Characterization and clinical use of inflammatory cerebrospinal fluid protein markers in Alzheimer's disease.

BACKGROUND: Neuroinflammation has gained increasing attention as a potential contributing factor in Alzheimer's disease (AD) pathology. A clinical cerebrospinal fluid biomarker capable of monitoring this process during the course of the disease has yet to emerge, chiefly owing to contradictory research findings. In this study, we sought to clarify the utility of inflammatory biomarkers in diagnostic procedures of AD in three steps: (1) to screen for proteins that are robustly detectable in cerebrospinal fluid; (2) based on this analysis, to explore any associations between the analytically robust markers and salient pathological features of AD; and (3) to determine the discriminative power of these markers in the clinical diagnosis of AD. METHODS: From a total of 46 proteins, 15 that were robustly detectable in cerebrospinal fluid were identified. A subsequent analysis of these markers in a cohort of 399 patients (nondemented subjects, patients with mild cognitive impairment [MCI], and patients with AD, supplemented by smaller cohorts of other diseases) was conducted. Fluid biomarker data were related to AD pathology and neuropsychological markers and adjusted for confounders such as age, sex, apolipoprotein E genotype, and biobank storage time. RESULTS: Cerebrospinal fluid levels of C-reactive protein and soluble TREM2 differed between nondemented subjects, patients with MCI, or patients with AD and were associated with amyloid and tau pathology. Several markers were associated with tau pathology only or with other neurodegenerative diseases. Correlations between neuropsychological performance and inflammatory markers were weak, but they were most prominent in AD and for the most challenging cognitive tests. All investigated covariates had significant influence, with varying effects across the markers. Still, none of the markers achieved discriminative power of more than 70% to distinguish between patient groups defined by clinical or neuropathological categories. CONCLUSIONS: Basic analytical considerations proved indispensable for this type of study because only one-third of the tested markers were robustly detectable in cerebrospinal fluid. Detectable inflammatory protein markers were associated in multiple ways with AD pathology. Yet, even significantly associated markers were not powerful enough in terms of effect strength, sensitivity, and specificity, and hence they were not suited for direct use in clinical diagnostic practice. Targets other than those most commonly considered in this field of research might provide results with better clinical applicability.

CXCR3 promotes plaque formation and behavioral deficits in an Alzheimer's disease model.

Chemokines are important modulators of neuroinflammation and neurodegeneration. In the brains of Alzheimer's disease (AD) patients and in AD animal models, the chemokine CXCL10 is found in high concentrations, suggesting a pathogenic role for this chemokine and its receptor, CXCR3. Recent studies aimed at addressing the role of CXCR3 in neurological diseases indicate potent, but diverse, functions for CXCR3. Here, we examined the impact of CXCR3 in the amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic mouse model of AD. We found that, compared with control APP/PSI animals, plaque burden and Aß levels were strongly reduced in CXCR3-deficient APP/PS1 mice. Analysis of microglial phagocytosis in vitro and in vivo demonstrated that CXCR3 deficiency increased the microglial uptake of Aß. Application of a CXCR3 antagonist increased microglial Aß phagocytosis, which was associated with reduced TNF-α secretion. Moreover, in CXCR3-deficient APP/PS1 mice, microglia exhibited morphological activation and reduced plaque association, and brain tissue from APP/PS1 animals lacking CXCR3 had reduced concentrations of proinflammatory cytokines compared with controls. Further, loss of CXCR3 attenuated the behavioral deficits observed in APP/PS1 mice. Together, our data indicate that CXCR3 signaling mediates development of AD-like pathology in APP/PS1 mice and suggest that CXCR3 has potential as a therapeutic target for AD.

Postoperative ileus involves interleukin-1 receptor signaling in enteric glia.

BACKGROUND & AIMS: Postoperative ileus (POI) is a common consequence of abdominal surgery that increases the risk of postoperative complications and morbidity. We investigated the cellular mechanisms and immune responses involved in the pathogenesis of POI. METHODS: We studied a mouse model of POI in which intestinal manipulation leads to inflammation of the muscularis externa and disrupts motility. We used C57BL/6 (control) mice as well as mice deficient in Toll-like receptors (TLRs) and cytokine signaling components (TLR-2(-/-), TLR-4(-/-), TLR-2/4(-/-), MyD88(-/-), MyD88/TLR adaptor molecule 1(-/-), interleukin-1 receptor [IL-1R1](-/-), and interleukin (IL)-18(-/-) mice). Bone marrow transplantation experiments were performed to determine which cytokine receptors and cell types are involved in the pathogenesis of POI. RESULTS: Development of POI did not require TLRs 2, 4, or 9 or MyD88/TLR adaptor molecule 2 but did require MyD88, indicating a role for IL-1R1. IL-1R1(-/-) mice did not develop POI; however, mice deficient in IL-18, which also signals via MyD88, developed POI. Mice given injections of an IL-1 receptor antagonist (anakinra) or antibodies to deplete IL-1α and IL-1ß before intestinal manipulation were protected from POI. Induction of POI activated the inflammasome in muscularis externa tissues of C57BL6 mice, and IL-1α and IL-1ß were released in ex vivo organ bath cultures. In bone marrow transplantation experiments, the development of POI required activation of IL-1 receptor in nonhematopoietic cells. IL-1R1 was expressed by enteric glial cells in the myenteric plexus layer, and cultured primary enteric glia cells expressed IL-6 and the chemokine monocyte chemotactic protein 1 in response to IL-1ß stimulation. Immunohistochemical analysis of human small bowel tissue samples confirmed expression of IL-1R1 in the ganglia of the myenteric plexus. CONCLUSIONS: IL-1 signaling, via IL-1R1 and MyD88, is required for development of POI after intestinal manipulation in mice. Agents that interfere with the IL-1 signaling pathway are likely to be effective in the treatment of POI.

Mrp14 deficiency ameliorates amyloid ß burden by increasing microglial phagocytosis and modulation of amyloid precursor protein processing.

Neuroinflammation plays a fundamental role in the pathogenesis of Alzheimer's disease (AD), resulting in the extensive activation of microglial and astroglial cells. Here we describe the role of myeloid-related protein Mrp14, a recently described amplifier of inflammation, in Alzheimer's disease and in the related amyloid precursor protein/presenilin1 (APP/PS1) mouse model. Detection of Mrp14 in control, mildly cognitive impaired, and AD patients revealed a strong induction of Mrp14 in protein extracts as well as in the cerebrospinal fluid, but not in blood plasma. In APP/PS1 mice, Mrp14 and its heterodimeric partner Mrp8 was found to be upregulated in microglial cells surrounding amyloid plaques. Functionally, loss of Mrp14 led to increased phagocytosis of fibrillar amyloid ß (Aß) in microglia cells in vitro and in vivo. Generating APP/PS1-transgenic mice deficient for Mrp14, we observed a decrease of key cytokines involved in APP processing, a reduction of BACE1 expression and activity, and consequently overall Aß deposition. We therefore conclude that Mrp14 promotes APP processing and Aß accumulation under neuroinflammatory conditions.

Nitric oxide decreases the enzymatic activity of insulin degrading enzyme in APP/PS1 mice.

Nitric oxide has been implicated in the regulation of enzyme activity, particularly the activity of metalloproteinases. Since the inducible form of the nitric oxide synthase (NOS2), is upregulated in Alzheimer's disease, we investigated the activity of two amyloid ß degrading enzymes, IDE and neprilysin. In vitro we demonstrated that the activity of IDE was inhibited by *NO donor Sin-1, whereas activity of neprilysin remained unaffected. In vivo the activity of insulin-degrading enzyme was lowered in APP/PS1 mice, but not in APP/PS1/NOS2(-/-) mice. These data suggest that NOS2 upregulation impairs amyloid ß degradation through negative regulation of IDE activity and thus loss of NOS2 activity will positively influence amyloid ß clearance.

Nitration of tyrosine 10 critically enhances amyloid ß aggregation and plaque formation.

Part of the inflammatory response in Alzheimer's disease (AD) is the upregulation of the inducible nitric oxide synthase (NOS2) resulting in increased NO production. NO contributes to cell signaling by inducing posttranslational protein modifications. Under pathological conditions there is a shift from the signal transducing actions to the formation of protein tyrosine nitration by secondary products like peroxynitrite and nitrogen dioxide. We identified amyloid ß (Aß) as an NO target, which is nitrated at tyrosine 10 (3NTyr(10)-Aß). Nitration of Aß accelerated its aggregation and was detected in the core of Aß plaques of APP/PS1 mice and AD brains. NOS2 deficiency or oral treatment with the NOS2 inhibitor L-NIL strongly decreased 3NTyr(10)-Aß, overall Aß deposition and cognitive dysfunction in APP/PS1 mice. Further, injection of 3NTyr(10)-Aß into the brain of young APP/PS1 mice induced ß-amyloidosis. This suggests a disease modifying role for NOS2 in AD and therefore represents a potential therapeutic target.

Distinct and non-redundant roles of microglia and myeloid subsets in mouse models of Alzheimer's disease.

Mononuclear phagocytes are important modulators of Alzheimer's disease (AD), but the specific functions of resident microglia, bone marrow-derived mononuclear cells, and perivascular macrophages have not been resolved. To elucidate the spatiotemporal roles of mononuclear phagocytes during disease, we targeted myeloid cell subsets from different compartments and examined disease pathogenesis in three different mouse models of AD (APP(swe/PS1), APP(swe), and APP23 mice). We identified chemokine receptor 2 (CCR2)-expressing myeloid cells as the population that was preferentially recruited to ß-amyloid (Aß) deposits. Unexpectedly, AD brains with dysfunctional microglia and devoid of parenchymal bone marrow-derived phagocytes did not show overt changes in plaque pathology and Aß load. In contrast, restriction of CCR2 deficiency to perivascular myeloid cells drastically impaired ß-amyloid clearance and amplified vascular Aß deposition, while parenchymal plaque deposition remained unaffected. Together, our data advocate selective functions of CCR2-expressing myeloid subsets, which could be targeted specifically to modify disease burden in AD.

Induced LC degeneration in APP/PS1 transgenic mice accelerates early cerebral amyloidosis and cognitive deficits.

Degeneration of locus ceruleus neurons and subsequent reduction of norepinephrine concentration in locus ceruleus projection areas represent an early pathological indicator of Alzheimer's disease. In order to model the pathology of the human disease and to study the effects of norepinephrine-depletion on amyloid precursor protein processing, behaviour, and neuroinflammation, locus ceruleus degeneration was induced in mice coexpressing the swedish mutant of the amyloid precursor protein and the presenilin 1 DeltaExon 9 mutant (APP/PS1) using the neurotoxin N-(2-chloroethyl)-N-ethyl-bromo-benzylamine (dsp4) starting treatment at 3 months of age. Norepinephrine transporter immunolabelling demonstrated severe loss of locus ceruleus neurons and loss of cortical norepinephrine transporter starting as early as 4.5 months of age and aggravating over time. Of note, dsp4-treated transgenic mice showed elevated amyloid beta levels and impaired spatial memory performance at 6.5 months of age compared to control-treated APP/PS1 transgenic mice, indicating an accelerating effect on cerebral amyloidosis and cognitive deficits. Likewise, norepinephrine-depletion increased neuroinflammation compared to transgenic controls as verified by macrophage inflammatory protein-1alpha and -1beta gene expression analysis. Exploratory activity and memory retention was compromised by age in APP/PS1 transgenic mice and further aggravated by induced noradrenergic deficiency. In contrast, novel object recognition was not influenced by norepinephrine deficiency, but by the APP/PS1 transgene at 12 months. Overall, our data indicate that early loss of noradrenergic innervation promotes amyloid deposition and modulates the activation state of inflammatory cells. This in turn could have had impact on the acceleration of cognitive deficits observed over time.

PPARgamma and RXRgamma ligands act synergistically as potent antineoplastic agents in vitro and in vivo glioma models.

Glioblastoma represent the most common primary brain tumor in adults and are currently considered incurable. We investigated antiproliferative and anti-invasive mechanisms of 6-OH-11-O-hydroxyfenantrene (IIF), a retinoid X receptor ligand, and pioglitazone (PGZ), a peroxisome proliferator-activated receptor gamma activator, in three different glioblastoma cell lines. A dose-dependent reduction of tumor invasion and strong decrease of matrix metalloproteinases 2 and 9 expression was observed, especially when a combination therapy of IIF and PGZ was administered. Combined treatment also markedly reduced proliferation and induced apoptosis in all glioma cell lines tested. This was in particular accompanied by decrease of antiapoptotic proteins Bcl2 and p53, while simultaneously pro-apoptotic cytochrome c, cleaved caspase 3, Bax and Bad levels increased. These in vitro findings were further substantiated in a murine glioma model in vivo, where oral administration of PGZ and IIF resulted in significantly reduced tumor volume and proliferation. Of note, treatment with nuclear receptor ligands was not only effective when the treatment was initiated shortly after the intraparenchymal seeding of the glioma cells, but even when initiated in the last third of the observation period. Collectively, our results demonstrate the effectiveness of a combined treatment of ligands of proliferator-activated receptor and retinoid X receptor against glioblastoma.

Formation of Pmel17 amyloid is regulated by juxtamembrane metalloproteinase cleavage, and the resulting C-terminal fragment is a substrate for gamma-secretase.

The formation of insoluble cross beta-sheet amyloid is pathologically associated with disorders such as Alzheimer, Parkinson, and Huntington diseases. One exception is the nonpathological amyloid derived from the protein Pmel17 within melanosomes to generate melanin pigment. Here we show that the formation of insoluble MalphaC intracellular fragments of Pmel17, which are the direct precursors to Pmel17 amyloid, depends on a novel juxtamembrane cleavage at amino acid position 583 between the furin-like proprotein convertase cleavage site and the transmembrane domain. The resulting Pmel17 C-terminal fragment is then processed by the gamma-secretase complex to release a short-lived intracellular domain fragment. Thus, by analogy to the Notch receptor, we designate this cleavage the S2 cleavage site, whereas gamma-secretase mediates proteolysis at the intramembrane S3 site. Substitutions or deletions at this S2 cleavage site, the use of the metalloproteinase inhibitor TAPI-2, as well as small interfering RNA-mediated knock-down of the metalloproteinases ADAM10 and 17 reduced the formation of insoluble Pmel17 fragments. These results demonstrate that the release of the Pmel17 ectodomain, which is critical for melanin amyloidogenesis, is initiated by S2 cleavage at a juxtamembrane position.

Sepsis causes neuroinflammation and concomitant decrease of cerebral metabolism.

BACKGROUND: Septic encephalopathy is a severe brain dysfunction caused by systemic inflammation in the absence of direct brain infection. Changes in cerebral blood flow, release of inflammatory molecules and metabolic alterations contribute to neuronal dysfunction and cell death. METHODS: To investigate the relation of electrophysiological, metabolic and morphological changes caused by SE, we simultaneously assessed systemic circulation, regional cerebral blood flow and cortical electroencephalography in rats exposed to bacterial lipopolysaccharide. Additionally, cerebral glucose uptake, astro- and microglial activation as well as changes of inflammatory gene transcription were examined by small animal PET using [18F]FDG, immunohistochemistry, and real time PCR. RESULTS: While the systemic hemodynamic did not change significantly, regional cerebral blood flow was decreased in the cortex paralleled by a decrease of alpha activity of the electroencephalography. Cerebral glucose uptake was reduced in all analyzed neocortical areas, but preserved in the caudate nucleus, the hippocampus and the thalamus. Sepsis enhanced the transcription of several pro- and anti-inflammatory cytokines and chemokines including tumor necrosis factor alpha, interleukin-1 beta, transforming growth factor beta, and monocot chemoattractant protein 1 in the cerebrum. Regional analysis of different brain regions revealed an increase in ED1-positive microglia in the cortex, while total and neuronal cell counts decreased in the cortex and the hippocampus. CONCLUSION: Together, the present study highlights the complexity of sepsis induced early impairment of neuronal metabolism and activity. Since our model uses techniques that determine parameters relevant to the clinical setting, it might be a useful tool to develop brain specific therapeutic strategies for human septic encephalopathy.

Endoplasmic reticulum-localized amyloid beta-peptide is degraded in the cytosol by two distinct degradation pathways.

The paradigm of endoplasmic reticulum (ER)-associated degradation (ERAD) holds that misfolded secretory and membrane proteins are translocated back to the cytosol and degraded by the proteasome in a coupled process. Analyzing the degradation of ER-localized amyloid beta-peptide (Abeta), we found a divergence from this general model. Cell-free reconstitution of the export in biosynthetically loaded ER-derived brain microsomes showed that the export was mediated by the Sec61p complex and required a cytosolic factor but was independent of ATP. In contrast to the ERAD substrates known so far, the exported Abeta was degraded by both, a proteasome-dependent and a proteasome-independent pathway. RNA interference experiments in Abeta-transfected cells identified the protease of the proteasome-independent pathway as insulin-degrading enzyme (IDE). The IDE-mediated clearance mechanism for ER-localized Abeta represents an as yet unknown type of ERAD which is not entirely dependent on the proteasome.