator (NCR) that is involved in T-cell quiescence, inhibition of T-cell activation, and in myeloid cells regulates cytokine production, chemotaxis, phagocytosis, and tolerance induction. In the central nervous system (CNS), VISTA is expressed by microglia, the resident macrophage of the parenchyma, and expression is decreased during neuroinflammation; however, the function of VISTA in microglia is unknown. Here, we extensively analyzed VISTA expression in different MS lesion stages and characterized the function of VISTA in the CNS by deleting VISTA in microglia. VISTA is differentially expressed in distinct MS lesion stages. In mice, VISTA deletion in Cx3Cr1-expressing cells induced a more amoeboid microglia morphology, indicating an immune-activated phenotype. Expression of genes associated with cell cycle and immune-activation was increased in VISTA KO microglia. In response to LPS and during experimental autoimmune encephalomyelitis (EAE), VISTA KO and WT microglia shared similar transcriptional profiles and VISTA deletion did not affect EAE disease progression or microglia responses. VISTA KO in microglia in vitro decreased the uptake of myelin. This study demonstrates that VISTA is involved in microglia function, which likely affects healthy CNS homeostasis and neuroinflammation.

Regionally diverse astrocyte subtypes and their heterogeneous response to EAE.

Astrocytes fulfil many functions in the central nervous system (CNS), including contribution to the blood brain barrier, synapse formation, and trophic support. In addition, they can mount an inflammatory response and are heterogeneous in morphology and function. To extensively characterize astrocyte subtypes, we FACS-isolated and gene expression profiled distinct astrocyte subtypes from three central nervous system regions; forebrain, hindbrain and spinal cord. Astrocyte subpopulations were separated based on GLAST/SLC1A3 and ACSA-2/ATP1B2 cell surface expression. The local brain environment proved key in establishing different transcriptional programs in astrocyte subtypes. Transcriptional differences between subtypes were also apparent in experimental autoimmune encephalomyelitis (EAE) mice, where these astrocyte subtypes showed distinct responses. While gene expression signatures associated with blood-brain barrier maintenance were lost, signatures involved in neuroinflammation and neurotoxicity were increased in spinal cord astrocytes, especially during acute disease stages. In chronic stages of EAE, this reactive astrocyte signature was slightly decreased, while obtaining a more proliferative profile, which might be relevant for glia scar formation and tissue regeneration. Morphological heterogeneity of astrocytes previously indicated the presence of astrocyte subtypes, and here we show diversity based on transcriptome variation associated with brain regions and differential responsiveness to a neuroinflammatory insult (EAE).

Intrinsic DNA damage repair deficiency results in progressive microglia loss and replacement.

The DNA excision repair protein Ercc1 is important for nucleotide excision, double strand DNA break, and interstrand DNA crosslink repair. In constitutive Ercc1-knockout mice, microglia display increased phagocytosis, proliferation and an enhanced responsiveness to lipopolysaccharide (LPS)-induced peripheral inflammation. However, the intrinsic effects of Ercc1-deficiency on microglia are unclear. In this study, Ercc1 was specifically deleted from Cx3cr1-expressing cells and changes in microglia morphology and immune responses at different times after deletion were determined. Microglia numbers were reduced with approximately 50% at 2-12 months after Ercc1 deletion. Larger and more ramified microglia were observed following Ercc1 deletion both in vivo and in organotypic hippocampal slice cultures. Ercc1-deficient microglia were progressively lost, and during this period, microglia proliferation was transiently increased. Ercc1-deficient microglia were gradually replaced by nondeficient microglia carrying a functional Ercc1 allele. In contrast to constitutive Ercc1-deficient mice, microglia-specific deletion of Ercc1 did not induce microglia activation or increase their responsiveness to a systemic LPS challenge. Gene expression analysis suggested that Ercc1 deletion in microglia induced a transient aging signature, which was different from a priming or disease-associated microglia gene expression profile.

Characterization and comparison of osteoblasts derived from mouse embryonic stem cells and induced pluripotent stem cells.

New developments in stem cell biology offer alternatives for the reconstruction of critical-sized bone defects. One of these developments is the use of induced pluripotent stem (iPS) cells. These stem cells are similar to embryonic stem (ES) cells, but can be generated from adult somatic cells and therefore do not raise ethical concerns. Proper characterization of iPS-derived osteoblasts is important for future development of safe clinical applications of these cells. For this reason, we differentiated mouse ES and iPS cells toward osteoblasts using osteogenic medium and compared their functionality. Immunocytochemical analysis showed significant expression of bone markers (osteocalcin and collagen type I) in osteoblasts differentiated from ES and iPS cells on days 7 and 30. An in vitro mineralization assay confirmed the functionality of osteogenically differentiated ES and iPS cells. Gene expression arrays focusing on osteogenic differentiation were performed in order to compare the gene expression pattern in both differentiated and undifferentiated ES cells and iPS cells. We observed a significant upregulation of osteogenesis-related genes such as Runx2, osteopontin, collagen type I, Tnfsf11, Csf1, and alkaline phosphatase upon osteogenic differentiation of the ES and iPS cells. We further validated the expression of key osteogenic genes Runx2, osteopontin, osteocalcin, collagen type I, and osterix in both differentiated and undifferentiated ES and iPS cells by means of quantified real-time polymerase chain reaction. We conclude that ES and iPS cells are similar in their osteogenic differentiation capacities, as well as in their gene expression patterns.

Oncostatin M promotes excitotoxicity by inhibiting glutamate uptake in astrocytes: implications in HIV-associated neurotoxicity.

BACKGROUND: Elevated levels of oncostatin M (OSM), an interleukin-6 cytokine family member, have been observed in HIV-1-associated neurocognitive disorders (HAND) and Alzheimer's disease. However, the function of OSM in these disease conditions is unclear. Since deficient glutamate uptake by astrocytes is instrumental in HAND-associated neurotoxicity, we hypothesized that OSM impairs glutamate uptake in astrocytes and thereby promotes neuronal excitotoxicity. METHODS: Primary cultures of mouse cortical astrocytes, neurons, microglia, and BV2 cell line were used. The expression of glutamate transporters (GLAST/EAAT1 and GLT-1/EAAT2) was investigated using real-time PCR and Western blot, and their activity was assessed by measuring (3)H-D-aspartate uptake. Neuronal toxicity was measured using the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl-) 2,5-diphenyltetrazolium bromide) assay and immunocytochemistry. A chimeric HIV-1 that infects murine cells (EcoHIV/NL4-3-GFP virus (EcoHIV)) was used to investigate whether the virus induces OSM, OSM receptor (OSMR)-ß, glycoprotein 130 (gp130), GLT-1, GLAST (mRNA and protein), and OSM release (ELISA) in cultured BV2 cells, primary microglia, or astrocytes. Statistical analyses of the data were performed using one-way ANOVA (to allow multiple comparisons) and two-tailed Student's t test. RESULTS: OSM treatment (10 ng/mL) time-dependently reduced GLAST and GLT-1 expression and inhibited (3)H-D-aspartate uptake in cultured astrocytes in a concentration-dependent manner, an effect prevented by the Janus kinase (JAK)/signal transducers and activators of transcription (STAT)3 inhibitor AG490. Down-regulation of astrocytic glutamate transport by OSM resulted in NMDA receptor-dependent excitotoxicity in cortical neurons. Infection with EcoHIV induced OSM gene expression and protein release in BV2 cells and microglia, but not in astrocytes. Conversely, EcoHIV caused a fivefold increase in OSMR-ß mRNA (but not gp130) and protein in astrocytes, but not in microglia, which did not express OSMR-ß protein. Finally, astrocytic expression of GLAST gene was unaffected by EcoHIV, whereas GLT-1 mRNA was increased by twofold. CONCLUSIONS: We provide first evidence that activation of JAK/STAT3 signaling by OSM inhibits glutamate uptake in astrocytes, which results in neuronal excitotoxicity. Our findings with EcoHIV suggest that targeting OSMR-ß signaling in astrocytes might alleviate HIV-1-associated excitotoxicity.

Enhanced microglial pro-inflammatory response to lipopolysaccharide correlates with brain infiltration and blood-brain barrier dysregulation in a mouse model of telomere shortening.

Microglia are a proliferative population of resident brain macrophages that under physiological conditions self-renew independent of hematopoiesis. Microglia are innate immune cells actively surveying the brain and are the earliest responders to injury. During aging, microglia elicit an enhanced innate immune response also referred to as 'priming'. To date, it remains unknown whether telomere shortening affects the proliferative capacity and induces priming of microglia. We addressed this issue using early (first-generation G1 mTerc(-/-) )- and late-generation (third-generation G3 and G4 mTerc(-/-) ) telomerase-deficient mice, which carry a homozygous deletion for the telomerase RNA component gene (mTerc). Late-generation mTerc(-/-) microglia show telomere shortening and decreased proliferation efficiency. Under physiological conditions, gene expression and functionality of G3 mTerc(-/-) microglia are comparable with microglia derived from G1 mTerc(-/-) mice despite changes in morphology. However, after intraperitoneal injection of bacterial lipopolysaccharide (LPS), G3 mTerc(-/-) microglia mice show an enhanced pro-inflammatory response. Nevertheless, this enhanced inflammatory response was not accompanied by an increased expression of genes known to be associated with age-associated microglia priming. The increased inflammatory response in microglia correlates closely with increased peripheral inflammation, a loss of blood-brain barrier integrity, and infiltration of immune cells in the brain parenchyma in this mouse model of telomere shortening.

Multipotent stem cell factor UGS148 is a marker for tanycytes in the adult hypothalamus.

The present study describes for the first time the neural expression and distribution of UGS148, a protein encoded by the RIKEN cDNA63330403K07 gene that has been shown to be prominently and characteristically expressed in neural stem cells (NSCs). Based on its molecular structure, UGS148 is an intracellular protein expected to be involved in intracellular sorting, trafficking, exocytosis and membrane insertion of proteins. We demonstrate that UGS148 is highly expressed in embryonic NSCs as well as, albeit at low level, in the adult neurogenic niches, the subventricular zone and the hippocampal dentate gyrus. Interestingly, the highest expression level of UGS148 in the adult mouse brain was observed specifically in the neurogenic cells lining the third ventricle, the tanycytes. Our in vitro studies show the involvement of UGS148 in the regulation of the proliferation of NSCs.

Detailed analysis of the genetic and epigenetic signatures of iPSC-derived mesodiencephalic dopaminergic neurons.

Induced pluripotent stem cells (iPSCs) hold great promise for in vitro generation of disease-relevant cell types, such as mesodiencephalic dopaminergic (mdDA) neurons involved in Parkinson's disease. Although iPSC-derived midbrain DA neurons have been generated, detailed genetic and epigenetic characterizations of such neurons are lacking. The goal of this study was to examine the authenticity of iPSC-derived DA neurons obtained by established protocols. We FACS purified mdDA (Pitx3 (Gfp/+) ) neurons derived from mouse iPSCs and primary mdDA (Pitx3 (Gfp/+) ) neurons to analyze and compare their genetic and epigenetic features. Although iPSC-derived DA neurons largely adopted characteristics of their in vivo counterparts, relevant deviations in global gene expression and DNA methylation were found. Hypermethylated genes, mainly involved in neurodevelopment and basic neuronal functions, consequently showed reduced expression levels. Such abnormalities should be addressed because they might affect unambiguous long-term functionality and hamper the potential of iPSC-derived DA neurons for in vitro disease modeling or cell-based therapy.

Adenosine A2B receptor-mediated leukemia inhibitory factor release from astrocytes protects cortical neurons against excitotoxicity.

BACKGROUND: Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF) have been widely reported. In the central nervous system (CNS), astrocytes are the major source for LIF, expression of which is enhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however, has remained an unanswered question. Since neuronal stress is associated with production of extracellular adenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptor signaling. METHODS: Mouse cortical neuronal and astrocyte cultures from wild-type and adenosine A(2B) receptor knock-out animals, as well as adenosine receptor agonists/antagonists and various enzymatic inhibitors, were used to study LIF expression and release in astrocytes. When needed, a one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test was used for statistical analysis. RESULTS: We show here that glutamate-stressed cortical neurons induce LIF expression through activation of adenosine A(2B) receptor subtype in cultured astrocytes and require signaling of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs: p38 and ERK1/2), and the nuclear transcription factor (NF)-κB. Moreover, LIF concentration in the supernatant in response to 5'-N-ethylcarboxamide (NECA) stimulation was directly correlated to de novo protein synthesis, suggesting that LIF release did not occur through a regulated release pathway. Immunocytochemistry experiments show that LIF-containing vesicles co-localize with clathrin and Rab11, but not with pHogrin, Chromogranin (Cg)A and CgB, suggesting that LIF might be secreted through recycling endosomes. We further show that pre-treatment with supernatants from NECA-treated astrocytes increased survival of cultured cortical neurons against glutamate, which was absent when the supernatants were pre-treated with an anti-LIF neutralizing antibody. CONCLUSIONS: Adenosine from glutamate-stressed neurons induces rapid LIF release in astrocytes. This rapid release of LIF promotes the survival of cortical neurons against excitotoxicity.

Interleukin-6-type cytokines in neuroprotection and neuromodulation: oncostatin M, but not leukemia inhibitory factor, requires neuronal adenosine A1 receptor function.

Neuroprotection is one of the prominent functions of the interleukin (IL)-6-type cytokine family, for which the underlying mechanism(s) are not fully understood. We have previously shown that neuroprotection and neuromodulation mediated by IL-6 require neuronal adenosine A(1) receptor (A(1) R) function. We now have investigated whether two other IL-6-type cytokines [oncostatin M (OSM) and leukemia inhibitory factor (LIF)] use a similar mechanism. It is presented here that OSM but not LIF, enhanced the expression of A(1) Rs (both mRNA and protein levels) in cultured neurons. Whereas the neuroprotective effect of LIF was unchanged in A(1) R deficient neurons, OSM failed to protect neurons in the absence of A(1) R. In addition, OSM pre-treatment for 4 h potentiated the A(1) R-mediated inhibition of electrically evoked excitatory post-synaptic currents recorded from hippocampal slices either under normal or hypoxic conditions. No such effect was observed after LIF pre-treatment. Our findings thus strongly suggest that, despite known structural and functional similarities, OSM and LIF use different mechanisms to achieve neuroprotection and neuromodulation.

Expression, transport, and axonal sorting of neuronal CCL21 in large dense-core vesicles.

Neurons are highly polarized cells, and neuron-neuron communication is based on directed transport and release of neurotransmitters, neuropeptides, and neurotrophins. Directed communication may also be attributed to neuron-microglia signaling, since neuronal damage can induce a microglia reaction at specific sites only. However, the mechanism underlying this site-specific microglia reaction is not yet understood. Neuronal CCL21 is a microglia-activating chemokine, which in brain is solely found in endangered neurons and is therefore a candidate for neuron-microglia signaling. Here we present that neuronal CCL21 is sorted into large dense-core vesicles, the secretory granules of the regulated release pathway of neurons. Live-cell imaging studies show preferential sorting of CCL21-containing vesicles into axons, indicating its directed transport. Thus, mouse neurons express and transport a microglia activating factor very similar to signaling molecules used in neuron-neuron communication. These data show for the first time the directed transport of a microglia activating factor in neurons and corroborate the function of neuronal CCL21 in directed neuron-microglia communication.

Expression of CXCL4 in microglia in vitro and in vivo and its possible signaling through CXCR3.

Signaling through chemokine receptor CXCR3 in the brain has been implicated in various brain diseases, as CXCR3 and its ligands are found under these conditions. Recently, a new chemokine ligand for CXCR3 was reported. In humans, an alternatively spliced variant of CXCR3 expressed on microvascular endothelial cells, named CXCR3b, was shown to bind CXCL4. In the periphery, the cellular expression and functions of CXCL4 are well described but in the brain its expression and function are unknown. Here, we show that brain microglia are a cellular source of CXCL4 in vitro and in vivo under neurodegenerating conditions. Microglial migration induced by CXCL4 is absent in CXCR3-deficient microglia, indicating a role of CXCR3. CXCL4 furthermore attenuates lipopolysaccharide-induced microglial phagocytosis and nitric oxide production in microglia and BV-2 cells. Based on these findings, it is proposed that locally released CXCL4 may control microglia responses.