Analyzing microglial-associated Aß in Alzheimer's disease transgenic mice with a novel mid-domain Aß-antibody.

The mechanisms of amyloid-ß (Aß)-degradation and clearance in Alzheimer's disease (AD) pathogenesis have been relatively little studied. Short Aß-fragments form by enzymatic cleavage and alternate amyloid-beta precursor protein (APP)-processing. Here we characterized a novel polyclonal Aß-antibody raised against an Aß mid-domain and used it to investigate microglial Aß-uptake in situ by microscopy at the light- and ultrastructural levels. The rabbit Aß-mid-domain antibody (ab338), raised against the mid-domain amino acids 21-34 (Aß21-34), was characterized with biochemical and histological techniques. To identify the epitope in Aß recognized by ab338, solid phase and solution binding data were compared with peptide folding scores as calculated with the Tango software. The ab338 antibody displayed high average affinity (KD: 6.2 × 10-10 M) and showed preference for C-terminal truncated Aß-peptides ending at amino acid 34 and Aß-mid domain peptides with high scores of ß-turn structure. In transgenic APP-mouse brain, ab338 labelled amyloid plaques and detected Aß-fragments in microglia at the ultra- and light microscopic levels. This reinforces a role of microglia/macrophages in Aß-clearance in vivo. The ab338 antibody might be a valuable tool to study Aß-clearance by microglial uptake and Aß-mid-domain peptides generated by enzymatic degradation and alternate production.

Systemic LPS-induced Aß-solubilization and clearance in AßPP-transgenic mice is diminished by heparanase overexpression.

Amyloid-ß (Aß) is the main constituent of amyloid deposits in Alzheimer's disease (AD). The neuropathology is associated with neuroinflammation. Here, we investigated effects of systemic lipopolysaccharide (LPS)-treatment on neuroinflammation and Aß deposition in AßPP-mice and double-transgenic mice with brain expression of AßPP and heparanase, an enzyme that degrades HS and generates an attenuated LPS-response. At 13 months of age, the mice received a single intraperitoneal injection of 50 µg LPS or vehicle, and were sacrificed 1.5 months thereafter. Aß in the brain was analyzed histologically and biochemically after sequential detergent extraction. Neuroinflammation was assessed by CD45 immunostaining and mesoscale cytokine/chemokine ELISA. In single-transgenic mice, LPS-treatment reduced total Aß deposition and increased Tween-soluble Aß. This was associated with a reduced CXCL1, IL-1ß, TNF-α-level and microgliosis, which correlated with amyloid deposition and total Aß. In contrast, LPS did not change Aß accumulation or inflammation marker in the double-transgenic mice. Our findings suggest that a single pro-inflammatory LPS-stimulus, if given sufficient time to act, triggers Aß-clearance in AßPP-transgenic mouse brain. The effects depend on HS and heparanase.

An improved CPRG colorimetric ligand-receptor signal transduction assay based on beta-galactosidase activity in mammalian BWZ-reporter cells.

INTRODUCTION: Reporter cells expressing a chimeric receptor that activates a reporter can be used for screening ligand-mediated signal transduction. In this study, we used reporter cells harboring an NFAT/lacZ construct that express ß-galactosidase when the chimeric receptor is stimulated. A colorimetric ß-galactosidase substrate, chlorophenol-red ß-d-galactopyranoside (CPRG), was used to detect enzymatic activity. Sub-optimal conditions have unfortunately extensively been reported with such reporter-based ß-galactosidase assays. Here, we aimed to improve the CPRG-based colorimetric assay such that receptor ligands could be effectively screened with reporter cells. METHODS: After stimulation of reporter cells, we determined ß-galactosidase activity by absorbance measurement of ß-galactosidase-dependent CPRG hydrolysis. We systematically examined each component in a standard lysis buffer most commonly reported for this type of reporter cells. Furthermore, we evaluated literature in the field. RESULTS: An increased CPRG substrate concentration combined with a different detergent, Saponin, and an optimal wavelength recording markedly increased the sensitivity for the detection of ß-galactosidase activity (≈4-fold increase). Moreover, the improved protocol resulted in increased linear time-dependent recording of enzymatic activity once cells had been lysed, and a more stable and reproducible assay to detect a ligand-stimulus with the reporter cells. The optimal time length of exposure to a stimulus was ligand-dependent. DISCUSSION: In conclusion, we provide an improved protocol with an optimized lysis buffer that gives up to a six-fold higher and more robust specific signal when NFAT/lacZ-based receptor-expressing reporter cells are exposed to a stimulus.

The Alzheimer's disease risk factors apolipoprotein E and TREM2 are linked in a receptor signaling pathway.

BACKGROUND: Triggering receptor expressed on myeloid cells 2 (TREM2) and apolipoprotein E (APOE) are genetically linked to Alzheimer's disease. Here, we investigated whether human ApoE mediates signal transduction through human and murine TREM2 and sought to identify a TREM2-binding domain in human ApoE. METHODS: To investigate cell signaling through TREM2, a cell line was used which expressed an NFAT-inducible ß-galactosidase reporter and human or murine TREM2, fused to CD8 transmembrane and CD3ζ intracellular signaling domains. ELISA-based binding assays were used to determine binding affinities of human ApoE isoforms to human TREM2 and to identify a TREM2-binding domain in ApoE. RESULTS: ApoE was found to be an agonist to human TREM2 with EC50 in the low nM range, and to murine TREM2 with reduced potency. In the reporter cells, TREM2 expression was lower than in nontransgenic mouse brain. Human ApoE isoforms ε2, ε3, and ε4 bound to human TREM2 with K d in the low nM range. The binding was displaced by an ApoE-mimetic peptide (amino acids 130-149). CONCLUSIONS: An ApoE-mediated dose-dependent signal transduction through TREM2 in reporter cells was demonstrated, and a TREM2-binding region in ApoE was identified. The relevance of an ApoE-TREM2 receptor signaling pathway to Alzheimer's disease is discussed.

Overexpression of heparanase lowers the amyloid burden in amyloid-ß precursor protein transgenic mice.

Heparan sulfate (HS) and HS proteoglycans (HSPGs) colocalize with amyloid-ß (Aß) deposits in Alzheimer disease brain and in Aß precursor protein (AßPP) transgenic mouse models. Heparanase is an endoglycosidase that specifically degrades the unbranched glycosaminoglycan side chains of HSPGs. The aim of this study was to test the hypothesis that HS and HSPGs are active participators of Aß pathogenesis in vivo. We therefore generated a double-transgenic mouse model overexpressing both human heparanase and human AßPP harboring the Swedish mutation (tgHpa*Swe). Overexpression of heparanase did not affect AßPP processing because the steady-state levels of Aß1-40, Aß1-42, and soluble AßPP ß were the same in 2- to 3-month-old double-transgenic tgHpa*Swe and single-transgenic tgSwe mice. In contrast, the Congo red-positive amyloid burden was significantly lower in 15-month-old tgHpa*Swe brain than in tgSwe brain. Likewise, the Aß burden, measured by Aßx-40 and Aßx-42 immunohistochemistry, was reduced significantly in tgHpa*Swe brain. The intensity of HS-stained plaques correlated with the Aßx-42 burden and was reduced in tgHpa*Swe mice. Moreover, the HS-like molecule heparin facilitated Aß1-42-aggregation in an in vitro Thioflavin T assay. The findings suggest that HSPGs contribute to amyloid deposition in tgSwe mice by increasing Aß fibril formation because heparanase-induced fragmentation of HS led to a reduced amyloid burden. Therefore, drugs interfering with Aß-HSPG interactions might be a potential strategy for Alzheimer disease treatment.

Systemic and Cerebral Vascular Endothelial Growth Factor Levels Increase in Murine Cerebral Malaria along with Increased Calpain and Caspase Activity and Can be Reduced by Erythropoietin Treatment.

The pathogenesis of cerebral malaria (CM) includes compromised microvascular perfusion, increased inflammation, cytoadhesion, and endothelial activation. These events cause blood-brain barrier disruption and neuropathology and associations with the vascular endothelial growth factor (VEGF) signaling pathway have been shown. We studied this pathway in mice infected with Plasmodium berghei ANKA causing murine CM with or without the use of erythropoietin (EPO) as adjunct therapy. ELISA and western blotting was used for quantification of VEGF and relevant proteins in brain and plasma. CM increased levels of VEGF in brain and plasma and decreased plasma levels of soluble VEGF receptor 2. EPO treatment normalized VEGF receptor 2 levels and reduced brain VEGF levels. Hypoxia-inducible factor (HIF)-1α was significantly upregulated whereas cerebral HIF-2α and EPO levels remained unchanged. Furthermore, we noticed increased caspase-3 and calpain activity in terminally ill mice, as measured by protease-specific cleavage of α-spectrin and p35. In conclusion, we detected increased cerebral and systemic VEGF as well as HIF-1α, which in the brain were reduced to normal in EPO-treated mice. Also caspase and calpain activity was reduced markedly in EPO-treated mice.