Journal: bioRxiv

Article Title: Identification of cellular and molecular risk signatures for progression to late-stage age-related macular degeneration using the 9-step Minnesota Grading System

doi: 10.1101/2025.07.10.664209

Figure Lengend Snippet: A . Flowchart describing selection of gene markers associated with a high-risk of progression to late AMD for validation, based on bulk RNAseq of the macular RPE/choroid. B. A heatmap showing high-risk marker gene expression in the macular RPE/choroid in samples of AMD1, AMD3L, and AMD3H. The levels of each mRNA are expressed as log 2 (TPM+1), where TPM stands for transcript per million, red indicates positive value and blue negative. C . Representative RNAscope images of C3 mRNA in AMD1, AMD3L, AMD3H, and AMD4/GA macula sections, with arrows indicating C3 mRNA + signals. C3 increases in the RPE-adjacent region with disease severity. D. Representative RNAscope images of TREM2 mRNA in AMD1, AMD3L, AMD3H, and AMD4/GA macula sections, with arrows indicating TREM2 mRNA+ signals. TREM2 mRNA becomes apparent in the choroid at AMD3H and persists in AMD4. E. ELISA measurement of TREM2 protein in AMD1 (n = 8 samples) and AMD4 (n = 6-8) in serum, macular and peripheral RPE/choroid tissue and neuroretina, vitreous, and aqueous humor. Donor information is in . The pM label indicates picomolar concentrations, with p value determined by unpaired Student’s t-test (* p < 0.05, *** P <0.001). F . Representative RNAscope images of OLR1 mRNA in AMD1, AMD3L, AMD3H, and AMD4/GA macula sections, with arrows indicating OLR1 mRNA+ signals. OLR1 increases in the RPE-adjacent region with disease severity. G . Representative RNAscope images of NGFR mRNA in AMD1, AMD3L, AMD3H, and AMD4/GA macula sections, with arrows indicating NGFR mRNA+ signals. Elevated NGFR signal is observed in AMD3H, proximal to the RPE.

Article Snippet: Total TREM2 protein levels were measured using a TREM2 Duoset (R&D Systems, Minneapolis, MN) ELISA kit for MSD-based assays (MSD, Rockville, MD) and read on a Sector S 600 imager.

Techniques: Selection, Biomarker Discovery, Marker, Gene Expression, RNAscope, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Identification of cellular and molecular risk signatures for progression to late-stage age-related macular degeneration using the 9-step Minnesota Grading System

doi: 10.1101/2025.07.10.664209

Figure Lengend Snippet: Fibroblasts predominantly express C3, immune cells show C3, OLR1 and TREM2 expression, and GFAP, NGFR, SPP1 and GPX2 are expressed in Muller glia and astrocytes.

Article Snippet: Total TREM2 protein levels were measured using a TREM2 Duoset (R&D Systems, Minneapolis, MN) ELISA kit for MSD-based assays (MSD, Rockville, MD) and read on a Sector S 600 imager.

Techniques: Expressing

Journal: bioRxiv

Article Title: Identification of cellular and molecular risk signatures for progression to late-stage age-related macular degeneration using the 9-step Minnesota Grading System

doi: 10.1101/2025.07.10.664209

Figure Lengend Snippet: A . A dot plot of TREM2 and OLR1 expression in all detected RPE/choroid cell types. Both TREM2 and OLR2 are preferentially expressed in choroidal macrophages. Of the three clusters of macrophages, TREM2 is expressed in macrophage 1 and 3 while OLR1 is expressed in macrophage 2 and 3. B . Dual genes plot of all 3 macrophage populations expressing TREM2 (green dots) versus OLR1 (red dots). There are very few macrophages (yellow dots) expressing both TREM2 and OLR1 .

Article Snippet: Total TREM2 protein levels were measured using a TREM2 Duoset (R&D Systems, Minneapolis, MN) ELISA kit for MSD-based assays (MSD, Rockville, MD) and read on a Sector S 600 imager.

Techniques: Expressing

Journal: bioRxiv

Article Title: Identification of cellular and molecular risk signatures for progression to late-stage age-related macular degeneration using the 9-step Minnesota Grading System

doi: 10.1101/2025.07.10.664209

Figure Lengend Snippet: In AMD3H, complement C3 can be induced in multiple neuronal and choroidal cell-types including fibroblasts, macrophages, and Müller/astrocytes. Macrophages expressing either TREM2 or OLR1 are observed near the RPE/choriocapillaris complex in AMD3H, possibly originating from the choroid and/or from the systemic circulation, and later in AMD3H/4 outer retina. Activated Müller/astrocytes cells expressing NGFR and GFAP expand from the inner retina to outer retina and RPE in AMD3H. Activation of complement, macrophages, and Müller/astrocytes may signal the onset of the end stage AMD. Graphic was created with Biorender ( biorender.com ).

Article Snippet: Total TREM2 protein levels were measured using a TREM2 Duoset (R&D Systems, Minneapolis, MN) ELISA kit for MSD-based assays (MSD, Rockville, MD) and read on a Sector S 600 imager.

Techniques: Expressing, Activation Assay

Journal: Cells

Article Title: TonEBP Haploinsufficiency Attenuates Microglial Activation and Memory Deficits in Middle-Aged and Amyloid β Oligomer-Treated Mice.

doi: 10.3390/cells12222612

Figure Lengend Snippet: Figure 1. TonEBP and microglial activation increase with age in the mouse hippocampus. (a) Western blot and quantitative analysis of TonEBP protein level in the hippocampi of 6-, 12-, and 24-month-old mice (n = 3–7). (b) Representative images of the ProteoStat staining assay in the CA1 region of hippocampal sections. The intensity of the ProteoStat dye is presented as fold changes. DAPI was used to stain nuclei. Scale bar = 10 µm. (c) Western blot and quantitative analysis of TREM2 and Iba-1 protein expression in the hippocampus (n = 3–7). (d) Representative microglial morphology and schemes graphically illustrate the calculation bases for the intersections (green spots) and ramification indices (red circles) for microglial activation. Scale bar = 10 µm. (e,f) Sholl analysis of intersections (e) and ramification indices (f). Data are presented as the mean ± SEM. * p < 0.05 vs. 6-month- old mice.

Article Snippet: Proteins were immunoblotted with cyclooxygenase-2 (COX-2, Santa Cruz), iba-1 (Wako), TonEBP (Santa Cruz), and TREM2 (Santa Cruz) antibodies. β-actin (Sigma Aldrich, St. Louis, MO, USA) was used as a control to normalize total protein levels.

Techniques: Activation Assay, Western Blot, Staining, Expressing

Journal: Cells

Article Title: TonEBP Haploinsufficiency Attenuates Microglial Activation and Memory Deficits in Middle-Aged and Amyloid β Oligomer-Treated Mice.

doi: 10.3390/cells12222612

Figure Lengend Snippet: Figure 2. TonEBP haploinsufficiency attenuates microglial activation in the hippocampi of middle- aged mice. (a) Relative telomere length in the hippocampi of 6- and 12-month-old WT and TonEBP (+/−) mice. Telomere length (n = 4–6) was measured by qRT-PCR. (b) Western blot and quantitative analysis of TonEBP, COX-2, TREM2, and Iba-1 protein expression in the hippocampi of 12-month- old WT and TonEBP (+/−) mice (n = 4–5). (c) Effects of TonEBP haploinsufficiency on Tnf-α and Il-6 mRNA expression using qRT-PCR. (d) Representative microglial morphology and schemes graphically illustrate the calculation bases for the intersections (green spots) and ramification indices (red circles) for microglial activation. Scale bar = 10 µm. (e,f) Sholl analysis of intersections (e) and ramification indices (f). Data are presented as the mean ± SEM. * p < 0.05 vs. WT.

Article Snippet: Proteins were immunoblotted with cyclooxygenase-2 (COX-2, Santa Cruz), iba-1 (Wako), TonEBP (Santa Cruz), and TREM2 (Santa Cruz) antibodies. β-actin (Sigma Aldrich, St. Louis, MO, USA) was used as a control to normalize total protein levels.

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot, Expressing

Journal: Cells

Article Title: TonEBP Haploinsufficiency Attenuates Microglial Activation and Memory Deficits in Middle-Aged and Amyloid β Oligomer-Treated Mice.

doi: 10.3390/cells12222612

Figure Lengend Snippet: Figure 5. TonEBP haploinsufficiency inhibits neuroinflammation and cognitive decline in the hip- pocampi of AβO-treated mice. (a) Western blot and quantitative analysis of TonEBP, COX-2, TREM2, and Iba-1 expression in the hippocampi of AβO-injected WT and TonEBP (+/−) mice (n = 4–5). (b) Percentage of spontaneous alternations and total arm entries in the Y-maze. (c) Representa- tive microphotographs of Golgi-stained hippocampi. Scale bar = 500 µm (top), 20 µm (bottom). (d) Representative, higher-resolution microphotographs of Golgi-stained neurons (red boxes in c). Scale bar = 10 µm. (e) Analysis of the basal and apical spine densities. (f) Analysis of the numbers of basal and apical spines according to spine shape. Data are presented as mean ± SEM. * p < 0.05 vs. AβO-treated WT mice.

Article Snippet: Proteins were immunoblotted with cyclooxygenase-2 (COX-2, Santa Cruz), iba-1 (Wako), TonEBP (Santa Cruz), and TREM2 (Santa Cruz) antibodies. β-actin (Sigma Aldrich, St. Louis, MO, USA) was used as a control to normalize total protein levels.

Techniques: Western Blot, Expressing, Injection, Staining

Journal: Human Molecular Genetics

Article Title: Behavioral and transcriptomic analysis of Trem2-null mice: not all knockout mice are created equal

doi: 10.1093/hmg/ddx366

Figure Lengend Snippet: Treml1 expression varies depending on the Trem2 knockout strategy. ( A ) Schematic of the relationship between Trem2 and Treml1 on Chromosome 17. Scale bar is indicated in red, transparent blue boxes indicate regions containing exons of the respective genes, and black arrows indicate direction of transcription. Trem2 targeting strategies for the ( B ) Velocigene, ( C ) Colonna, and ( D ) CRISPR/Cas9 approaches are depicted. TREM2 protein levels were determined by Meso Scale Discovery ELISA from Triton-X fractions of hippocampal tissues in ( E ) Velocigene, ( F ) Colonna, and ( G ) CRISPR/Cas9 derived Trem2 −/− male mice, confirming that each line has no TREM2 (knockout verification). Shown are the averages ± S.E.M from N = 3–4 animals per group (Unpaired two-tailed t -test, **** P ≤ 0.0001). (G– I ) Hippocampal RNA was isolated from respective male wild-type (WT) and Trem2 −/− mice (KO) for RT-qPCR analysis of Treml1 expression in (H) Trem2 −/− mice created using the Velocigene strategy and their WT C57BL/6 N controls, (I) Trem2 −/− mice from the Colonna targeting, and ( J ) Trem2 −/− mice from CRISPR/Cas9 targeting and controls. Shown are the averages ± S.E.M. from N = 6–7 animals per group. (Unpaired two-tailed t -test, **** P ≤ 0.0001).

Article Snippet: Using a sensitive MesoScale Discovery sandwich ELISA, we measured TREM2 protein levels and found that each deletion strategy resulted in ablation of TREM2 expression relative to controls ( ).

Techniques: Expressing, Knock-Out, CRISPR, Enzyme-linked Immunosorbent Assay, Derivative Assay, Two Tailed Test, Isolation, Quantitative RT-PCR

Journal: Journal of Neuroinflammation

Article Title: Long-term running exercise improves cognitive function and promotes microglial glucose metabolism and morphological plasticity in the hippocampus of APP/PS1 mice

doi: 10.1186/s12974-022-02401-5

Figure Lengend Snippet: Running exercise maintained TREM2 levels and reduced sTREM2 levels in APP/PS1 mice. a Relative expression of several top DEGs among WT_Sed mice, AD_Sed mice, and AD_Run mice. b Representative z-stack images (0.22 μm steps) of TREM2 (red) and IBA1 (white) microglia in the DG of WT_Sed, AD_Sed, WT_Run, and AD_Run mice and TREM2-positive puncta outside IBA1 were masked. Scale bar: 25 μm. c, d Levels of TREM2 and sTREM2 in the hippocampus of WT_Sed, AD_Sed, WT_Run, and AD_Run mice were determined by ELISA. n = 7–9. e The level of sTREM2 in the plasma of WT_Sed, AD_Sed, WT_Run, and AD_Run mice was determined by ELISA. n = 10. f Pearson’s correlation between plasma sTREM2 levels and hippocampal FDG uptake. g The expression of SPP1, TYROBP, ADAM10, p-SYK (Tyr525/526), and SYK in the hippocampus of WT_Sed, AD_Sed, WT_Run, and AD_Run mice was detected by western blotting. h–k Quantification results of SPP1/β-tubulin, TYROBP/β-tubulin, ADAM10/β-actin, and p-SYK/SYK. n = 3. Data are shown as the mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: These protein solutions from nine mice in each group were used to detect the levels of TREM2 and sTREM2 in the hippocampus with a mouse TREM2 ELISA kit (EIAab, E12753m, China) and a mouse sTREM2 ELISA kit (J&L, JL20435, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Western Blot

Journal: Cells

Article Title: TonEBP Haploinsufficiency Attenuates Microglial Activation and Memory Deficits in Middle-Aged and Amyloid β Oligomer-Treated Mice

doi: 10.3390/cells12222612

Figure Lengend Snippet: TonEBP and microglial activation increase with age in the mouse hippocampus. ( a ) Western blot and quantitative analysis of TonEBP protein level in the hippocampi of 6-, 12-, and 24-month-old mice ( n = 3–7). ( b ) Representative images of the ProteoStat staining assay in the CA1 region of hippocampal sections. The intensity of the ProteoStat dye is presented as fold changes. DAPI was used to stain nuclei. Scale bar = 10 µm. ( c ) Western blot and quantitative analysis of TREM2 and Iba-1 protein expression in the hippocampus ( n = 3–7). ( d ) Representative microglial morphology and schemes graphically illustrate the calculation bases for the intersections (green spots) and ramification indices (red circles) for microglial activation. Scale bar = 10 μm. ( e , f ) Sholl analysis of intersections ( e ) and ramification indices ( f ). Data are presented as the mean ± SEM. * p < 0.05 vs. 6-month-old mice.

Article Snippet: Proteins were immunoblotted with cyclooxygenase-2 (COX-2, Santa Cruz), iba-1 (Wako), TonEBP (Santa Cruz), and TREM2 (Santa Cruz) antibodies. β-actin (Sigma Aldrich, St. Louis, MO, USA) was used as a control to normalize total protein levels.

Techniques: Activation Assay, Western Blot, Staining, Expressing

Journal: Cells

Article Title: TonEBP Haploinsufficiency Attenuates Microglial Activation and Memory Deficits in Middle-Aged and Amyloid β Oligomer-Treated Mice

doi: 10.3390/cells12222612

Figure Lengend Snippet: TonEBP haploinsufficiency attenuates microglial activation in the hippocampi of middle-aged mice. ( a ) Relative telomere length in the hippocampi of 6- and 12-month-old WT and TonEBP (+/−) mice. Telomere length ( n = 4–6) was measured by qRT-PCR. ( b ) Western blot and quantitative analysis of TonEBP, COX-2, TREM2, and Iba-1 protein expression in the hippocampi of 12-month-old WT and TonEBP (+/−) mice ( n = 4–5). ( c ) Effects of TonEBP haploinsufficiency on Tnf-α and Il-6 mRNA expression using qRT-PCR. ( d ) Representative microglial morphology and schemes graphically illustrate the calculation bases for the intersections (green spots) and ramification indices (red circles) for microglial activation. Scale bar = 10 μm. ( e , f ) Sholl analysis of intersections ( e ) and ramification indices ( f ). Data are presented as the mean ± SEM. * p < 0.05 vs. WT.

Article Snippet: Proteins were immunoblotted with cyclooxygenase-2 (COX-2, Santa Cruz), iba-1 (Wako), TonEBP (Santa Cruz), and TREM2 (Santa Cruz) antibodies. β-actin (Sigma Aldrich, St. Louis, MO, USA) was used as a control to normalize total protein levels.

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot, Expressing

Journal: Cells

Article Title: TonEBP Haploinsufficiency Attenuates Microglial Activation and Memory Deficits in Middle-Aged and Amyloid β Oligomer-Treated Mice

doi: 10.3390/cells12222612

Figure Lengend Snippet: TonEBP haploinsufficiency inhibits neuroinflammation and cognitive decline in the hippocampi of AβO-treated mice. ( a ) Western blot and quantitative analysis of TonEBP, COX-2, TREM2, and Iba-1 expression in the hippocampi of AβO-injected WT and TonEBP (+/−) mice ( n = 4–5). ( b ) Percentage of spontaneous alternations and total arm entries in the Y-maze. ( c ) Representative microphotographs of Golgi-stained hippocampi. Scale bar = 500 μm (top), 20 μm (bottom). ( d ) Representative, higher-resolution microphotographs of Golgi-stained neurons (red boxes in c ). Scale bar = 10 μm. ( e ) Analysis of the basal and apical spine densities. ( f ) Analysis of the numbers of basal and apical spines according to spine shape. Data are presented as mean ± SEM. * p < 0.05 vs. AβO-treated WT mice.

Article Snippet: Proteins were immunoblotted with cyclooxygenase-2 (COX-2, Santa Cruz), iba-1 (Wako), TonEBP (Santa Cruz), and TREM2 (Santa Cruz) antibodies. β-actin (Sigma Aldrich, St. Louis, MO, USA) was used as a control to normalize total protein levels.

Techniques: Western Blot, Expressing, Injection, Staining

Journal: Cell reports

Article Title: The microglial response to inhibition of Colony-stimulating-factor-1 receptor by PLX3397 differs by sex in adult mice.

doi: 10.1016/j.celrep.2024.115176

Figure Lengend Snippet: Figure 4. Sex-dependent differences in microglial Galectin-3 expression at baseline and under CSF1R inhibition (A and B) Representative 203 confocal images showing microglia immunolabeled with Iba1 (green) and Galectin-3 (magenta) in control (A) and PLX-treated mice (B). For ease of visualization, representative images were subjected to altered brightness/contrast that masks the different Galectin-3 fluorescence intensity. (C) The percentage of Galectin-3+ microglia was higher in females compared to males in both cortex (left) and hippocampus (right), although the effect did not reach statistical significance. (D and E) PLX treatment increased the proportion (D) and density (E) of Galectin-3+ microglia in both males and females, also increasing the level of expression in individual microglia. n = 6–8 mice/sex/treatment (C), n = 3–8 mice/sex/dose (D and E), Student’s t test (C), two-way ANOVA with Bonferroni correction (D and E), p values are reported in Tables S4 and S5. Scale bar: 200 mm. All data are expressed as the mean ± SEM. See also Figure S6.

Article Snippet: PLX treatment reduced CSF1R expression in bo Galectin 3+ microglia generally had higher expression of CSF1R than Galectin-3 (J–L) PLX treatment led to increased TREM2 expression in both male and female higher levels of TREM2 compared to Galectin-3 microglia in females (K) and m n = 3–4 mice/sex/treatment; two-way ANOVA with Bonferroni correction, *p < 0.0 also Figure S6. our transcriptomic analysis showing significant downregulation of Csf1r in male but not in female microglia (Table S1).

Techniques: Expressing, Inhibition, Immunolabeling, Control

Journal: Cell reports

Article Title: The microglial response to inhibition of Colony-stimulating-factor-1 receptor by PLX3397 differs by sex in adult mice.

doi: 10.1016/j.celrep.2024.115176

Figure Lengend Snippet: Figure 5. Expression patterns of Galectin-3+ microglia at baseline and under CSF1R inhibition (A) Experimental paradigm depicting duration of PLX treatment and subsequent microglia gating and analysis. (B) PLX treatment increases the proportion of microglia expressing Galectin-3 to a greater extent in males compared to females. (C) PLX treatment increases the expression levels of Galectin-3 in Galectin-3+ microglia to a greater extent in males compared to females. (D–F) PLX treatment led to increased mitochondrial mass in female and male microglia, as estimated by expression of MitoTracker green. This effect was larger in males than in females (D). In both control and PLX groups, Galectin-3+ microglia appear to have a higher mitochondrial mass compared to Galectin-3 microglia in females (E) and males (F).

Article Snippet: PLX treatment reduced CSF1R expression in bo Galectin 3+ microglia generally had higher expression of CSF1R than Galectin-3 (J–L) PLX treatment led to increased TREM2 expression in both male and female higher levels of TREM2 compared to Galectin-3 microglia in females (K) and m n = 3–4 mice/sex/treatment; two-way ANOVA with Bonferroni correction, *p < 0.0 also Figure S6. our transcriptomic analysis showing significant downregulation of Csf1r in male but not in female microglia (Table S1).

Techniques: Expressing, Inhibition, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Neurotensin stimulates sortilin and mTOR in human microglia inhibitable by methoxyluteolin, a potential therapeutic target for autism

doi: 10.1073/pnas.1604992113

Figure Lengend Snippet: NTR3/sortilin gene expression and cellular localization in HM, and its release in response to NT. (A) Primary HM (2.5 × 105 cells) and immortalized HM-SV40 were stimulated with NT for 12 or 24 h to measure gene expression levels of NTR3/sortilin in control and NT-stimulated (10 or 100 nM) microglia by qRT-PCR. All conditions were performed in triplicate for each dataset and were repeated three times (n = 3). Results were normalized against the endogenous GAPDH and expressed relative to the mean of the control for the gene of interest, with significance of comparisons denoted by *P < 0.05, **P < 0.001, or ***P < 0.0001. Microglia-SV40 (5 × 103 cells per four-well chamber–coated slide) were stimulated with NT (10 nM) for 24 h, and then fixed and permeabilized to stain for nuclei using DAPI (blue), with specific antibodies for Alexa 488-NTR3/sortilin (green) or Alexa 594–Iba-1, a microglial marker protein (red), whereas rabbit IgG was used for the negative control. The cell surface and cytosolic distribution of NTR3/sortilin protein is shown in control and NT-stimulated primary HM (B) and immortalized HM-SV40 (C), where colocalization with Iba-1 is also apparent (white arrows). Qualitative analysis was done using images from triplicates, and representative images are shown. (D) HM-SV40 (1 × 106 cells) were stimulated with NT (10 nM) for 24 or 48 h to measure release of soluble NTR3/sortilin in culture media by ELISA. All conditions were performed in triplicate for each dataset and repeated three times (n = 3). Significance of comparisons is denoted by *P < 0.05 or **P < 0.001. (E) HM-SV40 (1 × 106 cells) were stimulated with NT (10 or 100 nM) for 24 or 48 h to measure total cellular NTR3/sortilin levels by Western blot analysis, where TREM-2 served as a microglial marker protein and β-actin as the loading control. All conditions were performed in triplicate for each dataset and were repeated three times (n = 3), and a representative image is shown.

Article Snippet: Protein levels of NTR1, NTR2, and NTR3/sortilin were determined by Western blot analysis on cellular lysates harvested from microglia (1 × 10 6 cells) stimulated with NT (1–100 nM) for 24 or 48 h. The membranes were probed with the following primary antibodies: NTR1 G9 (Santa Cruz Biotechnology); NTR2 (EMD Millipore Corporation); SORT1 (Sigma–Aldrich); NTR3 C20, triggering receptor expressed on myeloid cells-2 (TREM-2), and Iba-1 (Santa Cruz Biotechnology); and β-actin for the loading control (Cell Signaling Technology).

Techniques: Expressing, Quantitative RT-PCR, Staining, Marker, Negative Control, Enzyme-linked Immunosorbent Assay, Western Blot