Ceruloplasmin-deficient mice show changes in PTM profiles of proteins involved in messenger RNA processing and neuronal projections and synaptic processes.

Ceruloplasmin (Cp) is a multicopper oxidase with ferroxidase properties being of importance to the mobilisation and export of iron from cells and its ability to bind copper. In ageing humans, Cp deficiency is known to result in aceruloplasminemia, which among other is characterised by neurological symptoms. To obtain novel information about the functions of Cp in the central nervous system (CNS) we compared the brain proteome in forebrains from asymptomatic 4-6-month-old Cp-deficient (B6N(Cg)-Cptm1b(KOMP)Wtsi /J) and wild-type mice. Of more than 5600 quantified proteins, 23 proteins, were regulated, whereas more than 1200 proteins had regulated post-translational modifications (PTMs). The genes of the regulated proteins, glycoproteins and phosphoproteins appeared mostly to be located to neurons and oligodendrocyte precursor cells. Cp deficiency especially affected the function of proteins involved in the extension of neuronal projections, synaptic signalling and cellular mRNA processing and affected the expression of proteins involved in neurodegenerative disease and diabetes. Iron concentration and transferrin saturation were reduced in the blood of even younger, 3- to 5-month-old, Cp-deficient mice. Iron act as cofactor in many enzymatic processes and reactions. Changes in iron availability and oxidation as consequence of Cp deficiency could therefore affect the synthesis of proteins and lipids. This proteomic characterisation is to our knowledge the first to document the changes taking place in the CNS-proteome and its phosphorylation and glycosylation state in Cp-deficient mice.

A Comparison of PKD2L1-Expressing Cerebrospinal Fluid Contacting Neurons in Spinal Cords of Rodents, Carnivores, and Primates.

Cerebrospinal fluid contacting neurons (CSF-cNs) are a specific type of neurons located around the ventricles in the brain and the central canal in the spinal cord and have been demonstrated to be intrinsic sensory neurons in the central nervous system. One of the important channels responsible for the sensory function is the polycystic kidney disease 2-like 1 (PKD2L1) channel. Most of the studies concerning the distribution and function of the PKD2L1-expressing CSF-cNs in the spinal cord have previously been performed in non-mammalian vertebrates. In the present study immunohistochemistry was performed to determine the distribution of PKD2L1-immunoreactive (IR) CSF-cNs in the spinal cords of four mammalian species: mouse, rat, cat, and macaque monkey. Here, we found that PKD2L1-expressing CSF-cNs were present at all levels of the spinal cord in these animal species. Although the distribution pattern was similar across these species, differences existed. Mice and rats presented a clear PKD2L1-IR cell body labeling, whereas in cats and macaques the PKD2L1-IR cell bodies were more weakly labeled. Ectopic PKD2L1-IR neurons away from the ependymal layer were observed in all the animal species although the abundance and the detailed locations varied. The apical dendritic protrusions with ciliated fibers were clearly seen in the lumen of the central canal in all the animal species, but the sizes of protrusion bulbs were different among the species. PKD2L1-IR cell bodies/dendrites were co-expressed with doublecortin, MAP2 (microtubule-associated protein 2), and aromatic L-amino acid decarboxylase, but not with NeuN (neuronal nuclear protein), indicating their immature properties and ability to synthesize monoamine transmitters. In addition, in situ hybridization performed in rats revealed PKD2L1 mRNA expression in the cells around the central canal. Our results indicate that the intrinsic sensory neurons are conserved across non-mammalian and mammalian vertebrates. The similar morphology of the dendritic bulbs with ciliated fibers (probably representing stereocilia and kinocilia) protruding into the central canal across different animal species supports the notion that PKD2L1 is a chemo- and mechanical sensory channel that responds to mechanical stimulations and maintains homeostasis of the spinal cord. However, the differences of PKD2L1 distribution and expression between the species suggest that PKD2L1-expressing neurons may receive and process sensory signals differently in different animal species.

Proteomic signatures of neuroinflammation in Alzheimer's disease, multiple sclerosis and ischemic stroke.

Introduction: Inflammation is integral in the neuropathology of both chronic and acute neurological disorders. Knowing the inflammatory profile is important for clarification of disease mechanisms, diagnostic purposes, and ultimately treatment options. Areas covered: A systematic review was performed on literature from PubMed using the search terms 'Alzheimer's disease' (AD) or "multiple sclerosis" (MS) or "ischemic stroke" and 'proteomics'. Inflammatory proteins were assessed in blood, cerebrospinal fluid (CSF), and post-mortem brain tissue. Regulated inflammatory proteins across compartments and disorders mainly consisted of innate immune proteins, acute phase proteins and oxidative stress response proteins. In addition, immunoglobulin chains were signature proteins of MS, reflecting additional involvement of adaptive immunity. The Chitinase 3-like protein 1 was increased in ten original articles on MS and in three on AD supporting its implication in these diseases. Furthermore, CNS/CSF AD inflammatory proteins were matched to a CNS myeloid cell proteome implicating Alpha-2-Macroglobulin and Annexin A1 in AD pathogenesis. Expert opinion: Proteomics is an excellent technique for profiling inflammatory proteins in tissues and cells, but still targeted approaches are required for profiling of very low abundance proteins and peptides. Knowing the inflammatory signature of brain tissue, CSF, blood, and CNS myeloid cells holds the potential to point to novel mechanistic aspects of neurological diseases.

Post-stroke inflammation-target or tool for therapy?

Inflammation is currently considered a prime target for the development of new stroke therapies. In the acute phase of ischemic stroke, microglia are activated and then circulating immune cells invade the peri-infarct and infarct core. Resident and infiltrating cells together orchestrate the post-stroke inflammatory response, communicating with each other and the ischemic neurons, through soluble and membrane-bound signaling molecules, including cytokines. Inflammation can be both detrimental and beneficial at particular stages after a stroke. While it can contribute to expansion of the infarct, it is also responsible for infarct resolution, and influences remodeling and repair. Several pre-clinical and clinical proof-of-concept studies have suggested the effectiveness of pharmacological interventions that target inflammation post-stroke. Experimental evidence shows that targeting certain inflammatory cytokines, such as tumor necrosis factor, interleukin (IL)-1, IL-6, and IL-10, holds promise. However, as these cytokines possess non-redundant protective and immunoregulatory functions, their neutralization or augmentation carries a risk of unwanted side effects, and clinical translation is, therefore, challenging. This review summarizes the cell biology of the post-stroke inflammatory response and discusses pharmacological interventions targeting inflammation in the acute phase after a stroke that may be used alone or in combination with recanalization therapies. Development of next-generation immune therapies should ideally aim at selectively neutralizing pathogenic immune signaling, enhancing tissue preservation, promoting neurological recovery and leaving normal function intact.

TNF deficiency causes alterations in the spatial organization of neurogenic zones and alters the number of microglia and neurons in the cerebral cortex.

BACKGROUND: Although tumor necrosis factor (TNF) inhibitors are used to treat chronic inflammatory diseases, there is little information about how long-term inhibition of TNF affects the homeostatic functions that TNF maintains in the intact CNS. MATERIALS AND METHODS: To assess whether developmental TNF deficiency causes alterations in the naïve CNS, we estimated the number of proliferating cells, microglia, and neurons in the developing neocortex of E13.5, P7 and adult TNF knock out (TNF-/-) mice and wildtype (WT) littermates. We also measured changes in gene and protein expression and monoamine levels in adult WT and TNF-/- mice. To evaluate long-term effects of TNF inhibitors, we treated healthy adult C57BL/6 mice with either saline, the selective soluble TNF inhibitor XPro1595, or the nonselective TNF inhibitor etanercept. We estimated changes in cell number and protein expression after two months of treatment. We assessed the effects of TNF deficiency on cognition by testing adult WT and TNF-/- mice and mice treated with saline, XPro1595, or etanercept with specific behavioral tasks. RESULTS: TNF deficiency decreased the number of proliferating cells and microglia and increased the number of neurons. At the same time, TNF deficiency decreased the expression of WNT signaling-related proteins, specifically Collagen Triple Helix Repeat Containing 1 (CTHRC1) and Frizzled receptor 6 (FZD6). In contrast to XPro1595, long-term inhibition of TNF with etanercept in adult C57BL/6 mice decreased the number of BrdU+ cells in the granule cell layer of the dentate gyrus. Etanercept, but not XPro1595, also impaired spatial learning and memory in the Barnes maze memory test. CONCLUSION: TNF deficiency impacts the organization of neurogenic zones and alters the cell composition in brain. Long-term inhibition of TNF with the nonselective TNF inhibitor etanercept, but not the soluble TNF inhibitor XPro1595, decreases neurogenesis in the adult mouse hippocampus and impairs learning and memory after two months of treatment.

Protective effect of ibuprofen in a rat model of chronic oxaliplatin-induced peripheral neuropathy.

Despite extensive preclinical and clinical investigations, a clinically relevant neuroprotective agent against oxaliplatin-induced peripheral neuropathy, which affects the quality of life following chemotherapy, has not been identified. Epidemiological data suggest that ibuprofen may reduce the risk of neuropathy. Male rats were treated with oxaliplatin (n = 6), oxaliplatin and ibuprofen (n = 5) or vehicle (n = 5) every second day for 15 days. Neuropathy was evaluated using mechanical detection thresholds (MDT) at the hind paw and sensory nerve conduction velocity (SNCV) in the tail nerve at baseline, right after and 3 weeks after the end of treatment. Intraepidermal nerve fibre density (IENFD) was evaluated in the hind paw and inflammation in the dorsal root ganglia 3 weeks after treatment. Inflammation in the dorsal root ganglia was assessed using quantitative real-time RT-PCR (qPCR) of the mRNA levels for the pro-inflammatory cytokines, TNF-α and IL-1ß, and by immunohistochemical staining for Iba1+ macrophages. SNCV was reduced in rats treated with oxaliplatin and with oxaliplatin and ibuprofen compared to control rats 3 weeks after treatment. No differences were found for MDT 3 weeks after treatment. IENFD was reduced in rats treated with oxaliplatin. There was a trend towards up-regulation of TNF-α mRNA levels in rats treated with oxaliplatin and with oxaliplatin and ibuprofen. Morphological changes of Iba1+ macrophages suggested activation, but no differences were found in area fraction or size of macrophage cell bodies. The results did not support a neuroprotective effect of ibuprofen but indicated that inflammation may play a role in oxaliplatin-induced peripheral neuropathy.

Natural RNA circles function as efficient microRNA sponges.

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so-called competing endogenous RNA in humans and target mimicry in plants. We previously identified a highly expressed circular RNA (circRNA) in human and mouse brain. Here we show that this circRNA acts as a miR-7 sponge; we term this circular transcript ciRS-7 (circular RNA sponge for miR-7). ciRS-7 contains more than 70 selectively conserved miRNA target sites, and it is highly and widely associated with Argonaute (AGO) proteins in a miR-7-dependent manner. Although the circRNA is completely resistant to miRNA-mediated target destabilization, it strongly suppresses miR-7 activity, resulting in increased levels of miR-7 targets. In the mouse brain, we observe overlapping co-expression of ciRS-7 and miR-7, particularly in neocortical and hippocampal neurons, suggesting a high degree of endogenous interaction. We further show that the testis-specific circRNA, sex-determining region Y (Sry), serves as a miR-138 sponge, suggesting that miRNA sponge effects achieved by circRNA formation are a general phenomenon. This study serves as the first, to our knowledge, functional analysis of a naturally expressed circRNA.

Characterizing disease-associated changes in post-translational modifications by mass spectrometry.

INTRODUCTION: Exploring post-translational modifications (PTMs) with the use of mass spectrometry (PTMomics) is a rapidly developing area, with methods for discovery/quantification being developed and advanced on a regular basis. PTMs are highly important for the regulation of protein function, interaction and activity, both in physiological and disease states. Changes in PTMs can either cause, or be the result of a disease, making them central for biomarker studies and studies of disease pathogenesis. Recently, it became possible to study multiple PTMs simultaneously from low amount of sample material, thereby increasing coverage of the PTMome obtainable from a single sample. Thus, quantitative PTMomics holds great potential to discover biomarkers from tissue and body fluids as well as elucidating disease mechanisms through characterization of signaling pathways. Areas covered: Recent mass spectrometry-based methods for assessment of the PTMome, with focus on the most studied PTMs, are highlighted. Furthermore, both data dependent and data independent acquisition methods are evaluated. Finally, current challenges in the field are discussed. Expert commentary: PTMomics holds great potential for clinical and biomedical research, especially with the generation of spectral libraries of peptides and PTMs from individual patients (permanent PTM maps) for use in personalized medicine.

Neuron and neuroblast numbers and cytogenesis in the dentate gyrus of aged APPswe/PS1dE9 transgenic mice: Effect of long-term treatment with paroxetine.

Altered neurogenesis may influence hippocampal functions such as learning and memory in Alzheimer's disease. Selective serotonin reuptake inhibitors enhance neurogenesis and have been reported to reduce cerebral amyloidosis in both humans and transgenic mice. We have used stereology to assess the longitudinal changes in the number of doublecortin-expressing neuroblasts and number of granular neurons in the dentate gyrus of APPswe/PS1dE9 transgenic mice. Furthermore, we investigated the effect of long-term paroxetine treatment on the number of neuroblasts and granular neurons, hippocampal amyloidosis, and spontaneous alternation behaviour, a measure of spatial working memory, in transgenic mice. We observed no difference in granular neurons between transgenic and wild type mice up till 18months of age, and no differences with age in wild type mice. The number of neuroblasts and the performance in the spontaneous alternation task was reduced in aged transgenic mice. Paroxetine treatment from 9 to 18months of age reduced hippocampal amyloidosis without affecting the number of neuroblasts or granular neurons. These findings suggest that the amyloidosis affects the differentiation of neuroblasts and spatial working memory, independent of changes in total granular neurons. Furthermore, while long-term paroxetine treatment may be able to reduce hippocampal amyloidosis, it appears to have no effect on total number of granular neurons or spatial working memory.

Beneficial potential of intravenously administered IL-6 in improving outcome after murine experimental stroke.

Interleukin-6 (IL-6) is a pleiotropic cytokine with neuroprotective properties. Still, the therapeutic potential of IL-6 after experimental stroke has not yet been investigated in a clinically relevant way. Here, we investigated the therapeutic use of intravenously administered IL-6 and the soluble IL-6 receptor (sIL-6R) alone or in combination, early after permanent middle cerebral artery occlusion (pMCAo) in mice. IL-6 did not affect the infarct volume in C57BL/6 mice, at neither 24 nor 72h after pMCAo but reduced the infarct volume in IL-6 knockout mice at 24h after pMCAo. Assessment of post-stroke behavior showed an improved grip strength after a single IL-6 injection and also improved rotarod endurance after two injections, in C57BL/6 mice at 24h. An improved grip strength and a better preservation of sensory functions was also observed in IL-6 treated IL-6 knockout mice 24h after pMCAo. Co-administration of IL-6 and sIL-6R increased the infarct volume, the number of infiltrating polymorphonuclear leukocytes and impaired the rotarod endurance of C57BL/6 mice 24h after pMCAo. IL-6 administration to naïve C57BL/6 mice lead after 45min to increased plasma-levels of CXCL1 and IL-10, whereas IL-6 administration to C57BL/6 mice lead to a reduction in the ischemia-induced increase in IL-6 and CXCL1 at both mRNA and protein level in brain, and of IL-6 and CXCL1 in serum. We also investigated the expression of IL-6 and IL-6R after pMCAo and found that cortical neurons upregulated IL-6 mRNA and protein, and upregulated IL-6R after pMCAo. In conclusion, the results show a complex but potentially beneficial effect of intravenously administered IL-6 in experimental stroke.

CD8+ T cells complement antibodies in protecting against yellow fever virus.

The attenuated yellow fever (YF) vaccine (YF-17D) was developed in the 1930s, yet little is known about the protective mechanisms underlying its efficiency. In this study, we analyzed the relative contribution of cell-mediated and humoral immunity to the vaccine-induced protection in a murine model of YF-17D infection. Using different strains of knockout mice, we found that CD4(+) T cells, B cells, and Abs are required for full clinical protection of vaccinated mice, whereas CD8(+) T cells are dispensable for long-term survival after intracerebral challenge. However, by analyzing the immune response inside the infected CNS, we observed an accelerated T cell influx into the brain after intracerebral challenge of vaccinated mice, and this T cell recruitment correlated with improved virus control in the brain. Using mice deficient in B cells we found that, in the absence of Abs, YF vaccination can still induce some antiviral protection, and in vivo depletion of CD8(+) T cells from these animals revealed a pivotal role for CD8(+) T cells in controlling virus replication in the absence of a humoral response. Finally, we demonstrated that effector CD8(+) T cells also contribute to viral control in the presence of circulating YF-specific Abs. To our knowledge, this is the first time that YF-specific CD8(+) T cells have been demonstrated to possess antiviral activity in vivo.

Functional organization of an Mbp enhancer exposes striking transcriptional regulatory diversity within myelinating glia.

In mammals, large caliber axons are ensheathed by myelin, a glial specialization supporting axon integrity and conferring accelerated and energy-efficient action potential conduction. Myelin basic protein (MBP) is required for normal myelin elaboration with maximal mbp transcription in oligodendrocytes requiring the upstream M3 enhancer. To further characterize the mechanism regulating mbp transcription, we defined M3 structure/function relationships by evaluating its evolutionary conservation, DNA footprints and the developmental programing conferred in mice by M3 derivatives. Multiple M3 regulatory element combinations were found to drive expression in oligodendrocytes and Schwann cells with a minimal 129 bp sequence conferring expression in oligodendrocytes throughout myelin elaboration, maintenance and repair. Unexpectedly, M3 derivatives conferred markedly different spatial and temporal expression programs thus illuminating striking transcriptional heterogeneity within post-mitotic oligodendrocytes. Finally, one M3 derivative engaged only during primary myelination, not during adult remyelination, demonstrating that transcriptional regulation in the two states is not equivalent.

Myelin-specific T cells induce interleukin-1beta expression in lesion-reactive microglial-like cells in zones of axonal degeneration.

Infiltration of myelin-specific T cells into the central nervous system induces the expression of proinflammatory cytokines in patients with multiple sclerosis (MS). We have previously shown that myelin-specific T cells are recruited into zones of axonal degeneration, where they stimulate lesion-reactive microglia. To gain mechanistic insight, we used RNA microarray analysis to compare the transcript profile in hippocampi from perforant pathway axonal-lesioned mice with and without adoptively transferred myelin-specific T cells 2 days postlesion, when microglia are clearly lesion reactive. Pathway analysis revealed that, among the 1,447 differently expressed transcripts, the interleukin (IL)-1 pathway including all IL-1 receptor ligands was upregulated in the presence of myelin-specific T cells. Quantitative polymerase chain reaction showed increased mRNA levels of IL-1ß, IL-1α, and IL-1 receptor antagonist in the T-cell-infiltrated hippocampi from axonal-lesioned mice. In situ hybridization and immunohistochemistry showed a T-cell-enhanced lesion-specific expression of IL-1ß mRNA and protein, respectively, and induction of the apoptosis-associated speck-like protein, ASC, in CD11b(+) cells. Double in situ hybridization showed colocalization of IL-1ß mRNA in a subset of CD11b mRNA(+) cells, of which many were part of cellular doublets or clusters, characteristic of proliferating, lesion-reactive microglia. Double-immunofluorescence showed a T-cell-enhanced colocalization of IL-1ß to CD11b(+) cells, including lesion-reactive CD11b(+) ramified microglia. These results suggest that myelin-specific T cells stimulate lesion-reactive microglial-like cells to produce IL-1ß. These findings are relevant to understand the consequences of T-cell infiltration in white and gray matter lesions in patients with MS.

Cell therapy centered on IL-1Ra is neuroprotective in experimental stroke.

Cell-based therapies are emerging as new promising treatments in stroke. However, their functional mechanism and therapeutic potential during early infarct maturation has so far received little attention. Here, we asked if cell-based delivery of the interleukin-1 receptor antagonist (IL-1Ra), a known neuroprotectant in stroke, can promote neuroprotection, by modulating the detrimental inflammatory response in the tissue at risk. We show by the use of IL-1Ra-overexpressing and IL-1Ra-deficient mice that IL-1Ra is neuroprotective in stroke. Characterization of the cellular and spatiotemporal production of IL-1Ra and IL-1α/ß identifies microglia, not infiltrating leukocytes, as the major sources of IL-1Ra after experimental stroke, and shows IL-1Ra and IL-1ß to be produced by segregated subsets of microglia with a small proportion of these cells co-expressing IL-1α. Reconstitution of whole body irradiated mice with IL-1Ra-producing bone marrow cells is associated with neuroprotection and recruitment of IL-1Ra-producing leukocytes after stroke. Neuroprotection is also achieved by therapeutic injection of IL-1Ra-producing bone marrow cells 30 min after stroke onset, additionally improving the functional outcome in two different stroke models. The IL-1Ra-producing bone marrow cells increase the number of IL-1Ra-producing microglia, reduce the availability of IL-1ß, and modulate mitogen-activated protein kinase (MAPK) signaling in the ischemic cortex. The importance of these results is underlined by demonstration of IL-1Ra-producing cells in the human cortex early after ischemic stroke. Taken together, our results attribute distinct neuroprotective or neurotoxic functions to segregated subsets of microglia and suggest that treatment strategies increasing the production of IL-1Ra by infiltrating leukocytes or microglia may also be neuroprotective if applied early after stroke onset in patients.

Suppressors of cytokine signaling 1 and 3 are upregulated in brain resident cells in response to virus-induced inflammation of the central nervous system via at least two distinctive pathways.

UNLABELLED: Suppressors of cytokine signaling (SOCS) proteins are intracellular proteins that inhibit cytokine signaling in a variety of cell types. A number of viral infections have been associated with SOCS upregulation; however, not much is known about the mechanisms regulating SOCS expression during viral infection. In this study, we used two pathologically distinct intracerebral (i.c.) infection models to characterize temporal and spatial aspects of SOCS expression in the virus-infected central nervous system (CNS), and by employing various knockout mouse models, we sought to identify regulatory mechanisms that may underlie a virus induced upregulation of SOCS in the CNS. We found that i.c. infection with either lymphocytic choriomeningitis virus (LCMV) or yellow fever virus (YF) results in gradual upregulation of SOCS1/3 mRNA expression peaking at day 7 postinfection (p.i.). In the LCMV model, SOCS mRNA was expressed in brain resident cells, including astrocytes and some neurons, and for SOCS1 in particular this upregulation was almost entirely mediated by gamma interferon (IFN-γ) produced by infiltrating T cells. After infection with YF, we also found SOCS expression to be upregulated in brain resident cells with a peak on day 7 p.i., but in this model, the upregulation was only partially dependent on IFN-γ and T cells, indicating that at least one other mediator was involved in the upregulation of SOCS following YF infection. We conclude that virus-induced inflammation of the CNS is associated with upregulation of SOCS1/3 mRNA expression in brain resident cells and that at least two distinctive pathways can lead to this upregulation. IMPORTANCE: In the present report, we have studied the induction of SOCS1 and SOCS3 expression in the context of virus-induced CNS infection. We found that both a noncytolytic and a cytolytic virus induce marked upregulation of SOCS1 and -3 expression. Notably, the kinetics of the observed upregulation follows that of activity within proinflammatory signaling pathways and, interestingly, type II interferon (IFN), which is also a key inducer of inflammatory mediators, seems to be essential in initiating this counterinflammatory response. Another key observation is that not only cells of the immune system but also CNS resident cells are actively involved in both the pro- and the counterinflammatory immune circuits; thus, for example, astrocytes upregulate both C-X-C-motif chemokine 10 (CXCL10) and SOCS when exposed to type II IFN in vivo.

Cytokine-producing microglia have an altered beta-amyloid load in aged APP/PS1 Tg mice.

Beta-amyloid (Aß) plaques and chronic neuroinflammation are significant neuropathological features of Alzheimer's disease. Microglial cells in aged brains have potential to produce cytokines such as TNF and IL-1 family members (IL-1α, IL-1ß, and IL-1Ra) and to phagocytose Aß in Alzheimer's disease, however the inter-relationship between these processes is poorly understood. Here we show that % Aß plaque load followed a sigmoidal trajectory with age in the neocortex of APPswe/PS1ΔE9 Tg mice, and correlated positively with soluble Aß40 and Aß42. Aß measures were moderately correlated with mRNA levels of CD11b, TNF, and IL-1Ra. Cytokine production and Aß load were assessed in neocortical CD11b(+)(CD45(+)) microglia by flow cytometry. Whereas most microglia in aged mice produced IL-1Ra, relatively low proportions of microglia produced TNF, IL-1α, and IL-1ß. However, microglial production of these latter cytokines was generally increased in APP/PS1 Tg mice. Microglia that phagocytosed endogenously-produced Aß were only observed in APP/PS1 Tg mice. Differences in phagocytic index and total Aß load were observed in microglia with specific cytokine profiles. Both phagocytic index and total Aß load were higher in IL-1α(+) and IL-1Ra(+) microglia, than microglia that did not produce these cytokines. In contrast, total Aß load was lower in IL-1ß(+) and TNF(+) microglia, compared to IL-1ß(-) and TNF(-) microglia, and TNF(+) microglia also had a lower phagocytic index. Using GFP bone marrow chimeric mice, we confirmed that the majority of neocortical CD11b(+)(CD45(+)) microglia were resident cells (GFP(-)) in APP/PS1 Tg mice, even after selectively analysing CD11b(+)CD45(high) cells, which are typically considered to be infiltrating cells. Together, our data demonstrate that cytokine expression is selectively correlated with age and Aß pathology, and is associated with an altered Aß load in phagocytic microglia from APP/PS1 Tg mice. These findings have implications for understanding the regulation of microglial cytokine production and phagocytosis of Aß in Alzheimer's disease.

No effect of ablation of surfactant protein-D on acute cerebral infarction in mice.

BACKGROUND: Crosstalk between the immune system in the brain and the periphery may contribute to the long-term outcome both in experimental and clinical stroke. Although, the immune defense collectin surfactant protein-D (SP-D) is best known for its role in pulmonary innate immunity, SP-D is also known to be involved in extrapulmonary modulation of inflammation in mice. We investigated whether SP-D affected cerebral ischemic infarction and ischemia-induced inflammatory responses in mice. METHODS: The effect of SP-D was studied by comparing the size of ischemic infarction and the inflammatory and astroglial responses in SP-D knock out (KO) and wild type (WT) mice subjected to permanent middle cerebral artery occlusion. SP-D mRNA production was assessed in isolated cerebral arteries and in the whole brain by PCR, and SP-D protein in normal appearing and ischemic human brain by immunohistochemistry. Changes in plasma SP-D and TNF were assessed by ELISA and proximity ligation assay, respectively. RESULTS: Infarct volumetric analysis showed that ablation of SP-D had no effect on ischemic infarction one and five days after induction of ischemia. Further, ablation of SP-D had no effect on the ischemia-induced increase in TNF mRNA production one day after induction of ischemia; however the TNF response to the ischemic insult was affected at five days. SP-D mRNA was not detected in parenchymal brain cells in either naïve mice or in mice subjected to focal cerebral ischemia. However, SP-D mRNA was detected in middle cerebral artery cells in WT mice and SP-D protein in vascular cells both in normal appearing and ischemic human brain tissue. Measurements of the levels of SP-D and TNF in plasma in mice suggested that levels were unaffected by the ischemic insult. Microglial-leukocyte and astroglial responses were comparable in SP-D KO and WT mice. CONCLUSIONS: SP-D synthesis in middle cerebral artery cells is consistent with SP-D conceivably leaking into the infarcted area and affecting local cytokine production. However, there was no SP-D synthesis in parenchymal brain cells and ablation of SP-D had no effect on ischemic cerebral infarction.

Systemically administered anti-TNF therapy ameliorates functional outcomes after focal cerebral ischemia.

BACKGROUND: The innate immune system contributes to the outcome after stroke, where neuroinflammation and post-stroke systemic immune depression are central features. Tumor necrosis factor (TNF), which exists in both a transmembrane (tm) and soluble (sol) form, is known to sustain complex inflammatory responses associated with stroke. We tested the effect of systemically blocking only solTNF versus blocking both tmTNF and solTNF on infarct volume, functional outcome and inflammation in focal cerebral ischemia. METHODS: We used XPro1595 (a dominant-negative inhibitor of solTNF) and etanercept (which blocks both solTNF and tmTNF) to test the effect of systemic administration on infarct volume, functional recovery and inflammation after focal cerebral ischemia in mice. Functional recovery was evaluated after one, three and five days, and infarct volumes at six hours, 24 hours and five days after ischemia. Brain inflammation, liver acute phase response (APR), spleen and blood leukocyte profiles, along with plasma microvesicle analysis, were evaluated. RESULTS: We found that both XPro1595 and etanercept significantly improved functional outcomes, altered microglial responses, and modified APR, spleen T cell and microvesicle numbers, but without affecting infarct volumes. CONCLUSIONS: Our data suggest that XPro1595 and etanercept improve functional outcome after focal cerebral ischemia by altering the peripheral immune response, changing blood and spleen cell populations and decreasing granulocyte infiltration into the brain. Blocking solTNF, using XPro1595, was just as efficient as blocking both solTNF and tmTNF using etanercept. Our findings may have implications for future treatments with anti-TNF drugs in TNF-dependent diseases.

Alternative polyadenylation and miR-34 family members regulate tau expression.

Tau pathologically aggregates in Alzheimer's disease, and evidence suggests that reducing tau expression may be safe and beneficial for the prevention or treatment of this disease. We sought to examine the role of the 3'-untranslated region (3'-UTR) of human tau mRNA in regulating tau expression. Tau expresses two 3'-UTR isoforms, long and short, as a result of alternative polyadenylation. Using luciferase reporter constructs, we found that expression from these isoforms is differentially controlled in human neuroblastoma cell lines M17D and SH-SY5Y. Several microRNAs were computationally identified as candidates that might bind the long, but not short, tau 3'-UTR isoform. A hit from a screen of candidates, miR-34a, was subsequently shown to repress the expression of endogenous tau protein in M17D cells. Conversely, inhibition of endogenously expressed miR-34 family members leads to increased endogenous tau expression. In addition, through an unbiased screen of fragments of the human tau 3'-UTR using a luciferase reporter assay, we identified several other regions in the long tau 3'-UTR isoform that contain regulatory cis-elements. Improved understanding of the regulation of tau expression by its 3'-UTR may ultimately lead to the development of novel therapeutic strategies for the treatment of Alzheimer's disease and other tauopathies. mRNA 3'-untranslated regions (3'-UTR) often regulate transcript stability or translation. Despite the centrality of the tau protein in Alzheimer's and other neurodegenerative diseases, the human tau 3'-UTR has been little studied. This report identifies regions of the tau 3'-UTR that influence expression and shows that microRNA (miR)-34a targets this 3'-UTR to lower expression, which is considered an important therapeutic goal.

Differential impact of interferon regulatory factor 7 in initiation of the type I interferon response in the lymphocytic choriomeningitis virus-infected central nervous system versus the periphery.

Interferon (IFN) regulatory factors (IRFs) are a family of transcription factors involved in regulating type I IFN genes and other genes participating in the early antiviral host response. To better understand the mechanisms involved in virus-induced central nervous system (CNS) inflammation, we studied the influence of IRF1, -3, -7, and -9 on the transcriptional activity of key genes encoding antiviral host factors in the CNS of mice infected with lymphocytic choriomeningitis virus (LCMV). A key finding is that neither IRF3 nor IRF7 is absolutely required for induction of a type I IFN response in the LCMV-infected CNS, whereas concurrent elimination of both factors markedly reduces the virus-induced host response. This is unlike the situation in the periphery, where deficiency of IRF7 almost eliminates the LCMV-induced production of the type I IFNs. This difference is seemingly related to the local environment, as peripheral production of type I IFNs is severely reduced in intracerebrally (i.c.) infected IRF7-deficient mice, which undergo a combined infection of the CNS and peripheral organs, such as spleen and lymph nodes. Interestingly, despite the redundancy of IRF7 in initiating the type I IFN response in the CNS, the response is not abolished in IFN-ß-deficient mice, as might have been expected. Collectively, these data demonstrate that the early type I IFN response to LCMV infection in the CNS is controlled by a concerted action of IRF3 and -7. Consequently this work provides strong evidence for differential regulation of the type I IFN response in the CNS versus the periphery during viral infection.