human trem2 fc standard  (R&D Systems)

Journal: Journal of Neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: BCAAs and BCKAs impact the response of microglial TREM2 and autophagy to Aβ. A Western blot analysis of TREM2-related protein levels in cortex of APP/PS1 mice and the corresponding quantification results (n = 4). B Relative mRNA levels of DAP12 and CD68 in cortex (n = 5). C Western blot analysis of autophagy-related protein levels in cortex and the corresponding quantification results (n = 4). D Relative mRNA levels of BECLIN1 and LAMP1 in cortex (n = 5). Western blot analysis of TREM2-related E and autophagy-related F Protein levels in BV2 cell treated with or without BCAAs/ BCKAs for 4 h in the presence of Aβ and the corresponding quantification results (n = 3). Western blot analysis of TREM2-related G and autophagy-related H protein levels in BV2 cell treated with or without BCAAs/ BCKAs for 12 h in the presence of Aβ and the corresponding quantification results (n = 3). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: The recombinant human TREM2 protein was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot

Journal: Journal of Neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: BCAAs and BCKAs suppress TREM2 activation through binding to TREM2. A Molecular docking results of L-Leucine and KICA binding to TREM2 extracellular domain. B Biolayer interferometry (BLI) analysis of interaction of BCAAs or BCKAs with TREM2. BCAAs and BCKAs was titrated from 15.63 to 500 μM and 31.25 to 500 μM, respectively. C LC–MS/MS spectra of TREM2 immunoprecipitated L-Leucine- 13 C of BV2 cells after BCAAs isotope treatment for 1 h. D Thermal stabilization of TREM2 protein evaluated by cellular thermal shift assay (CETSA) after treating BV2 cells with BCAAs/ BCKAs for 4 h (n = 3). E Representative images of binding of fluorescein labeled-Aβ to cell surface TREM2 in BV2 cells after treatment of BCAAs/ BCKAs for 2 h (n = 3, Scale bars, 40 μm). F Representative images of microglial phagocytosis of fluorescein labeled-Aβ in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of S1P for 4 h (n = 3, Scale bars, 40 μm). G Western blot analysis of phosphorylation levels of SYK in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of sphingosine-1-phosphate (S1P) for 4 h (n = 3). Data in D , E , F and G are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: The recombinant human TREM2 protein was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Thermal Shift Assay, Labeling, Western Blot, Phospho-proteomics

Journal: Journal of Neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: BCAAs and BCKAs reduce the expression and recycling of microglial TREM2 via inhibiting autophagy. Western blot analysis of TREM2-related A and autophagy-related B protein levels in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3). C Representative images of microglial phagocytosis of fluorescein labeled-Aβ in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3, Scale bars, 40 μm). D Representative images of TREM2 recycling in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3, Scale bars, 40 μm). Western blot analysis of TREM2-related E and autophagy-related F protein levels in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of chloroquine for 12 h (n = 3). G Representative images of TREM2 recycling in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of chloroquine for 12 h (n = 3, Scale bars, 40 μm). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test or Student’s t test

Article Snippet: The recombinant human TREM2 protein was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Labeling

Journal: Alzheimer's research & therapy

Article Title: Preclinical and first-in-human evaluation of AL002, a novel TREM2 agonistic antibody for Alzheimer's disease.

doi: 10.1186/s13195-024-01599-1

Figure Lengend Snippet: Fig. 5 Pharmacodynamics of single ascending dose AL002 in CSF of healthy volunteers. (A-B) sTREM2, (C) SPP1 protein, (D) sCSF1R, and (E) IL1RN protein were measured in CSF samples collected at the indicated time points. Data are presented as unadjusted mean ± SD percent change from baseline. (A), (C), (D), and (E) n = 6 in placebo, 6 mg/kg, 15 mg/kg, 30 mg/kg cohorts; n = 5 in 45 mg/kg cohort; n = 5–6 in 60 mg/kg cohorts I and N. (B) In 60 mg/kg cohort N, n = 2 at Day 13, n = 5 at Day 30 and 43, n = 3 at Day 57. Nudging was used to differentiate overlapping datasets. CSF, cerebrospinal fluid; CSF1R, colony-stimulating factor 1 receptor; LLOQ, lower limit of quantification; NHP, nonhuman primate; sCSF1R, soluble CSF1R; SEM, standard error of the mean; sTREM2, soluble TREM2; TREM2, triggering receptor expressed on myeloid cells-2

Article Snippet: As previously described [32], sTREM2 in human CSF and plasma were measured using a custom AL002-tolerant ECLA method using recombinant human TREM2 Fc Chimera (R&D Systems cat# 1828-T2-05) as a standard. sCSF1R in human CSF was measured using a commercial ELISA assay by R&D Systems.

Techniques: Drug discovery

Journal: Alzheimer's research & therapy

Article Title: Preclinical and first-in-human evaluation of AL002, a novel TREM2 agonistic antibody for Alzheimer's disease.

doi: 10.1186/s13195-024-01599-1

Figure Lengend Snippet: Fig. 6 Pharmacodynamics of single ascending dose AL002 in plasma of healthy volunteers. (A) IL1RN protein and (B) sTREM2 were measured in plasma samples collected at the indicated time points. Data are presented as unadjusted mean ± SEM percent change from baseline. (A), (B), n = 11 in placebo, n = 3 in 0.003-0.2 mg/kg, n = 6 in each of 0.6 mg/kg, 2 mg/kg, 6 mg/kg, 15 mg/kg, 30 mg/kg, 45 mg/kg; n = 11–14 in 60 mg/kg cohorts I and N. EOI, end of injection; h, hours; SEM, standard error of the mean; sTREM2, soluble TREM2; TREM2, triggering receptor expressed on myeloid cells-2

Article Snippet: As previously described [32], sTREM2 in human CSF and plasma were measured using a custom AL002-tolerant ECLA method using recombinant human TREM2 Fc Chimera (R&D Systems cat# 1828-T2-05) as a standard. sCSF1R in human CSF was measured using a commercial ELISA assay by R&D Systems.

Techniques: Drug discovery, Clinical Proteomics, Injection

Journal: Alzheimer's research & therapy

Article Title: Preclinical and first-in-human evaluation of AL002, a novel TREM2 agonistic antibody for Alzheimer's disease.

doi: 10.1186/s13195-024-01599-1

Figure Lengend Snippet: Fig. 7 Proposed mechanism of action of AL002. (A) TREM2 is constitutively shed into sTREM2. (B) AL002 binding promotes TREM2 clustering and ac tivation via DAP12 and SYK phosphorylation, thus leading to SPP1 and IL1RN protein production. (C) AL002-mediated TREM2/DAP12 phosphorylation induces activation-mediated internalization of TREM2, thus leading to TREM2 degradation. This mechanism is typical of ITAM-containing receptors, thus preventing prolonged and massive stimulation of the receptor. ITAM, immunoreceptor tyrosine-based activation motif; sTREM2, soluble TREM2; TREM2, triggering receptor expressed on myeloid cells-2

Article Snippet: As previously described [32], sTREM2 in human CSF and plasma were measured using a custom AL002-tolerant ECLA method using recombinant human TREM2 Fc Chimera (R&D Systems cat# 1828-T2-05) as a standard. sCSF1R in human CSF was measured using a commercial ELISA assay by R&D Systems.

Techniques: Binding Assay, Phospho-proteomics, Activation Assay