Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: Primary antibodies used in this study

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques:

Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: CMKLR1 deficiency decreases mortality and improves cognition in APP/PS1 mice and/or ICV-STZ mice. A, Survival curves for WT, APP/PS1, CMKLR1−/−, and APP/PS1-CMKLR1−/− mice. Data are mean ± SEM, with 22-29 mice in each group. ***p < 0.001 compared with WT mice. $$p < 0.01 compared with APP/PS1 mice. B–F, Spatial reference learning and memory were determined in Morris water maze. Mice were trained in Morris water maze for consecutive 5 d, four trials per day. The distance traveled (B) and time spent (C) to reach the escape platform are shown. The probe trials were tested on the seventh day after a day off. The percentage of the distance traveled (D) and the time spent (E) within each quadrant are shown. F, Representative swimming paths of a mouse on day 7. Data are mean ± SEM, with 6-10 mice in each group (WT, n = 9; APP/PS1, n = 9; CMKLR1−/−, n = 10; APP/PS1-CMKLR1−/−, n = 6). *p < 0.05; **p < 0.01; compared with WT mice. $p < 0.05 compared with APP/PS1 mice. G, Schematic representation of study design. The ICV-STZ mice and ICV-saline mice were produced by ICV injection of a single dose of 3.0 mg/kg STZ or equal volume of 0.9% saline into both lateral ventricles of the mice brain, respectively. The body weight of these mice was measured every day in the first 7 d, and then once every 7 d. Six weeks after ICV injection, all mice were subjected to a battery of behavioral tests, and then killed for biochemical studies. H, The body weight of WT and CMKLR1−/− mice after receiving STZ or saline was recorded. I–M, Spatial reference learning and memory were examined in Morris water maze. The distance traveled (I) and the time spent (J) to reach the escape platform are shown. The probe trials were tested on seventh day after a day off. The percentage of the distance traveled (K) and the time spent (L) within each quadrant are shown. M, Representative swimming paths of a mouse on day 7. Data are mean ± SEM, with 4-8 mice in each group (WT ICV-saline, n = 8; WT ICV-STZ, n = 7; CMKLR1−/− ICV-saline, n = 8; CMKLR1−/− ICV-STZ, n = 4). *p < 0.05; ***p < 0.001; compared with WT ICV-saline mice. ##p < 0.01 compared with CMKLR1−/− ICV-saline mice. $$p < 0.01 compared with WT ICV-STZ mice.

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques: Produced, Injection

Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: CMKLR1 deficiency is associated with increased Aβ deposition in APP/PS1 mice. A, Representative immunofluorescence images of Aβ (Aβ deposits, green) frozen in the hippocampus and the cortex of WT, APP/PS1, CMKLR1−/−, and APP/PS1-CMKLR1−/− mice aged 6, 9, and 13 months. Scale bar, 500 μm. Quantification of the fluorescence intensity of Aβ deposits, showing that CMKLR1 deficiency significantly increases Aβ deposition in APP/PS1 mice at the age of 6 months (B), 9 months (C), and 13 months (D). Data are mean ± SEM, based on three fields for each region, using at least 3 mice in each group. *p < 0.05; **p < 0.01; compared with WT mice. ###p < 0.001 compared with CMKLR1−/− mice. $$p < 0.01; $$$p < 0.001; compared with APP/PS1 mice. E, Insoluble Aβ1-42 from the hippocampus of 9-month-old mice were measured using ELISA. Data are mean ± SEM, with at least 3 mice in each group. *p < 0.05 compared with WT mice. ###p < 0.001 compared with CMKLR1−/− mice. $$$p < 0.001 compared with APP/PS1 mice. F, Real-time PCR quantification of the transcripts for APP of the mice at the age of 9 months. Data are mean ± SEM, with at least 3 mice in each group. *p < 0.05 compared with WT mice. #p < 0.05 compared with CMKLR1−/− mice. G, Representative Western blots showing the expression of APP protein in the brain of 9-month-old mice. H, Quantification of the immunoreactivity of the blots, normalized against β-actin. Data are mean ± SEM, with at least 3 mice in each group. *p < 0.05 compared with WT mice. #p < 0.05 compared with CMKLR1−/− mice.

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques: Immunofluorescence, Fluorescence, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: CMKLR1 deficiency has no effect on the locomotion, spontaneous exploratory, and anxiety of APP/PS1 mice aged 9 months. A, The locomotion of mice was tested on Rotarod, and the fall latency was recorded. B, The spontaneous exploratory activity of mice was examined in an open field, and the distance traveled during 15 min was recorded. The anxiety of mice was tested in an elevated plus maze (C–E) and an open field (F–H). The distance traveled (C) and the time spent (D) in the open arm were recorded. E, Representative traces of a mouse's movements during the elevated plus maze. The distance traveled (F) and the time spent (G) in the center were recorded. H, Representative traces of a mouse's movements during the open field test. Data are mean ± SEM, with 6-10 mice in each group (WT, n = 9; APP/PS1, n = 7; CMKLR1−/−, n = 10; APP/PS1-CMKLR1−/−, n = 6). *p < 0.05; **p < 0.01; compared with WT mice. #p < 0.05 compared with CMKLR1−/− mice.

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques: Activity Assay

Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: CMKLR1 deficiency attenuates hyperphosphorylation of tau in the brain of APP/PS1 mice and ICV-STZ mice. A, Representative Western blots showing tau phosphorylation at Ser199, Thr205, Ser396, and Ser404 in the hippocampus of WT, APP/PS1, CMKLR1−/−, and APP/PS1-CMKLR1−/− mice at the age of 9 months. The levels of nonphosphorylated tau (Tau1) and total tau (Tau5) were also measured. Quantification of the immunoreactivity of the blots, normalized against total tau (B) and β-actin (C). Data are mean ± SEM, with 6 mice in each group. *p < 0.05 compared with WT mice. $p < 0.05 compared with APP/PS1 mice. D, Representative Western blots showing tau phosphorylation in the brain of WT and CMKLR1−/− mice receiving ICV injection of STZ or saline. Quantification of the immunoreactivity of the blots, normalized against total tau (E) and β-actin (F). Data are mean ± SEM, with 6 mice in each group. *p < 0.05; **p < 0.01; ***p < 0.001; compared with WT ICV-saline mice. $p < 0.05; $$p < 0.01; compared with WT ICV-STZ mice.

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques: Western Blot, Injection

Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: Upregulation of CMKLR1 and colocalization with MAP2, Iba1, and GFAP in the brain of APP/PS1 mice. A, Representative immunofluorescence images of CMKLR1 (green) in CA1, CA3, and DG regions of the hippocampus and in the cortex of WT and APP/PS1 mice aged 6 months and 9 months. Scale bar, 50 µm. Quantification of the fluorescence intensity of CMKLR1 in each of the regions, showing that CMKLR1 was significantly increased in the hippocampus and the cortex of APP/PS1 mice at the age of 6 months (B) and 9 months (C). Data are mean ± SEM, with at least 3 mice in each group. *p < 0.05; **p < 0.01; compared with WT mice. D–F, Colocalization of CMKLR1 (green) with neurons (anti-MAP2, red, D), microglia (anti-Iba1, red, E), and astrocytes (anti-GFAP, red, F) in the hippocampus and the cortex of APP/PS1 mice aged 9 months. Scale bar, 50 μm.

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques: Immunofluorescence, Fluorescence

Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: Inhibition of neuronal CMKLR1 attenuates tau hyperphosphorylation induced by high glucose treatment in SH-SY5Y cells. A, Representative immunofluorescence images of CMKLR1 (green) in SH-SY5Y cells. Scale bar, 50 μm. B, Representative Western blots showing tau hyperphosphorylation at Ser199, Thr205, and Ser396 from SH-SY5Y treated with 5, 15, or 30 mm glucose for 12 or 24 h. Quantification of the immunoreactivity of the blots, normalized against total tau (C) and β-actin (D). E, Representative Western blots showing tau hyperphosphorylation at Ser199 and Ser396 from SH-SY5Y incubated with 5 or 30 mm glucose for 24 h, with or without a 1 h pretreatment with C15 (1 or 10 µm). Quantification of the immunoreactivity of the blots, normalized against total tau (F) and β-actin (G). Data are mean ± SEM from three separate experiments, each in duplicate or triplicate. *p < 0.05; **p < 0.01; ***p < 0.001; compared with 5 mM glucose treatment group. $p < 0.05; $$p < 0.01; compared with 30 mm glucose treatment group. Glu: glucose.

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques: Inhibition, Immunofluorescence, Western Blot, Incubation

Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: CMKLR1 deficiency is associated with tau spreading in ICV-Tau model mice. A, Schematic representation of ICV-Tau model mouse construction. The hippocampus extracts containing hyperphosphorylated tau (from 9-month-old APP/PS1 mice) or normal phosphorylated tau (from age-matched WT mice) were centrifuged at 5,500 rpm for 5 min in 4°C, and then the supernatant was injected into the ventricles of 6-month-old WT mice and age-matched CMKLR1−/− mice to establish the ICV-hyper-pTau mice and ICV-nor-pTau mice. Mice were killed 3 weeks after the injection. B, Representative Western blots showing tau hyperphosphorylation at Ser199, Thr205, and Ser396 in the hippocampus of WT and CMKLR1−/− mice receiving ICV injection of the extracts containing hyper-pTau or nor-pTau. Quantification of the immunoreactivity of the blots, normalized against total tau (C) and β-actin (D). E, Representative Western blots showing tau hyperphosphorylation in the cortex of WT and CMKLR1−/− mice receiving ICV injection of the extracts containing hyper-pTau or nor-pTau. Quantification of the immunoreactivity of the blots, normalized against total tau (F) and β-actin (G). Data are mean ± SEM, with at least 2 mice in each group (WT ICV-nor-pTau, n = 2; WT ICV-hyper-pTau, n = 3; CMKLR1−/− ICV-nor-pTau, n = 3; CMKLR1−/− ICV-hyper-pTau, n = 2). *p < 0.05; **p < 0.01; compared with WT ICV-nor-pTau mice. $p < 0.05; $$p < 0.01; compared with WT ICV-hyper-pTau mice.

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques: Injection, Western Blot

Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: Neuronal CMKLR1 affects tau phosphorylation through regulating tau seeding. A, Tau protein in the supernatant from SH-SY5Y stimulated with 5 or 30 mm glucose was examined using ELISA. Data are mean ± SEM from three separate experiments, each in duplicate or triplicate. **p < 0.01 compared with DMEM medium without glucose treatment. ##p < 0.01 compared with 5 mm glucose treatment group. B, Representative Western blots showing the human specific tau protein in the supernatant from SH-SY5Y stimulated with 5 or 30 mm glucose. C, Representative immunofluorescence images of CMKLR1 (green) in primary cultures of mouse neurons. Scale bar, 50 μm. D–G, The supernatant from SH-SY5Y treated with 5 or 30 mm glucose was collected, purified, and then applied to primary cultures of neurons from WT mice and CMKLR1−/− mice for 3 d. Representative immunofluorescence images of human specific tau protein (red) and human and mouse phosphorylated tau at Thr205 site (green, D) and Ser396 site (green, E) in primary cultures of neurons from WT mice and CMKLR1−/− mice. The primary cultures of neurons were incubated with the purified supernatant form SH-SY5Y treated with 5 or 30 mm glucose. Scale bar, 50 μm. Quantification of the area of mouse phosphorylated tau at pT205 (F) and pS396 (G), calculated by subtracting the fluorescence area of human tau (yellow) from the fluorescence area of Hu and Ms tau (green), showing that human tau taken up by mouse neurons significantly accelerated the hyperphosphorylation of mouse tau in primary cultures of mouse neurons. Data are mean ± SEM from three separate experiments, each in duplicate or triplicate. ***p < 0.001 compared with primary cultures of neurons from WT mice treated with the purified supernatant stimulated with 5 mm glucose. $$$p < 0.001 compared with primary cultures of neurons from WT mice treated with the purified supernatant stimulated with 30 mm glucose. Glu: glucose.

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Purification, Incubation, Fluorescence

Journal: Molecular Medicine

Article Title: Low-dose pro-resolving mediators temporally reset the resolution response to microbial inflammation

doi: 10.1186/s10020-024-00877-w

Figure Lengend Snippet: Regulation of SPM receptors with human monocyte-derived macrophages

Article Snippet: Peripheral blood mononuclear cells were isolated, and MΦ were differentiated by culturing freshly isolated monocytes in RPMI media supplemented with 10% FBS and recombinant human GM-CSF (10 ng/ml; R&D Systems) for 7 d. Macrophages were then incubated with 10 nM of RvD1, RvD2, MaR1, RvE2 or vehicle control for 24 h. Cells were collected and receptor surface expression monitored by flow cytometry using the following specific antibodies and appropriate isotype controls: GPR18 Antibody [Alexa Fluor® 647] (NBP2-24918AF647, Novus Biologicals) and Rabbit IgG Isotype Control [Alexa Fluor® 647] (NBP2-36463AF647, Novus Biologicals), human ChemR23 PE-conjugated Antibody (FAB362P, R&D systems) and mouse IgG3 PE-conjugated Antibody (IC007P, R&D systems), LGR6 Polyclonal antibody (17658-1-AP, Proteintech) and PE-conjugated F(ab')2-Donkey anti-Rabbit IgG (H + L) Secondary Antibody (12-4739-81, eBioscience).

Techniques:

Journal: Arthritis Research & Therapy

Article Title: Chemerin activates fibroblast-like synoviocytes in patients with rheumatoid arthritis

doi: 10.1186/ar3475

Figure Lengend Snippet: Expression of ChemR23 in rheumatoid arthritis and osteoarthritis synovial tissue samples . ChemR23 expression in synovial tissue samples examined by immunohistochemistry that were obtained from five rheumatoid arthritis (RA) patients (A) and three osteoarthritis (OA) patients (B) . All sections were counterstained with hematoxylin. Sections of RA synovial tissues were also double-stained with CD68, CD1a, DC-LAMP or vimentin, and ChemR23 and were analyzed by fluorescence microscopy as follows: (C) CD68, (D) ChemR23, (E) merged image of (C) and (D), (F) CD1a, (G) ChemR23, (H) merged image of (F) and (G), (I) DC-LAMP, (J) ChemR23, (K) merged image of (I) and (J), (L) vimentin, (M) ChemR23 and (N) merged image of (L) and (M). Arrows indicate double-positive cells. Original magnification ×100 in (A) and (B) and ×400 in (C) through (N). Whole-mount specimens obtained from RA patients were stained with anti-ChemR23 pAb (green) or phalloidin (blue) and then analyzed by confocal laser-scanning microscopy. (O) x - y view of synovial tissue sectioning. x - z image (P) and y-z image (Q) of synovial tissue sectioning are displayed below and to the right of the x - y view in (O). Scale bars = 80 μm. White straight line boxes indicate identical cell in each direction (O-Q). Dashed line connects the boxes.

Article Snippet: Serial sections were then incubated for two hours at 4°C with 1 μg/ml rabbit anti-chemerin pAb (affinity-purified from rabbit serum immunized with glutathione S -transferase-chemerin (E 21 -S 157 ) fusion protein; provided by KAN Research Institute, Kobe, Japan), 1 μg/ml rabbit anti-ChemR23 pAb (Acris Antibodies, Herford, Germany) or normal rabbit immunoglobulin G (IgG) (Sigma-Aldrich, St Louis, MO, USA) as an isotype control.

Techniques: Expressing, Immunohistochemistry, Staining, Fluorescence, Microscopy, Confocal Laser Scanning Microscopy

Journal: Arthritis Research & Therapy

Article Title: Chemerin activates fibroblast-like synoviocytes in patients with rheumatoid arthritis

doi: 10.1186/ar3475

Figure Lengend Snippet: Expression of chemerin and ChemR23 in rheumatoid arthritis and osteoarthritis synovial tissue samples . (A) Western blots of chemerin protein expression in three rheumatoid arthritis (RA) and four osteoarthritis (OA) synovial tissues. M, protein molecular weight marker. (B) Relative protein expression of chemerin to β-actin in RA and OA synovial tissues. * P < 0.05 relative to RA synovial tissue. (C) Western blots of ChemR23 protein expression in three RA and four OA synovial tissues. M, protein molecular weight marker. (D) Relative expression of ChemR23 protein to β-actin. * P < 0.05 relative to RA synovial tissue.

Article Snippet: Serial sections were then incubated for two hours at 4°C with 1 μg/ml rabbit anti-chemerin pAb (affinity-purified from rabbit serum immunized with glutathione S -transferase-chemerin (E 21 -S 157 ) fusion protein; provided by KAN Research Institute, Kobe, Japan), 1 μg/ml rabbit anti-ChemR23 pAb (Acris Antibodies, Herford, Germany) or normal rabbit immunoglobulin G (IgG) (Sigma-Aldrich, St Louis, MO, USA) as an isotype control.

Techniques: Expressing, Western Blot, Molecular Weight, Marker

Journal: Arthritis Research & Therapy

Article Title: Chemerin activates fibroblast-like synoviocytes in patients with rheumatoid arthritis

doi: 10.1186/ar3475

Figure Lengend Snippet: Expression of chemerin and ChemR23 by rheumatoid arthritis fibroblast-like synoviocytes . (A) and (B) Chemerin expression stimulated by TNF-α (A) or IFN-γ (B) and evaluated by ELISA using supernatants of cultured fibroblast-like synoviocytes (FLSs) isolated from synovial tissue samples taken from rheumatoid arthritis (RA) patients ( n = 4). FLSs (2 × 10 4 cells/well) were stimulated at 37°C for 48 hours with TNF-α (0.1, 1 or 10 ng/ml) (A) or IFN-γ (1, 10 or 100 ng/ml) (A). Data in (A) and (B) are presented as means (± SEM) of one of four independent experiments analyzed in triplicate. * P < 0.05 relative to control. (C) through (E) Immunocytochemistry showing double-staining of cultured RA FLSs with (C) vimentin (D) ChemR23 and (E) a merged image of (C) and (D). (F) Western blot of ChemR23 protein expression in RA FLSs following TNF-α (10 ng/ml), IFN-γ (100 ng/ml), transforming growth factor (TGF)-β1 (1 ng/ml), IL-1β (5 ng/ml) or IL-6 (20 ng/ml) stimulation at 37°C for 24 hours.

Article Snippet: Serial sections were then incubated for two hours at 4°C with 1 μg/ml rabbit anti-chemerin pAb (affinity-purified from rabbit serum immunized with glutathione S -transferase-chemerin (E 21 -S 157 ) fusion protein; provided by KAN Research Institute, Kobe, Japan), 1 μg/ml rabbit anti-ChemR23 pAb (Acris Antibodies, Herford, Germany) or normal rabbit immunoglobulin G (IgG) (Sigma-Aldrich, St Louis, MO, USA) as an isotype control.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Isolation, Immunocytochemistry, Double Staining, Western Blot

Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: Primary antibodies used in this study

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques:

Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: CMKLR1 deficiency decreases mortality and improves cognition in APP/PS1 mice and/or ICV-STZ mice. A, Survival curves for WT, APP/PS1, CMKLR1−/−, and APP/PS1-CMKLR1−/− mice. Data are mean ± SEM, with 22-29 mice in each group. ***p < 0.001 compared with WT mice. $$p < 0.01 compared with APP/PS1 mice. B–F, Spatial reference learning and memory were determined in Morris water maze. Mice were trained in Morris water maze for consecutive 5 d, four trials per day. The distance traveled (B) and time spent (C) to reach the escape platform are shown. The probe trials were tested on the seventh day after a day off. The percentage of the distance traveled (D) and the time spent (E) within each quadrant are shown. F, Representative swimming paths of a mouse on day 7. Data are mean ± SEM, with 6-10 mice in each group (WT, n = 9; APP/PS1, n = 9; CMKLR1−/−, n = 10; APP/PS1-CMKLR1−/−, n = 6). *p < 0.05; **p < 0.01; compared with WT mice. $p < 0.05 compared with APP/PS1 mice. G, Schematic representation of study design. The ICV-STZ mice and ICV-saline mice were produced by ICV injection of a single dose of 3.0 mg/kg STZ or equal volume of 0.9% saline into both lateral ventricles of the mice brain, respectively. The body weight of these mice was measured every day in the first 7 d, and then once every 7 d. Six weeks after ICV injection, all mice were subjected to a battery of behavioral tests, and then killed for biochemical studies. H, The body weight of WT and CMKLR1−/− mice after receiving STZ or saline was recorded. I–M, Spatial reference learning and memory were examined in Morris water maze. The distance traveled (I) and the time spent (J) to reach the escape platform are shown. The probe trials were tested on seventh day after a day off. The percentage of the distance traveled (K) and the time spent (L) within each quadrant are shown. M, Representative swimming paths of a mouse on day 7. Data are mean ± SEM, with 4-8 mice in each group (WT ICV-saline, n = 8; WT ICV-STZ, n = 7; CMKLR1−/− ICV-saline, n = 8; CMKLR1−/− ICV-STZ, n = 4). *p < 0.05; ***p < 0.001; compared with WT ICV-saline mice. ##p < 0.01 compared with CMKLR1−/− ICV-saline mice. $$p < 0.01 compared with WT ICV-STZ mice.

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques: Produced, Injection

Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: CMKLR1 deficiency is associated with increased Aβ deposition in APP/PS1 mice. A, Representative immunofluorescence images of Aβ (Aβ deposits, green) frozen in the hippocampus and the cortex of WT, APP/PS1, CMKLR1−/−, and APP/PS1-CMKLR1−/− mice aged 6, 9, and 13 months. Scale bar, 500 μm. Quantification of the fluorescence intensity of Aβ deposits, showing that CMKLR1 deficiency significantly increases Aβ deposition in APP/PS1 mice at the age of 6 months (B), 9 months (C), and 13 months (D). Data are mean ± SEM, based on three fields for each region, using at least 3 mice in each group. *p < 0.05; **p < 0.01; compared with WT mice. ###p < 0.001 compared with CMKLR1−/− mice. $$p < 0.01; $$$p < 0.001; compared with APP/PS1 mice. E, Insoluble Aβ1-42 from the hippocampus of 9-month-old mice were measured using ELISA. Data are mean ± SEM, with at least 3 mice in each group. *p < 0.05 compared with WT mice. ###p < 0.001 compared with CMKLR1−/− mice. $$$p < 0.001 compared with APP/PS1 mice. F, Real-time PCR quantification of the transcripts for APP of the mice at the age of 9 months. Data are mean ± SEM, with at least 3 mice in each group. *p < 0.05 compared with WT mice. #p < 0.05 compared with CMKLR1−/− mice. G, Representative Western blots showing the expression of APP protein in the brain of 9-month-old mice. H, Quantification of the immunoreactivity of the blots, normalized against β-actin. Data are mean ± SEM, with at least 3 mice in each group. *p < 0.05 compared with WT mice. #p < 0.05 compared with CMKLR1−/− mice.

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques: Immunofluorescence, Fluorescence, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: CMKLR1 deficiency has no effect on the locomotion, spontaneous exploratory, and anxiety of APP/PS1 mice aged 9 months. A, The locomotion of mice was tested on Rotarod, and the fall latency was recorded. B, The spontaneous exploratory activity of mice was examined in an open field, and the distance traveled during 15 min was recorded. The anxiety of mice was tested in an elevated plus maze (C–E) and an open field (F–H). The distance traveled (C) and the time spent (D) in the open arm were recorded. E, Representative traces of a mouse's movements during the elevated plus maze. The distance traveled (F) and the time spent (G) in the center were recorded. H, Representative traces of a mouse's movements during the open field test. Data are mean ± SEM, with 6-10 mice in each group (WT, n = 9; APP/PS1, n = 7; CMKLR1−/−, n = 10; APP/PS1-CMKLR1−/−, n = 6). *p < 0.05; **p < 0.01; compared with WT mice. #p < 0.05 compared with CMKLR1−/− mice.

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques: Activity Assay

Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: CMKLR1 deficiency attenuates hyperphosphorylation of tau in the brain of APP/PS1 mice and ICV-STZ mice. A, Representative Western blots showing tau phosphorylation at Ser199, Thr205, Ser396, and Ser404 in the hippocampus of WT, APP/PS1, CMKLR1−/−, and APP/PS1-CMKLR1−/− mice at the age of 9 months. The levels of nonphosphorylated tau (Tau1) and total tau (Tau5) were also measured. Quantification of the immunoreactivity of the blots, normalized against total tau (B) and β-actin (C). Data are mean ± SEM, with 6 mice in each group. *p < 0.05 compared with WT mice. $p < 0.05 compared with APP/PS1 mice. D, Representative Western blots showing tau phosphorylation in the brain of WT and CMKLR1−/− mice receiving ICV injection of STZ or saline. Quantification of the immunoreactivity of the blots, normalized against total tau (E) and β-actin (F). Data are mean ± SEM, with 6 mice in each group. *p < 0.05; **p < 0.01; ***p < 0.001; compared with WT ICV-saline mice. $p < 0.05; $$p < 0.01; compared with WT ICV-STZ mice.

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques: Western Blot, Injection

Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: Upregulation of CMKLR1 and colocalization with MAP2, Iba1, and GFAP in the brain of APP/PS1 mice. A, Representative immunofluorescence images of CMKLR1 (green) in CA1, CA3, and DG regions of the hippocampus and in the cortex of WT and APP/PS1 mice aged 6 months and 9 months. Scale bar, 50 µm. Quantification of the fluorescence intensity of CMKLR1 in each of the regions, showing that CMKLR1 was significantly increased in the hippocampus and the cortex of APP/PS1 mice at the age of 6 months (B) and 9 months (C). Data are mean ± SEM, with at least 3 mice in each group. *p < 0.05; **p < 0.01; compared with WT mice. D–F, Colocalization of CMKLR1 (green) with neurons (anti-MAP2, red, D), microglia (anti-Iba1, red, E), and astrocytes (anti-GFAP, red, F) in the hippocampus and the cortex of APP/PS1 mice aged 9 months. Scale bar, 50 μm.

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques: Immunofluorescence, Fluorescence

Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: Inhibition of neuronal CMKLR1 attenuates tau hyperphosphorylation induced by high glucose treatment in SH-SY5Y cells. A, Representative immunofluorescence images of CMKLR1 (green) in SH-SY5Y cells. Scale bar, 50 μm. B, Representative Western blots showing tau hyperphosphorylation at Ser199, Thr205, and Ser396 from SH-SY5Y treated with 5, 15, or 30 mm glucose for 12 or 24 h. Quantification of the immunoreactivity of the blots, normalized against total tau (C) and β-actin (D). E, Representative Western blots showing tau hyperphosphorylation at Ser199 and Ser396 from SH-SY5Y incubated with 5 or 30 mm glucose for 24 h, with or without a 1 h pretreatment with C15 (1 or 10 µm). Quantification of the immunoreactivity of the blots, normalized against total tau (F) and β-actin (G). Data are mean ± SEM from three separate experiments, each in duplicate or triplicate. *p < 0.05; **p < 0.01; ***p < 0.001; compared with 5 mM glucose treatment group. $p < 0.05; $$p < 0.01; compared with 30 mm glucose treatment group. Glu: glucose.

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques: Inhibition, Immunofluorescence, Western Blot, Incubation

Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: CMKLR1 deficiency is associated with tau spreading in ICV-Tau model mice. A, Schematic representation of ICV-Tau model mouse construction. The hippocampus extracts containing hyperphosphorylated tau (from 9-month-old APP/PS1 mice) or normal phosphorylated tau (from age-matched WT mice) were centrifuged at 5,500 rpm for 5 min in 4°C, and then the supernatant was injected into the ventricles of 6-month-old WT mice and age-matched CMKLR1−/− mice to establish the ICV-hyper-pTau mice and ICV-nor-pTau mice. Mice were killed 3 weeks after the injection. B, Representative Western blots showing tau hyperphosphorylation at Ser199, Thr205, and Ser396 in the hippocampus of WT and CMKLR1−/− mice receiving ICV injection of the extracts containing hyper-pTau or nor-pTau. Quantification of the immunoreactivity of the blots, normalized against total tau (C) and β-actin (D). E, Representative Western blots showing tau hyperphosphorylation in the cortex of WT and CMKLR1−/− mice receiving ICV injection of the extracts containing hyper-pTau or nor-pTau. Quantification of the immunoreactivity of the blots, normalized against total tau (F) and β-actin (G). Data are mean ± SEM, with at least 2 mice in each group (WT ICV-nor-pTau, n = 2; WT ICV-hyper-pTau, n = 3; CMKLR1−/− ICV-nor-pTau, n = 3; CMKLR1−/− ICV-hyper-pTau, n = 2). *p < 0.05; **p < 0.01; compared with WT ICV-nor-pTau mice. $p < 0.05; $$p < 0.01; compared with WT ICV-hyper-pTau mice.

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques: Injection, Western Blot

Journal: The Journal of Neuroscience

Article Title: The Chemokine-like Receptor 1 Deficiency Improves Cognitive Deficits of AD Mice and Attenuates Tau Hyperphosphorylation via Regulating Tau Seeding

doi: 10.1523/JNEUROSCI.0455-20.2020

Figure Lengend Snippet: Neuronal CMKLR1 affects tau phosphorylation through regulating tau seeding. A, Tau protein in the supernatant from SH-SY5Y stimulated with 5 or 30 mm glucose was examined using ELISA. Data are mean ± SEM from three separate experiments, each in duplicate or triplicate. **p < 0.01 compared with DMEM medium without glucose treatment. ##p < 0.01 compared with 5 mm glucose treatment group. B, Representative Western blots showing the human specific tau protein in the supernatant from SH-SY5Y stimulated with 5 or 30 mm glucose. C, Representative immunofluorescence images of CMKLR1 (green) in primary cultures of mouse neurons. Scale bar, 50 μm. D–G, The supernatant from SH-SY5Y treated with 5 or 30 mm glucose was collected, purified, and then applied to primary cultures of neurons from WT mice and CMKLR1−/− mice for 3 d. Representative immunofluorescence images of human specific tau protein (red) and human and mouse phosphorylated tau at Thr205 site (green, D) and Ser396 site (green, E) in primary cultures of neurons from WT mice and CMKLR1−/− mice. The primary cultures of neurons were incubated with the purified supernatant form SH-SY5Y treated with 5 or 30 mm glucose. Scale bar, 50 μm. Quantification of the area of mouse phosphorylated tau at pT205 (F) and pS396 (G), calculated by subtracting the fluorescence area of human tau (yellow) from the fluorescence area of Hu and Ms tau (green), showing that human tau taken up by mouse neurons significantly accelerated the hyperphosphorylation of mouse tau in primary cultures of mouse neurons. Data are mean ± SEM from three separate experiments, each in duplicate or triplicate. ***p < 0.001 compared with primary cultures of neurons from WT mice treated with the purified supernatant stimulated with 5 mm glucose. $$$p < 0.001 compared with primary cultures of neurons from WT mice treated with the purified supernatant stimulated with 30 mm glucose. Glu: glucose.

Article Snippet: CMKLR1/AF488 , Polyclonal , CMKLR1 , , Bioss.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Purification, Incubation, Fluorescence