Journal: EMBO Molecular Medicine

Article Title: Impaired migration and lung invasion of human melanoma by a novel small molecule targeting the transmembrane domain of death receptor p75 NTR

doi: 10.1038/s44321-025-00297-1

Figure Lengend Snippet: ( A ) Volcano plot of AraTM screening assay of Screening Set v2010 (8482 compounds) from the Chemical Biology Consortium Sweden ( www.cbcs.se ). Fold change was calculated against vehicle (DMSO) and hits were defined as compounds resulting in greater than ±0.5 fold change in GFP/OD 630 signal without affecting OD 630 by greater than 0.3-fold across three independent runs (red dots). P values were calculated by one-way ANOVA ( N = 3). Compound Div17E5 is indicated. ( B ) Chemical structure of Div17E5. ( C ) Dose response of Div17E5 in the AraTM assay of p75 NTR TMD in comparison with unrelated TMDs from α2β3 integrin and Matrix-2 protein (M2) from the viral envelope of influenza A virus. Results are plotted as mean ± SD ( N = 3). ( D – F ) Comparison of wild-type (WT) p75 NTR TMD and I252A, P253G and V254A mutants in the AraTM assay in response to increasing doses of Div17E5. GFP/OD 630 signal for WT TMD without any drug was set at 100% and all other measurements are relative to that. Results are plotted as mean ± SD ( N = 3). * P = 0.03: ** P = 0.006 (one-way ANOVA followed by Tukey’s multiple comparisons). ( G ) Live cell homo-FRET anisotropy in response to Div17E5 of full-length, wild-type human p75 NTR expressed in COS cells. Shown are representative traces of average anisotropy change after addition of Div17E5 (10 μM) or vehicle. ( H ) Live cell homo-FRET anisotropy in response to Div17E5 of full-length, wild-type human p75 NTR in comparison to I252A p75 NTR TMD mutant. Shown are representative traces of average anisotropy change after addition of Div17E5 (10 μM). .

Article Snippet: Antibody against human p75 NTR extracellular domain used for immunoprecipitation was obtained from Alomone Labs (#ANT-007); the one used for immunofluorescence was from Promega (#G323A).

Techniques: Screening Assay, AraTM Assay, Comparison, Virus, Mutagenesis

Journal: EMBO Molecular Medicine

Article Title: Impaired migration and lung invasion of human melanoma by a novel small molecule targeting the transmembrane domain of death receptor p75 NTR

doi: 10.1038/s44321-025-00297-1

Figure Lengend Snippet: ( A ) Expression of p75 NTR in A875 control (NT, non-targeting) and knock-down (shp75) cells. ( B ) Dose response analysis of cleaved PARP induction by Div17E5 in A875 control (NT) and knock-down (shp75) cells. ( C ) Western blot analysis of cleaved PARP in A875 melanoma cells in response to Div17E5 (10 µM) in the presence or absence of pan-caspase inhibitor Q-VD-pOH (5 µM). Reprobing for GAPDH was used as loading control. The experiment was repeated three times with comparable results. ( D ) Dose-dependent cell viability of A875 control (NT) and knock-down (shp75) cells in response to Div17E5. Results are plotted as mean ± SD ( N = 3). * P = 0.04; ** P = 0.006 (one-way ANOVA followed by Tukey’s multiple comparisons). .

Article Snippet: Antibody against human p75 NTR extracellular domain used for immunoprecipitation was obtained from Alomone Labs (#ANT-007); the one used for immunofluorescence was from Promega (#G323A).

Techniques: Expressing, Control, Knockdown, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Impaired migration and lung invasion of human melanoma by a novel small molecule targeting the transmembrane domain of death receptor p75 NTR

doi: 10.1038/s44321-025-00297-1

Figure Lengend Snippet: ( A ) Chemical structure of Np75-4A22. ( B ) Dose response of Div17E5 and Np75-4A22 in the AraTM assay of p75 NTR TMD. Results are plotted as mean ± SD ( N = 3). ** P = 0.007 (one-way ANOVA followed by Tukey’s multiple comparisons). ( C – E ) Comparison of wild-type p75 NTR TMD and I252A, P253G and V254A mutants in the AraTM assay in response to increasing doses of Np75-4A22. GFP/OD 630 signal without any drug added was set at 100%. Results are plotted as mean ± SD ( N = 3). * P = 0.035; ** P = 0.008 (one-way ANOVA followed by Tukey’s multiple comparisons). ( F – H ) Specific interaction between Np75-4A22 (4 mM) and p75 NTR TMD (1 mM) in bicelles that mimic a lipid bilayer. ( F ) 1 H- 15 N TROSY-HSQC spectra of p75 NTR TMD in DMPC-DH6PC bicelles ( q = 0.5) in the absence (red) and presence (blue) of 4 molar excess Np75-4A22. Cross-peaks undergoing significant chemical shift are indicated with arrows and the corresponding residues labeled. ( G ) The same as ( F ) for the TMD of TrkB receptor tyrosine kinase, showing mostly non-specific interaction. ( H ) Same as ( F ) for the TMD of TNF receptor 2 (TNFR2), showing essentially no interaction. .

Article Snippet: Antibody against human p75 NTR extracellular domain used for immunoprecipitation was obtained from Alomone Labs (#ANT-007); the one used for immunofluorescence was from Promega (#G323A).

Techniques: AraTM Assay, Comparison, Labeling

Journal: EMBO Molecular Medicine

Article Title: Impaired migration and lung invasion of human melanoma by a novel small molecule targeting the transmembrane domain of death receptor p75 NTR

doi: 10.1038/s44321-025-00297-1

Figure Lengend Snippet: ( A ) Representative Western blot analysis (IB) of fascin in p75 NTR immunoprecipitates (IP) and whole cell lysates (WCL) of A875 melanoma cells after overnight treatment with NGF or Np75-4A22 as indicated. The histogram on the right shows quantification expressed as average of three independent experiments, each performed in triplicate ± SD. # P = 0.0091 vs. control (first bar); ** P = 0.0088 vs. NGF alone (second bar) (one-way ANOVA followed by Tukey’s multiple comparisons). ( B ) Representative Western blot analysis of fascin in Triton-insoluble extracts of A875 melanoma cells after overnight treatment with NGF or Np75-4A22 as indicated. The histogram on the right shows quantification expressed as average of three independent experiments, each performed in triplicate ± SD. # P = 0.0412 vs. control (first bar); ** P = 0.0082 vs. NGF alone (second bar) (one-way ANOVA followed by Tukey’s multiple comparisons). ( C ) Photomicrographs of cultured A875 control (NT) and knock-down (shp75) cells treated with NGF or Np75-4A22 as indicated and stained with fluorophor-conjugated phalloidin (green), to reveal actin filaments, and DAPI (blue). White arrows denote some of the filopodia in NGF-treated cells. Scale bar, 5 μM. ( D , E ) Quantification of filopodia per cell in A875 NT ( D ) and A875 shp75 ( E ) cells treated with 100 ng/ml NGF or 1 or 5 μM Np75-4A22 as indicated. Average of three independent experiments, each performed in triplicate, are shown ± SEM. ### P = 0.0004 vs. vehicle (first bar); *** P = 0.0001 vs. NGF alone (second bar). .

Article Snippet: Antibody against human p75 NTR extracellular domain used for immunoprecipitation was obtained from Alomone Labs (#ANT-007); the one used for immunofluorescence was from Promega (#G323A).

Techniques: Western Blot, Control, Cell Culture, Knockdown, Staining

Journal: EMBO Molecular Medicine

Article Title: Impaired migration and lung invasion of human melanoma by a novel small molecule targeting the transmembrane domain of death receptor p75 NTR

doi: 10.1038/s44321-025-00297-1

Figure Lengend Snippet: p75 NTR immunostaining (red) in lung metastasis induced by A875-NT ( A ) or shp75-A875 ( B ) cells counter-stained for human nucleolin (green) and DAPI (white). Scale bar, 100 μM.

Article Snippet: Antibody against human p75 NTR extracellular domain used for immunoprecipitation was obtained from Alomone Labs (#ANT-007); the one used for immunofluorescence was from Promega (#G323A).

Techniques: Immunostaining, Staining

Journal: bioRxiv

Article Title: Neuroprotective function of astrocyte p75 NTR in Alzheimer’s Disease through regulation of cholesterol metabolism

doi: 10.1101/2025.10.20.683521

Figure Lengend Snippet: (A) Representative micrographs of cultured astrocytes isolated from the hippocampus and cerebral cortex of wild type, p75 ΔDD and p75 C259A mice. Confluent monolayers were seeded with Aβ oligomers, washed and co-stained for Aβ (6E10) and GFAP to reveal uptake of Aβ plaques. Histograms show quantification of Aβ inclusions normalized to GFAP + cell number and expressed as percentage of wild type (WT) levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. *, p<0.05; **, p<0.01 vs. WT. (B) Representative micrographs of cultured astrocytes isolated from the hippocampus and cerebral cortex of wild type mice. Confluent monolayers were treated with the indicated neurotrophins (50ng/ml), seeded with Aβ oligomers, washed and co-stained for Aβ (6E10) and GFAP to reveal uptake of Aβ plaques. Histograms show quantification of Aβ inclusions normalized to GFAP + cell number and expressed as percentage of vehicle levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. *, p<0.05; **, p<0.01 vs. WT. (C) ELISA of Aβ-42 concentration in the supernatant of wild type hippocampal and cerebral cortex primary neuron cultures infected with AAV to express human AD mutant APP and treated with astrocyte conditioned medium (ACM) of the corresponding brain region derived from wild type, p75 ΔDD and p75 C259A mice as indicated. Results (mean ± SEM) are presented as percentage of astrocyte-free (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. *, p<0.05; ***, p<0.001; ****, p<0.0001 as indicated.

Article Snippet: The mouse lines utilized in this study are as follows: 5xFAD Aldh1l1-Cre ERT2 ; p75 NTR knock-out ; conditional knock-in p75 NTR ΔDD fl and C259A fl , generated by Cyagen Co., China; constitutive knock-in p75 NTR ΔDD and C259A ; constitutive knock-in p75 NTR -DHEA, generated by Taconic-Artemis, Germany; and constitutive knock-in p75 NTR -KKEA, generated at the Chinese Institute for Brain Research.

Techniques: Cell Culture, Isolation, Staining, Enzyme-linked Immunosorbent Assay, Concentration Assay, Infection, Mutagenesis, Derivative Assay

Journal: bioRxiv

Article Title: Neuroprotective function of astrocyte p75 NTR in Alzheimer’s Disease through regulation of cholesterol metabolism

doi: 10.1101/2025.10.20.683521

Figure Lengend Snippet: (A) Genes in the steroid and cholesterol biosynthesis gene sets enriched in p75 ΔDD and p75 C259A hippocampal and cortical astrocytes as revealed by RNA-Seq. Results form two duplicate samples of each condition are shown. Selected genes Dhcr7 , Hmgcr , Fdft1 and Msmo1 are highlighted. (B) Western blot analysis of SREBP2 and HMGCR expression in cultured hippocampal and cortical astrocytes from wild type and p75 ΔDD mice. Histograms show quantification of protein levels normalized to GAPDH and expressed as percentage of wild type (WT) levels. Results are presented as mean ± SEM (N=3) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. # , p=0.062; †, p=0.12;*, p<0.05; **** p<0.0001 vs. WT. (C) Western blot analysis of SREBP2 and HMGCR expression in cultured hippocampal and cortical astrocytes from wild type and p75 C259A mice. Histograms show quantification of protein levels normalized to GAPDH and expressed as percentage of wild type (WT) levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. *, p<0.05; ***, p<0.001 vs. WT. (D) Western blot analysis of SREBP2 expression in hippocampal extracts from wild type, 5xFAD, Aldh1l1-CreER T2 ;ΔDD fl/fl ;5xFAD and Aldh1l1-CreER T2 ;C259A fl/fl ;5xFAD mice. Histogram show quantification of protein levels normalized to GAPDH and expressed as percentage of wild type (WT) levels. Results are presented as mean ± SEM (N=3 mice per group) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. *, p<0.05; **, p<0.01 vs. 5xFAD.

Article Snippet: The mouse lines utilized in this study are as follows: 5xFAD Aldh1l1-Cre ERT2 ; p75 NTR knock-out ; conditional knock-in p75 NTR ΔDD fl and C259A fl , generated by Cyagen Co., China; constitutive knock-in p75 NTR ΔDD and C259A ; constitutive knock-in p75 NTR -DHEA, generated by Taconic-Artemis, Germany; and constitutive knock-in p75 NTR -KKEA, generated at the Chinese Institute for Brain Research.

Techniques: RNA Sequencing, Western Blot, Expressing, Cell Culture

Journal: bioRxiv

Article Title: Neuroprotective function of astrocyte p75 NTR in Alzheimer’s Disease through regulation of cholesterol metabolism

doi: 10.1101/2025.10.20.683521

Figure Lengend Snippet: (A) Representative micrographs showing Filipin staining and GFAP immunostaining of sagittal sections through the hippocampus of wild type, 5xFAD, Aldh1l1-CreER T2 ;ΔDD fl/fl ;5xFAD and Aldh1l1-CreER T2 ;C259A fl/fl ;5xFAD mice at 6 month of age. Mice were injected with tamoxifen at 2 month of age. Histogram shows quantification of Filipin area (as % of GFAP area) in hippocampus of wild type, 5xFAD, Aldh1l1-CreER T2 ;ΔDD fl/fl ;5xFAD and Aldh1l1-CreER T2 ;C259A fl/fl ;5xFAD mice injected with TMX at 2 month and analyzed at 6 month. Results are presented as mean ± SEM (N=7-11 mice per group) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. *, p<0.05; **, p<0.01 vs. 5xFAD. (B) Representative micrographs of cultured astrocytes isolated from the hippocampus of wild type, p75 ΔDD and p75 C259A mice stained with Filipin to reveal cholesterol content and anti-GFAP antibody. Histogram shows quantification of Filipin area normalized to GFAP + cell number and expressed as percentage of wild type (WT) levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. **, p<0.01 vs. WT. (C) Representative micrographs of cultured wild type hippocampal astrocytes treated with different neurotrophins at the indicated concentrations stained with Filipin to reveal cholesterol content and anti-GFAP antibody. Histogram shows quantification of Filipin area normalized to GFAP + cell number and expressed as percentage of vehicle levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. **, p<0.01; ***, p<0.001; ****, p<0.0001 vs. vehicle. (D) Quantification of free cholesterol in the supernatant of cultured hippocampal astrocytes isolated from wild type, p75 ΔDD and p75 C259A mice. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. *, p<0.05; **, p<0.01 vs. WT. (E) Representative micrographs of cultured wild type hippocampal neurons supplemented with conditioned medium from wild type, p75 ΔDD and p75 C259A astrocytes stained with Filipin to reveal cholesterol content and anti-MAP2 antibody. Control indicates normal unsupplemented neuron culture medium. Histogram shows quantification of Filipin area normalized to MAP2 area and expressed as percentage of wild type (WT) levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. ****, p<0.0001 vs. control. (F) Representative micrographs of cultured astrocytes isolated from the hippocampus of wild type, p75 DHEA and p75 KKEA mice stained with Filipin to reveal cholesterol content and anti-GFAP antibody. Histogram shows quantification of Filipin area normalized to GFAP + cell number and expressed as percentage of wild type (WT) levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. **, p<0.01 vs. WT. (G) Representative micrographs of cultured wild type hippocampal astrocytes treated with NF-kB inhibitor JSH23 (20ìM) stained with Filipin to reveal cholesterol content and anti-GFAP antibody. Histogram shows quantification of Filipin area normalized to GFAP + cell number and expressed as percentage of vehicle levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by Student’s t-test. *, p<0.05 vs. vehicle.

Article Snippet: The mouse lines utilized in this study are as follows: 5xFAD Aldh1l1-Cre ERT2 ; p75 NTR knock-out ; conditional knock-in p75 NTR ΔDD fl and C259A fl , generated by Cyagen Co., China; constitutive knock-in p75 NTR ΔDD and C259A ; constitutive knock-in p75 NTR -DHEA, generated by Taconic-Artemis, Germany; and constitutive knock-in p75 NTR -KKEA, generated at the Chinese Institute for Brain Research.

Techniques: Staining, Immunostaining, Injection, Cell Culture, Isolation, Control

Journal: bioRxiv

Article Title: Neuroprotective function of astrocyte p75 NTR in Alzheimer’s Disease through regulation of cholesterol metabolism

doi: 10.1101/2025.10.20.683521

Figure Lengend Snippet: (A) Representative micrographs of cultured astrocytes isolated from the hippocampus and cerebral cortex of wild type, p75 ΔDD and p75 C259A mice treated with MβCD (10mM) or vehicle and stained with Filipin to reveal cholesterol content and anti-GFAP antibody. Histogram shows quantification of Filipin area normalized to GFAP + cell number and expressed as percentage of vehicle-treated wild type (WT) levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by Student’s t-test. *, p<0.05; **, p<0.01: ***, p<0.001 vs. vehicle of corresponding genotype; #, p<0.05; ##, p<0.01; ###, p<0.001 vs. vehicle treated WT. (B) Representative micrographs of cultured astrocytes isolated from the hippocampus and cerebral cortex of wild type mice. Confluent monolayers were treated with MβCD (10mM) or vehicle, seeded with Aβ oligomers, washed and co-stained for Aβ (6E10) and GFAP to reveal uptake of Aβ plaques. Histograms show quantification of Aβ inclusions normalized to GFAP + cell number and expressed as percentage of wild type (WT) levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by Student’s t-test. *, p<0.05; **, p<0.01: ***, p<0.001 vs. vehicle of corresponding genotype; #, p<0.05; ##, p<0.01 vs. vehicle treated WT. (C) ELISA of Aβ-42 concentration in the supernatant of wild type hippocampal and cerebral cortex primary neuron cultures infected with AAV to express human AD mutant APP and treated with MβCD (10mM) or vehicle. Results (mean ± SEM) are presented as percentage of vehicle (N=3 independent experiments each performed in triplicate) and analyzed by Student’s t-test. *, p<0.05 vs. vehicle.

Article Snippet: The mouse lines utilized in this study are as follows: 5xFAD Aldh1l1-Cre ERT2 ; p75 NTR knock-out ; conditional knock-in p75 NTR ΔDD fl and C259A fl , generated by Cyagen Co., China; constitutive knock-in p75 NTR ΔDD and C259A ; constitutive knock-in p75 NTR -DHEA, generated by Taconic-Artemis, Germany; and constitutive knock-in p75 NTR -KKEA, generated at the Chinese Institute for Brain Research.

Techniques: Cell Culture, Isolation, Staining, Enzyme-linked Immunosorbent Assay, Concentration Assay, Infection, Mutagenesis

Journal: bioRxiv

Article Title: Neuroprotective function of astrocyte p75 NTR in Alzheimer’s Disease through regulation of cholesterol metabolism

doi: 10.1101/2025.10.20.683521

Figure Lengend Snippet: (A) Representative micrographs of cultured astrocytes isolated from the hippocampus and cerebral cortex of wild type, p75 ΔDD and p75 C259A mice. Confluent monolayers were seeded with Aβ oligomers, washed and co-stained for Aβ (6E10) and GFAP to reveal uptake of Aβ plaques. Histograms show quantification of Aβ inclusions normalized to GFAP + cell number and expressed as percentage of wild type (WT) levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. *, p<0.05; **, p<0.01 vs. WT. (B) Representative micrographs of cultured astrocytes isolated from the hippocampus and cerebral cortex of wild type mice. Confluent monolayers were treated with the indicated neurotrophins (50ng/ml), seeded with Aβ oligomers, washed and co-stained for Aβ (6E10) and GFAP to reveal uptake of Aβ plaques. Histograms show quantification of Aβ inclusions normalized to GFAP + cell number and expressed as percentage of vehicle levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. *, p<0.05; **, p<0.01 vs. WT. (C) ELISA of Aβ-42 concentration in the supernatant of wild type hippocampal and cerebral cortex primary neuron cultures infected with AAV to express human AD mutant APP and treated with astrocyte conditioned medium (ACM) of the corresponding brain region derived from wild type, p75 ΔDD and p75 C259A mice as indicated. Results (mean ± SEM) are presented as percentage of astrocyte-free (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. *, p<0.05; ***, p<0.001; ****, p<0.0001 as indicated.

Article Snippet: The mouse lines utilized in this study are as follows: 5xFAD Aldh1l1-Cre ERT2 ; p75 NTR knock-out ; conditional knock-in p75 NTR ΔDD fl and C259A fl , generated by Cyagen Co., China; constitutive knock-in p75 NTR ΔDD and C259A ; constitutive knock-in p75 NTR -DHEA, generated by Taconic-Artemis, Germany; and constitutive knock-in p75 NTR -KKEA, generated at the Chinese Institute for Brain Research.

Techniques: Cell Culture, Isolation, Staining, Enzyme-linked Immunosorbent Assay, Concentration Assay, Infection, Mutagenesis, Derivative Assay

Journal: bioRxiv

Article Title: Neuroprotective function of astrocyte p75 NTR in Alzheimer’s Disease through regulation of cholesterol metabolism

doi: 10.1101/2025.10.20.683521

Figure Lengend Snippet: (A) Genes in the steroid and cholesterol biosynthesis gene sets enriched in p75 ΔDD and p75 C259A hippocampal and cortical astrocytes as revealed by RNA-Seq. Results form two duplicate samples of each condition are shown. Selected genes Dhcr7 , Hmgcr , Fdft1 and Msmo1 are highlighted. (B) Western blot analysis of SREBP2 and HMGCR expression in cultured hippocampal and cortical astrocytes from wild type and p75 ΔDD mice. Histograms show quantification of protein levels normalized to GAPDH and expressed as percentage of wild type (WT) levels. Results are presented as mean ± SEM (N=3) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. # , p=0.062; †, p=0.12;*, p<0.05; **** p<0.0001 vs. WT. (C) Western blot analysis of SREBP2 and HMGCR expression in cultured hippocampal and cortical astrocytes from wild type and p75 C259A mice. Histograms show quantification of protein levels normalized to GAPDH and expressed as percentage of wild type (WT) levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. *, p<0.05; ***, p<0.001 vs. WT. (D) Western blot analysis of SREBP2 expression in hippocampal extracts from wild type, 5xFAD, Aldh1l1-CreER T2 ;ΔDD fl/fl ;5xFAD and Aldh1l1-CreER T2 ;C259A fl/fl ;5xFAD mice. Histogram show quantification of protein levels normalized to GAPDH and expressed as percentage of wild type (WT) levels. Results are presented as mean ± SEM (N=3 mice per group) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. *, p<0.05; **, p<0.01 vs. 5xFAD.

Article Snippet: The mouse lines utilized in this study are as follows: 5xFAD Aldh1l1-Cre ERT2 ; p75 NTR knock-out ; conditional knock-in p75 NTR ΔDD fl and C259A fl , generated by Cyagen Co., China; constitutive knock-in p75 NTR ΔDD and C259A ; constitutive knock-in p75 NTR -DHEA, generated by Taconic-Artemis, Germany; and constitutive knock-in p75 NTR -KKEA, generated at the Chinese Institute for Brain Research.

Techniques: RNA Sequencing, Western Blot, Expressing, Cell Culture

Journal: bioRxiv

Article Title: Neuroprotective function of astrocyte p75 NTR in Alzheimer’s Disease through regulation of cholesterol metabolism

doi: 10.1101/2025.10.20.683521

Figure Lengend Snippet: (A) Representative micrographs showing Filipin staining and GFAP immunostaining of sagittal sections through the hippocampus of wild type, 5xFAD, Aldh1l1-CreER T2 ;ΔDD fl/fl ;5xFAD and Aldh1l1-CreER T2 ;C259A fl/fl ;5xFAD mice at 6 month of age. Mice were injected with tamoxifen at 2 month of age. Histogram shows quantification of Filipin area (as % of GFAP area) in hippocampus of wild type, 5xFAD, Aldh1l1-CreER T2 ;ΔDD fl/fl ;5xFAD and Aldh1l1-CreER T2 ;C259A fl/fl ;5xFAD mice injected with TMX at 2 month and analyzed at 6 month. Results are presented as mean ± SEM (N=7-11 mice per group) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. *, p<0.05; **, p<0.01 vs. 5xFAD. (B) Representative micrographs of cultured astrocytes isolated from the hippocampus of wild type, p75 ΔDD and p75 C259A mice stained with Filipin to reveal cholesterol content and anti-GFAP antibody. Histogram shows quantification of Filipin area normalized to GFAP + cell number and expressed as percentage of wild type (WT) levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. **, p<0.01 vs. WT. (C) Representative micrographs of cultured wild type hippocampal astrocytes treated with different neurotrophins at the indicated concentrations stained with Filipin to reveal cholesterol content and anti-GFAP antibody. Histogram shows quantification of Filipin area normalized to GFAP + cell number and expressed as percentage of vehicle levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. **, p<0.01; ***, p<0.001; ****, p<0.0001 vs. vehicle. (D) Quantification of free cholesterol in the supernatant of cultured hippocampal astrocytes isolated from wild type, p75 ΔDD and p75 C259A mice. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. *, p<0.05; **, p<0.01 vs. WT. (E) Representative micrographs of cultured wild type hippocampal neurons supplemented with conditioned medium from wild type, p75 ΔDD and p75 C259A astrocytes stained with Filipin to reveal cholesterol content and anti-MAP2 antibody. Control indicates normal unsupplemented neuron culture medium. Histogram shows quantification of Filipin area normalized to MAP2 area and expressed as percentage of wild type (WT) levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. ****, p<0.0001 vs. control. (F) Representative micrographs of cultured astrocytes isolated from the hippocampus of wild type, p75 DHEA and p75 KKEA mice stained with Filipin to reveal cholesterol content and anti-GFAP antibody. Histogram shows quantification of Filipin area normalized to GFAP + cell number and expressed as percentage of wild type (WT) levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. **, p<0.01 vs. WT. (G) Representative micrographs of cultured wild type hippocampal astrocytes treated with NF-kB inhibitor JSH23 (20ìM) stained with Filipin to reveal cholesterol content and anti-GFAP antibody. Histogram shows quantification of Filipin area normalized to GFAP + cell number and expressed as percentage of vehicle levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by Student’s t-test. *, p<0.05 vs. vehicle.

Article Snippet: The mouse lines utilized in this study are as follows: 5xFAD Aldh1l1-Cre ERT2 ; p75 NTR knock-out ; conditional knock-in p75 NTR ΔDD fl and C259A fl , generated by Cyagen Co., China; constitutive knock-in p75 NTR ΔDD and C259A ; constitutive knock-in p75 NTR -DHEA, generated by Taconic-Artemis, Germany; and constitutive knock-in p75 NTR -KKEA, generated at the Chinese Institute for Brain Research.

Techniques: Staining, Immunostaining, Injection, Cell Culture, Isolation, Control

Journal: bioRxiv

Article Title: Neuroprotective function of astrocyte p75 NTR in Alzheimer’s Disease through regulation of cholesterol metabolism

doi: 10.1101/2025.10.20.683521

Figure Lengend Snippet: (A) Representative micrographs of cultured astrocytes isolated from the hippocampus and cerebral cortex of wild type, p75 ΔDD and p75 C259A mice treated with MβCD (10mM) or vehicle and stained with Filipin to reveal cholesterol content and anti-GFAP antibody. Histogram shows quantification of Filipin area normalized to GFAP + cell number and expressed as percentage of vehicle-treated wild type (WT) levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by Student’s t-test. *, p<0.05; **, p<0.01: ***, p<0.001 vs. vehicle of corresponding genotype; #, p<0.05; ##, p<0.01; ###, p<0.001 vs. vehicle treated WT. (B) Representative micrographs of cultured astrocytes isolated from the hippocampus and cerebral cortex of wild type mice. Confluent monolayers were treated with MβCD (10mM) or vehicle, seeded with Aβ oligomers, washed and co-stained for Aβ (6E10) and GFAP to reveal uptake of Aβ plaques. Histograms show quantification of Aβ inclusions normalized to GFAP + cell number and expressed as percentage of wild type (WT) levels. Results are presented as mean ± SEM (N=3 independent experiments each performed in triplicate) and analyzed by Student’s t-test. *, p<0.05; **, p<0.01: ***, p<0.001 vs. vehicle of corresponding genotype; #, p<0.05; ##, p<0.01 vs. vehicle treated WT. (C) ELISA of Aβ-42 concentration in the supernatant of wild type hippocampal and cerebral cortex primary neuron cultures infected with AAV to express human AD mutant APP and treated with MβCD (10mM) or vehicle. Results (mean ± SEM) are presented as percentage of vehicle (N=3 independent experiments each performed in triplicate) and analyzed by Student’s t-test. *, p<0.05 vs. vehicle.

Article Snippet: The mouse lines utilized in this study are as follows: 5xFAD Aldh1l1-Cre ERT2 ; p75 NTR knock-out ; conditional knock-in p75 NTR ΔDD fl and C259A fl , generated by Cyagen Co., China; constitutive knock-in p75 NTR ΔDD and C259A ; constitutive knock-in p75 NTR -DHEA, generated by Taconic-Artemis, Germany; and constitutive knock-in p75 NTR -KKEA, generated at the Chinese Institute for Brain Research.

Techniques: Cell Culture, Isolation, Staining, Enzyme-linked Immunosorbent Assay, Concentration Assay, Infection, Mutagenesis

Journal: EMBO Molecular Medicine

Article Title: Impaired migration and lung invasion of human melanoma by a novel small molecule targeting the transmembrane domain of death receptor p75 NTR

doi: 10.1038/s44321-025-00297-1

Figure Lengend Snippet: ( A ) Volcano plot of AraTM screening assay of Screening Set v2010 (8482 compounds) from the Chemical Biology Consortium Sweden ( www.cbcs.se ). Fold change was calculated against vehicle (DMSO) and hits were defined as compounds resulting in greater than ±0.5 fold change in GFP/OD 630 signal without affecting OD 630 by greater than 0.3-fold across three independent runs (red dots). P values were calculated by one-way ANOVA ( N = 3). Compound Div17E5 is indicated. ( B ) Chemical structure of Div17E5. ( C ) Dose response of Div17E5 in the AraTM assay of p75 NTR TMD in comparison with unrelated TMDs from α2β3 integrin and Matrix-2 protein (M2) from the viral envelope of influenza A virus. Results are plotted as mean ± SD ( N = 3). ( D – F ) Comparison of wild-type (WT) p75 NTR TMD and I252A, P253G and V254A mutants in the AraTM assay in response to increasing doses of Div17E5. GFP/OD 630 signal for WT TMD without any drug was set at 100% and all other measurements are relative to that. Results are plotted as mean ± SD ( N = 3). * P = 0.03: ** P = 0.006 (one-way ANOVA followed by Tukey’s multiple comparisons). ( G ) Live cell homo-FRET anisotropy in response to Div17E5 of full-length, wild-type human p75 NTR expressed in COS cells. Shown are representative traces of average anisotropy change after addition of Div17E5 (10 μM) or vehicle. ( H ) Live cell homo-FRET anisotropy in response to Div17E5 of full-length, wild-type human p75 NTR in comparison to I252A p75 NTR TMD mutant. Shown are representative traces of average anisotropy change after addition of Div17E5 (10 μM). .

Article Snippet: For immunoprecipitation, A875 cell extracts were incubated for 16 h at 4 °C on a rotating wheel with 0.8 μg of anti-p75 NTR antibody (ANT-007, Alomone) and then incubated with Sepharose protein-G beads (GE Healthcare).

Techniques: Screening Assay, AraTM Assay, Comparison, Virus, Mutagenesis

Journal: EMBO Molecular Medicine

Article Title: Impaired migration and lung invasion of human melanoma by a novel small molecule targeting the transmembrane domain of death receptor p75 NTR

doi: 10.1038/s44321-025-00297-1

Figure Lengend Snippet: ( A ) Expression of p75 NTR in A875 control (NT, non-targeting) and knock-down (shp75) cells. ( B ) Dose response analysis of cleaved PARP induction by Div17E5 in A875 control (NT) and knock-down (shp75) cells. ( C ) Western blot analysis of cleaved PARP in A875 melanoma cells in response to Div17E5 (10 µM) in the presence or absence of pan-caspase inhibitor Q-VD-pOH (5 µM). Reprobing for GAPDH was used as loading control. The experiment was repeated three times with comparable results. ( D ) Dose-dependent cell viability of A875 control (NT) and knock-down (shp75) cells in response to Div17E5. Results are plotted as mean ± SD ( N = 3). * P = 0.04; ** P = 0.006 (one-way ANOVA followed by Tukey’s multiple comparisons). .

Article Snippet: For immunoprecipitation, A875 cell extracts were incubated for 16 h at 4 °C on a rotating wheel with 0.8 μg of anti-p75 NTR antibody (ANT-007, Alomone) and then incubated with Sepharose protein-G beads (GE Healthcare).

Techniques: Expressing, Control, Knockdown, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Impaired migration and lung invasion of human melanoma by a novel small molecule targeting the transmembrane domain of death receptor p75 NTR

doi: 10.1038/s44321-025-00297-1

Figure Lengend Snippet: ( A ) Chemical structure of Np75-4A22. ( B ) Dose response of Div17E5 and Np75-4A22 in the AraTM assay of p75 NTR TMD. Results are plotted as mean ± SD ( N = 3). ** P = 0.007 (one-way ANOVA followed by Tukey’s multiple comparisons). ( C – E ) Comparison of wild-type p75 NTR TMD and I252A, P253G and V254A mutants in the AraTM assay in response to increasing doses of Np75-4A22. GFP/OD 630 signal without any drug added was set at 100%. Results are plotted as mean ± SD ( N = 3). * P = 0.035; ** P = 0.008 (one-way ANOVA followed by Tukey’s multiple comparisons). ( F – H ) Specific interaction between Np75-4A22 (4 mM) and p75 NTR TMD (1 mM) in bicelles that mimic a lipid bilayer. ( F ) 1 H- 15 N TROSY-HSQC spectra of p75 NTR TMD in DMPC-DH6PC bicelles ( q = 0.5) in the absence (red) and presence (blue) of 4 molar excess Np75-4A22. Cross-peaks undergoing significant chemical shift are indicated with arrows and the corresponding residues labeled. ( G ) The same as ( F ) for the TMD of TrkB receptor tyrosine kinase, showing mostly non-specific interaction. ( H ) Same as ( F ) for the TMD of TNF receptor 2 (TNFR2), showing essentially no interaction. .

Article Snippet: For immunoprecipitation, A875 cell extracts were incubated for 16 h at 4 °C on a rotating wheel with 0.8 μg of anti-p75 NTR antibody (ANT-007, Alomone) and then incubated with Sepharose protein-G beads (GE Healthcare).

Techniques: AraTM Assay, Comparison, Labeling

Journal: EMBO Molecular Medicine

Article Title: Impaired migration and lung invasion of human melanoma by a novel small molecule targeting the transmembrane domain of death receptor p75 NTR

doi: 10.1038/s44321-025-00297-1

Figure Lengend Snippet: ( A ) Representative Western blot analysis (IB) of fascin in p75 NTR immunoprecipitates (IP) and whole cell lysates (WCL) of A875 melanoma cells after overnight treatment with NGF or Np75-4A22 as indicated. The histogram on the right shows quantification expressed as average of three independent experiments, each performed in triplicate ± SD. # P = 0.0091 vs. control (first bar); ** P = 0.0088 vs. NGF alone (second bar) (one-way ANOVA followed by Tukey’s multiple comparisons). ( B ) Representative Western blot analysis of fascin in Triton-insoluble extracts of A875 melanoma cells after overnight treatment with NGF or Np75-4A22 as indicated. The histogram on the right shows quantification expressed as average of three independent experiments, each performed in triplicate ± SD. # P = 0.0412 vs. control (first bar); ** P = 0.0082 vs. NGF alone (second bar) (one-way ANOVA followed by Tukey’s multiple comparisons). ( C ) Photomicrographs of cultured A875 control (NT) and knock-down (shp75) cells treated with NGF or Np75-4A22 as indicated and stained with fluorophor-conjugated phalloidin (green), to reveal actin filaments, and DAPI (blue). White arrows denote some of the filopodia in NGF-treated cells. Scale bar, 5 μM. ( D , E ) Quantification of filopodia per cell in A875 NT ( D ) and A875 shp75 ( E ) cells treated with 100 ng/ml NGF or 1 or 5 μM Np75-4A22 as indicated. Average of three independent experiments, each performed in triplicate, are shown ± SEM. ### P = 0.0004 vs. vehicle (first bar); *** P = 0.0001 vs. NGF alone (second bar). .

Article Snippet: For immunoprecipitation, A875 cell extracts were incubated for 16 h at 4 °C on a rotating wheel with 0.8 μg of anti-p75 NTR antibody (ANT-007, Alomone) and then incubated with Sepharose protein-G beads (GE Healthcare).

Techniques: Western Blot, Control, Cell Culture, Knockdown, Staining

Journal: EMBO Molecular Medicine

Article Title: Impaired migration and lung invasion of human melanoma by a novel small molecule targeting the transmembrane domain of death receptor p75 NTR

doi: 10.1038/s44321-025-00297-1

Figure Lengend Snippet: p75 NTR immunostaining (red) in lung metastasis induced by A875-NT ( A ) or shp75-A875 ( B ) cells counter-stained for human nucleolin (green) and DAPI (white). Scale bar, 100 μM.

Article Snippet: For immunoprecipitation, A875 cell extracts were incubated for 16 h at 4 °C on a rotating wheel with 0.8 μg of anti-p75 NTR antibody (ANT-007, Alomone) and then incubated with Sepharose protein-G beads (GE Healthcare).

Techniques: Immunostaining, Staining

Journal: Bone Reports

Article Title: p75 NTR regulates postnatal skeletal development via NGF-responsive JNK signaling

doi: 10.1016/j.bonr.2025.101854

Figure Lengend Snippet: Global p75 NTR -deficient mice exhibited an osteoporotic phenotype. (A) Photos of 4 and 12-week-old p75 NTR+/+ and p75 NTR−/− mice. Scale bar: 4.0 cm. (B) Body weight curve of p75 NTR+/+ and p75 NTR−/− mice from P0 - P28. Three-dimensional Micro-CT images of 4 (C) and 12 (E) weeks old p75 NTR+/+ and p75 NTR−/− mice's whole femurs, trabecular bones, and cortical bones. The BV/TV, Tb. Th, Tb.N, CtV/TV, Ct. Th and length of femurs of 4 (D) and 12 (F) weeks old p75 NTR+/+ and p75 NTR− /− mice were quantified by 3D Micro-CT. Scale bar (left panel): 1 mm, Scale bar (right panel): 0.5 mm. For 4-week-old mice, n = 5 for each group. For 12-week-old mice, p75 NTR+/+ ( n = 3) and p75 NTR−/− ( n = 4). Alcian blue staining images of the growth plate of 4 (G) and 12 (I) weeks old p75 NTR+/+ and p75 NTR−/− mice's tibias. The growth plate thickness (GP. Th) of 4 (H) and 12 (J) weeks old p75 NTR+/+ and p75 NTR− /− mice were quantified. For 4-week-old mice, n = 5 for each group. For 12-week-old mice, p75 NTR+/+ ( n = 6) and p75 NTR−/− (n = 5). Unpaired Student's t -test was used for analysis, with * p < 0.05, ** p < 0.01, *** p < 0.001. n represents the number of mice analyzed.

Article Snippet: p75 NTR−/− (Stock No: 002213), p75 NTR flox (p75 NTRf/f ) (Stock No: 031162), and paired related homeobox-Cre (Prx1-Cre) (Stock No: 005584) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and housed in the animal facility at the University of California, San Francisco (UCSF) according to institutional guidelines.

Techniques: Micro-CT, Staining

Journal: Bone Reports

Article Title: p75 NTR regulates postnatal skeletal development via NGF-responsive JNK signaling

doi: 10.1016/j.bonr.2025.101854

Figure Lengend Snippet: MSC-specific p75 NTR -deficient mice exhibited an osteoporotic phenotype. (A) PCR results of p75 NTR in p75 NTRf/f and Prx1-Cre; p75 NTR f/f spleen, heart, liver, kidney, muscle, and bone. (B) PCR results of p75 NTR in p75 NTRf/f and Prx1-Cre; p75 NTR f/f monocyte and BMSC. p75 NTR expression was not detected in BMSCs. (C) Photos of 4 and 12-week-old p75 NTRf/f and Prx1-Cre; p75 NTR f/f mice. Scale bar: 4 cm. (D) Body weight of 4 and 12-week-old p75 NTRf/f and Prx1-Cre; p75 NTR f/f mice. For 4-week-old mice, p75 NTRf/f ( n = 3) and Prx1-Cre; p75 NTR f/f ( n = 4). For 12-week-old mice, p75 NTRf/f (n = 5) and Prx1-Cre; p75 NTR f/f (n = 4). Three-dimensional Micro-CT images of 4 (E) and 12 (G) weeks old p75 NTRf/f and Prx1-Cre; p75 NTR f/f mice whole femurs, trabecular bones, and cortical bones. The BV/TV, Tb. Th, Tb.N, CtV/TV, Ct. Th, and length of femurs of 4 (F) and 12 (H) weeks old p75 NTRf/f and Prx1-Cre; p75 NTR f/f mice were quantified by 3D Micro-CT. Scale bar (left panel): 1 mm, Scale bar (right panel): 0.5 mm. For 4-week-old mice, p75 NTRf/f ( n = 4) and Prx1-Cre; p75 NTR f/f ( n = 7). For 12-week-old mice, p75 NTRf/f (n = 6) and Prx1-Cre; p75 NTR f/f (n = 6). Alcian blue staining images of the growth plate of 4 (I) and 12 (K) weeks old p75 NTRf/f and Prx1-Cre; p75 NTR f/f mice's Tibias. The growth plate thickness (GP. Th) of 4 (J) and 12 (L) weeks old p75 NTRf/f and Prx1-Cre; p75 NTR f/f mice were quantified. For both ages, n = 5 for each group. Unpaired Student's t -test was used for analysis, with * p < 0.05, ** p < 0.01, *** p < 0.001. n represents the number of mice analyzed.

Article Snippet: p75 NTR−/− (Stock No: 002213), p75 NTR flox (p75 NTRf/f ) (Stock No: 031162), and paired related homeobox-Cre (Prx1-Cre) (Stock No: 005584) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and housed in the animal facility at the University of California, San Francisco (UCSF) according to institutional guidelines.

Techniques: Expressing, Micro-CT, Staining

Journal: Bone Reports

Article Title: p75 NTR regulates postnatal skeletal development via NGF-responsive JNK signaling

doi: 10.1016/j.bonr.2025.101854

Figure Lengend Snippet: p75 NTR mediates osteogenic differentiation of MSCs. (A) Schematic representation of the osteogenic differentiation process of BMSCs derived from p75 NTR+/+ and p75 NTR−/− mice. Created in BioRender. Kadota, C. (2025) https://BioRender.com/a77c926 (B) Alizarin Red Staining (ARS) images of BMSCs harvested from p75 NTR+/+ and p75 NTR−/− mice cultured in GM and OIM for 21 days, along with quantification of Alizarin Red-positive areas. n = 4 for each group. (C) qRT-PCR analysis of osteoblast differentiation markers in BMSCs cultured in OIM. The expression of osteoblastic differentiation markers, including PTHrP , Runx2 , Dlx5 , Osx , and Alp , were compared, and p75 NTR−/− showed significant downregulation compared to p75 NTR+/+ . n = 3 for each group. Unpaired Student's t -test was used for analysis, with * p < 0.05, ** p < 0.01, *** p < 0.001. n represents the number of experimental replicates.

Article Snippet: p75 NTR−/− (Stock No: 002213), p75 NTR flox (p75 NTRf/f ) (Stock No: 031162), and paired related homeobox-Cre (Prx1-Cre) (Stock No: 005584) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and housed in the animal facility at the University of California, San Francisco (UCSF) according to institutional guidelines.

Techniques: Derivative Assay, Staining, Cell Culture, Quantitative RT-PCR, Expressing

Journal: Bone Reports

Article Title: p75 NTR regulates postnatal skeletal development via NGF-responsive JNK signaling

doi: 10.1016/j.bonr.2025.101854

Figure Lengend Snippet: p75 NTR -deficient BMSCs showed reduced NGF signaling and MAPK activation. (A) Volcano plot shows gene expression changes in bone marrow stem cells (BMSCs) derived from 4-week-old p75 NTR+/+ and p75 NTR−/− mice (n = 3 per group). Expression was profiled using the NanoString nCounter platform in a panel of 477 genes. The x-axis represents the log 2 fold change in p75 NTR−/− relative to p75 NTR+/+ and the y-axis is the –log 10 ( p -value). The dashed horizontal line indicates p = 0.05. Among the 637 genes tested, 15 were significantly downregulated (p < 0.05) in p75 NTR−/− BMSCs. Red dots indicate upregulated genes, while blue dots indicate downregulated genes in p75 NTR−/− . (B) Heatmap of the 15 differentially expressed genes. Red indicates higher expression, while blue indicates lower expression. (C)-(E) Bar graph of pathway enrichment analysis for the 15 differentially expressed genes. The analysis was performed using the BioPlanet 2019, (C) BioCarta 2016, and (E) Reactome pathways 2024. The y-axis lists the top 15 significantly enriched terms (p < 0.05), and the x-axis shows the –log 10 (p-value). n represents the number of experimental replicates.

Article Snippet: p75 NTR−/− (Stock No: 002213), p75 NTR flox (p75 NTRf/f ) (Stock No: 031162), and paired related homeobox-Cre (Prx1-Cre) (Stock No: 005584) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and housed in the animal facility at the University of California, San Francisco (UCSF) according to institutional guidelines.

Techniques: Activation Assay, Gene Expression, Derivative Assay, Expressing

Journal: Bone Reports

Article Title: p75 NTR regulates postnatal skeletal development via NGF-responsive JNK signaling

doi: 10.1016/j.bonr.2025.101854

Figure Lengend Snippet: KDM4B regulates p75 NTR -mediated osteogenesis via JNK signaling pathway. (A)Western blot of JNK signaling pathway (p-JNK and p-c-Jun) in p75 NTR+/+ and p75 NTR−/− BMSCs. (B) ARS images and quantification after 21 days treatment in OIM with NGF (10 ng/ml) and/or SP600125 (5 μM). n = 4 for each group. (C) qRT-PCR analysis of epigenetic genes in p75 NTR+/+ and p75 NTR−/− BMSCs with or without NGF treatment at 2 h. n = 3 for each group. (D) ARS images and quantification after 21 days treatment in OIM with pLV-empty or pLV- Kdm4b , and NGF (10 ng/ml). n = 4 for each group. (E) Upper panel: ARS images and quantification after 21 days treatment in OIM with pLV-empty or pLV- Kdm4b in p75 NTR+/+ and p75 NTR−/− BMSCs. Lower panel: gene expression analysis of Kdm4b and Dlx5 after treating with pLV-empty or pLV- Kdm4b . Dlx5 expression increased along with the upregulation of Kdm4b . n = 4 for each group. * p < 0.05, ** p < 0.01, *** p < 0.001. Two-way ANOVA with multiple comparison was used for c, d and e, followed by Tukey's post-hoc test. n represents the number of experimental replicates.

Article Snippet: p75 NTR−/− (Stock No: 002213), p75 NTR flox (p75 NTRf/f ) (Stock No: 031162), and paired related homeobox-Cre (Prx1-Cre) (Stock No: 005584) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and housed in the animal facility at the University of California, San Francisco (UCSF) according to institutional guidelines.

Techniques: Western Blot, Quantitative RT-PCR, Gene Expression, Expressing, Comparison

Journal: iScience

Article Title: Cholinotrophic basal forebrain connectome dysfunction in Down syndrome with and without dementia

doi: 10.1016/j.isci.2025.113041

Figure Lengend Snippet: Images and quantitation of cholinotrophic basal forebrain neuron pathology within the nucleus basalis of Meynert in DSD+, DSD- and AMC cases (A–N) Photomicrographs show p75 NTR (brown, A-C), ChAT (purple, D-F) positive cells in AMC, DSD-, and DSD+ cases. Note the decrease in the number and intensity of both p75 NTR and ChAT labeled neurons in DSD- cases compared to an even greater reduction in DSD+ and globose shaped cells (black arrows) in DSD+ cases (C, F). Arrows in (A–F) mark cells shown at a higher magnification in the boxed areas located at the lower right corner of each panel. Dark brown AT8 (G, I) and TauC3 (H, J) bearing neurofibrillary tangles (NFTs) were observed only in the DSD- and DSD+ cases. Black arrows indicate globose shaped NFTs (G, H, and I) that are shown at a higher magnification in boxed areas adjacent to the lower magnification images. Note that not all neurons within the nbM (thin black arrows) contained tau pathology (G, H) in DSD- cases. Moreover, TauC3 staining revealed two NFT phenotypes that displayed either peripherally located or intense labeling that filled the entire structure (see boxed images adjacent to (H) and (J)). Scale bar in F = 50 μm and inset = 20 μm applies to panels A-E. Scale bar in J = 20 μm and inset = 20 μm applies to (G–J). Histograms show a significant reduction in both p75 NTR (K) and ChAT (L) positive cells in DSD+ compared to AMC. Although no significant were found in number of AT8 or TauC3 NFT positive cells (M), there was an increase in NTs in DSD+ compared to DSD- (N). ACM n = 5, DSD- n = 5, DSD+ n = 10. Data shown are presented as mean ± SEM. Statistical significance was determined using the Kruskal–Wallis’s test followed by Dunn’s test for comparisons across three clinical groups, and the Mann–Whitney test for comparisons between two groups (DSD- vs. DSD+). Significance levels (∗) were set at: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Antibodies were added to blocking buffer and membranes incubated overnight (4°C) in mouse anti-p75 NTR ([1:150], Santa Cruz Biotechnology Cat# sc-271708, RRID: AB_10714958 ), rabbit anti-ChAT ([1:500] (Proteintech Cat# 20747-1-AP, RRID: AB_10898169 ), rabbit anti-TrkA ([1:150], Fitzgerald Industries International Cat# 20R-TR013, RRID: AB_1289098 ) or rabbit anti-proNGF ([1:250], Alomone Labs Cat# ANT-005, RRID: AB_2040021 ), washed, incubated with horseradish peroxidase-conjugated with either a goat anti-mouse IgG (1:200) or goat anti-rabbit IgG (1:200) secondary antibody at RT, visualized by chemiluminescence (Kodak Image Station 440CF; Perkin-Elmer, Wellesley, MA), and quantified with Kodak 1.

Techniques: Quantitation Assay, Labeling, Staining, MANN-WHITNEY

Journal: iScience

Article Title: Cholinotrophic basal forebrain connectome dysfunction in Down syndrome with and without dementia

doi: 10.1016/j.isci.2025.113041

Figure Lengend Snippet: Immunofluorescent images and quantification of p75 NTR neurons with and without AT8 and MAP2, as well as Thioflavin S single and AT8/Thioflavin S dual-labeled nbM cells in DSD+ and DSD-cases. (A–U) Immunofluorescent neurons labeled with p75 NTR (A, E, green), MAP2 (B, F, red), AT8 (C, G, Cyan), ThS (blue) (I, L) and merge images (D, H, K, N, R) in DSD- and DSD+ cases. In the DSD-cases there were greater p75 NTR cells AT8 negative neurons and p75 NTR MAP2 dual labeled neurons (A-D) compared to the DSD+ cases (E–H). Note that not all p75 NTR cells colocalize with AT8 or MAP2 in both DS groups (D, H). To determine the stage of a tangle tissue was stained for ThS, a marker of advanced pathology and the early stage AT8 phosphorylation antibody. Note that there are only a few ThS-labeled tangles that displayed AT8 in both DS groups (yellow arrows), compared to single ThS tangles, which were greater in DSD+ than in DSD- (white arrows) (I-L). Note that ThS positive NFTs that do not contain AT8 also do not colocalize with MAP2 (O-R). Scale bar in F = 25 μm applies to panels A-G, N = 25 μm applies to panels I-M and R = 10 μm and applies to O-Q. Graph showing a significant reduction in both p75 NTR AT8 immuno-negative, and p75 NTR MAP2 dual labeled neurons in DSD+ compared to DSD- (S). ThS-positive cells were greater (T), while the percentage of double-labeled cells with AT8 and ThS decreased (U) in DSD+ compared to individuals without dementia. DSD- n = 5, DSD+ n = 5. Data are presented as mean ± SEM. Statistical significance was determined using Mann–Whitney test for comparisons between DSD- and DSD+. Significance levels (∗) were set at: ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Antibodies were added to blocking buffer and membranes incubated overnight (4°C) in mouse anti-p75 NTR ([1:150], Santa Cruz Biotechnology Cat# sc-271708, RRID: AB_10714958 ), rabbit anti-ChAT ([1:500] (Proteintech Cat# 20747-1-AP, RRID: AB_10898169 ), rabbit anti-TrkA ([1:150], Fitzgerald Industries International Cat# 20R-TR013, RRID: AB_1289098 ) or rabbit anti-proNGF ([1:250], Alomone Labs Cat# ANT-005, RRID: AB_2040021 ), washed, incubated with horseradish peroxidase-conjugated with either a goat anti-mouse IgG (1:200) or goat anti-rabbit IgG (1:200) secondary antibody at RT, visualized by chemiluminescence (Kodak Image Station 440CF; Perkin-Elmer, Wellesley, MA), and quantified with Kodak 1.

Techniques: Labeling, Staining, Marker, Phospho-proteomics, MANN-WHITNEY

Journal: iScience

Article Title: Cholinotrophic basal forebrain connectome dysfunction in Down syndrome with and without dementia

doi: 10.1016/j.isci.2025.113041

Figure Lengend Snippet: FC cholinotrophic protein levels in AMC, DSD- and DSD+ (A–D) Representative immunoblots, and bar graphs show a significant upregulation of (A) proNGF and (B) p75 NTR , while (C) ChAT protein was downregulated between AMC and DSD+. (D) TrkA protein levels were stable across the groups analyzed. ACM n = 5, DSD- n = 5, DSD+ n = 13. Data are presented as mean ± SEM. Statistical significance was determined using the Kruskal–Wallis’s test followed by Dunn’s test for comparisons across three clinical groups. Significance levels (∗) were set at: ∗ p < 0.05.

Article Snippet: Antibodies were added to blocking buffer and membranes incubated overnight (4°C) in mouse anti-p75 NTR ([1:150], Santa Cruz Biotechnology Cat# sc-271708, RRID: AB_10714958 ), rabbit anti-ChAT ([1:500] (Proteintech Cat# 20747-1-AP, RRID: AB_10898169 ), rabbit anti-TrkA ([1:150], Fitzgerald Industries International Cat# 20R-TR013, RRID: AB_1289098 ) or rabbit anti-proNGF ([1:250], Alomone Labs Cat# ANT-005, RRID: AB_2040021 ), washed, incubated with horseradish peroxidase-conjugated with either a goat anti-mouse IgG (1:200) or goat anti-rabbit IgG (1:200) secondary antibody at RT, visualized by chemiluminescence (Kodak Image Station 440CF; Perkin-Elmer, Wellesley, MA), and quantified with Kodak 1.

Techniques: Western Blot

Journal: iScience

Article Title: Cholinotrophic basal forebrain connectome dysfunction in Down syndrome with and without dementia

doi: 10.1016/j.isci.2025.113041

Figure Lengend Snippet: Summary of changes in the cholinotrophic basal forebrain connectome in DS Diagrammatic sagittal view of the human brain (A) and a modified stacked bar graph (B) illustrating differences in the pathobiology of the cholinotrophic projection system between non-trisomy age-matched control (AMC), DS without dementia (DSD-) and DS with dementia (DSD+) individuals with DS. Frontal cortex protein levels for ChAT (pink), proNGF (green), p75 NTR (purple) and TrkA (light blue). Nucleus basalis NFTs of ThS (blue), AT8 (orange) and TauC3 (black) and counts of p75 NTR and ChAT (red) neurons. Created with BioRender.com .

Article Snippet: Antibodies were added to blocking buffer and membranes incubated overnight (4°C) in mouse anti-p75 NTR ([1:150], Santa Cruz Biotechnology Cat# sc-271708, RRID: AB_10714958 ), rabbit anti-ChAT ([1:500] (Proteintech Cat# 20747-1-AP, RRID: AB_10898169 ), rabbit anti-TrkA ([1:150], Fitzgerald Industries International Cat# 20R-TR013, RRID: AB_1289098 ) or rabbit anti-proNGF ([1:250], Alomone Labs Cat# ANT-005, RRID: AB_2040021 ), washed, incubated with horseradish peroxidase-conjugated with either a goat anti-mouse IgG (1:200) or goat anti-rabbit IgG (1:200) secondary antibody at RT, visualized by chemiluminescence (Kodak Image Station 440CF; Perkin-Elmer, Wellesley, MA), and quantified with Kodak 1.

Techniques: Modification, Control

Journal: iScience

Article Title: Cholinotrophic basal forebrain connectome dysfunction in Down syndrome with and without dementia

doi: 10.1016/j.isci.2025.113041

Figure Lengend Snippet: Images and quantitation of cholinotrophic basal forebrain neuron pathology within the nucleus basalis of Meynert in DSD+, DSD- and AMC cases (A–N) Photomicrographs show p75 NTR (brown, A-C), ChAT (purple, D-F) positive cells in AMC, DSD-, and DSD+ cases. Note the decrease in the number and intensity of both p75 NTR and ChAT labeled neurons in DSD- cases compared to an even greater reduction in DSD+ and globose shaped cells (black arrows) in DSD+ cases (C, F). Arrows in (A–F) mark cells shown at a higher magnification in the boxed areas located at the lower right corner of each panel. Dark brown AT8 (G, I) and TauC3 (H, J) bearing neurofibrillary tangles (NFTs) were observed only in the DSD- and DSD+ cases. Black arrows indicate globose shaped NFTs (G, H, and I) that are shown at a higher magnification in boxed areas adjacent to the lower magnification images. Note that not all neurons within the nbM (thin black arrows) contained tau pathology (G, H) in DSD- cases. Moreover, TauC3 staining revealed two NFT phenotypes that displayed either peripherally located or intense labeling that filled the entire structure (see boxed images adjacent to (H) and (J)). Scale bar in F = 50 μm and inset = 20 μm applies to panels A-E. Scale bar in J = 20 μm and inset = 20 μm applies to (G–J). Histograms show a significant reduction in both p75 NTR (K) and ChAT (L) positive cells in DSD+ compared to AMC. Although no significant were found in number of AT8 or TauC3 NFT positive cells (M), there was an increase in NTs in DSD+ compared to DSD- (N). ACM n = 5, DSD- n = 5, DSD+ n = 10. Data shown are presented as mean ± SEM. Statistical significance was determined using the Kruskal–Wallis’s test followed by Dunn’s test for comparisons across three clinical groups, and the Mann–Whitney test for comparisons between two groups (DSD- vs. DSD+). Significance levels (∗) were set at: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: p75 NTR , Santa Cruz Biotechnology , Cat# sc-271708; RRID: AB_10714958.

Techniques: Quantitation Assay, Labeling, Staining, MANN-WHITNEY

Journal: iScience

Article Title: Cholinotrophic basal forebrain connectome dysfunction in Down syndrome with and without dementia

doi: 10.1016/j.isci.2025.113041

Figure Lengend Snippet: Immunofluorescent images and quantification of p75 NTR neurons with and without AT8 and MAP2, as well as Thioflavin S single and AT8/Thioflavin S dual-labeled nbM cells in DSD+ and DSD-cases. (A–U) Immunofluorescent neurons labeled with p75 NTR (A, E, green), MAP2 (B, F, red), AT8 (C, G, Cyan), ThS (blue) (I, L) and merge images (D, H, K, N, R) in DSD- and DSD+ cases. In the DSD-cases there were greater p75 NTR cells AT8 negative neurons and p75 NTR MAP2 dual labeled neurons (A-D) compared to the DSD+ cases (E–H). Note that not all p75 NTR cells colocalize with AT8 or MAP2 in both DS groups (D, H). To determine the stage of a tangle tissue was stained for ThS, a marker of advanced pathology and the early stage AT8 phosphorylation antibody. Note that there are only a few ThS-labeled tangles that displayed AT8 in both DS groups (yellow arrows), compared to single ThS tangles, which were greater in DSD+ than in DSD- (white arrows) (I-L). Note that ThS positive NFTs that do not contain AT8 also do not colocalize with MAP2 (O-R). Scale bar in F = 25 μm applies to panels A-G, N = 25 μm applies to panels I-M and R = 10 μm and applies to O-Q. Graph showing a significant reduction in both p75 NTR AT8 immuno-negative, and p75 NTR MAP2 dual labeled neurons in DSD+ compared to DSD- (S). ThS-positive cells were greater (T), while the percentage of double-labeled cells with AT8 and ThS decreased (U) in DSD+ compared to individuals without dementia. DSD- n = 5, DSD+ n = 5. Data are presented as mean ± SEM. Statistical significance was determined using Mann–Whitney test for comparisons between DSD- and DSD+. Significance levels (∗) were set at: ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: p75 NTR , Santa Cruz Biotechnology , Cat# sc-271708; RRID: AB_10714958.

Techniques: Labeling, Staining, Marker, Phospho-proteomics, MANN-WHITNEY

Journal: iScience

Article Title: Cholinotrophic basal forebrain connectome dysfunction in Down syndrome with and without dementia

doi: 10.1016/j.isci.2025.113041

Figure Lengend Snippet: FC cholinotrophic protein levels in AMC, DSD- and DSD+ (A–D) Representative immunoblots, and bar graphs show a significant upregulation of (A) proNGF and (B) p75 NTR , while (C) ChAT protein was downregulated between AMC and DSD+. (D) TrkA protein levels were stable across the groups analyzed. ACM n = 5, DSD- n = 5, DSD+ n = 13. Data are presented as mean ± SEM. Statistical significance was determined using the Kruskal–Wallis’s test followed by Dunn’s test for comparisons across three clinical groups. Significance levels (∗) were set at: ∗ p < 0.05.

Article Snippet: p75 NTR , Santa Cruz Biotechnology , Cat# sc-271708; RRID: AB_10714958.

Techniques: Western Blot

Journal: iScience

Article Title: Cholinotrophic basal forebrain connectome dysfunction in Down syndrome with and without dementia

doi: 10.1016/j.isci.2025.113041

Figure Lengend Snippet: Summary of changes in the cholinotrophic basal forebrain connectome in DS Diagrammatic sagittal view of the human brain (A) and a modified stacked bar graph (B) illustrating differences in the pathobiology of the cholinotrophic projection system between non-trisomy age-matched control (AMC), DS without dementia (DSD-) and DS with dementia (DSD+) individuals with DS. Frontal cortex protein levels for ChAT (pink), proNGF (green), p75 NTR (purple) and TrkA (light blue). Nucleus basalis NFTs of ThS (blue), AT8 (orange) and TauC3 (black) and counts of p75 NTR and ChAT (red) neurons. Created with BioRender.com .

Article Snippet: p75 NTR , Santa Cruz Biotechnology , Cat# sc-271708; RRID: AB_10714958.

Techniques: Modification, Control