Journal: The FASEB Journal

Article Title: CD47 Promotes, While Exposure to Apoptotic Cells Destroys the Fusion Program of Differentiating Myoblasts

doi: 10.1096/fj.202503809R

Figure Lengend Snippet: CD47, SIRPα, and TSP‐1 are required for proper myoblast fusion. (a) Confocal microscopic images of 3‐day differentiated C2C12 cells stained for CD47 (red) and SIRPα (green) proteins, and DNA (blue), demonstrating accumulation of both CD47 and SIRPα in the finger‐like myoblast filopodia at the contact site between the fusing cells. The white arrow indicates the contact site between neighboring myoblasts. (b) Left panel: the average TPM values for Thbs1 in proliferating and 6‐day differentiated C2C12 cells detected by RNA sequencing. Right panel: the mRNA expression level of Thbs1 in proliferating and differentiating C2C12 cells determined by qRT‐PCR. Data are expressed as mean and individual values. (** p < 0.01, *** p < 0.001, Student's t ‐test). n = 5 and n = 3 samples for RNA sequencing and qRT‐PCR, respectively. (c) Fusion indexes and representative immunofluorescence images of myosin heavy chain 4 (green) and NucBlue (blue)‐stained 6‐day differentiated C2C12 cells growing in the absence or presence of neutralizing anti‐TSP‐1 antibodies during the last three days of their differentiation. (** p < 0.01, Student's t ‐test). n = 4 samples. (d) Proposed model for the involvement of CD47 in the regulation of myoblast fusion. CD47, acting in a cis‐manner with integrins, promotes cell fusion. TSP‐1 serves as a bridging molecule for these proteins to recognize PS on the surface of the other fusing myoblast . In addition, CD47 acting in a trans‐manner activates SIRPα signaling to prevent accidental viable cell uptake.

Article Snippet: Catalog numbers of the TaqMan assays used were the following: Csnk2a2 Mm00441242_m1, Myomaker Mm00481256_m1, MyoD1 Mm00440387_m1, Myog Mm00446194_m1, Myh1 Mm01332489_m1, Cd47 Mm00495011_m1, Cmas Mm00515534_m1, Pax7 Mm00834082_m1, Thbs1 Mm00449032_g1, and Sirpa Mm00455928_m1.

Techniques: Staining, RNA Sequencing, Expressing, Quantitative RT-PCR, Immunofluorescence

Journal: Aging Cell

Article Title: Astrocyte Senescence Impairs Synaptogenesis due to Thrombospondin‐1 Loss

doi: 10.1111/acel.70382

Figure Lengend Snippet: 6‐month‐old ACSA‐2+ SAMP8 primary cultured astrocytes and differentiated astrocytes derived from SAMP8 neural stem cells display loss in TSP‐1 levels. (A) RNA‐seq transcriptional profile of Thbs1 gene in astrocytes, neurons, endothelial cells, oligodendrocytes and microglia in P7 (postnatal day) mice. (B) RNA‐seq transcriptional profile of Thbs1 gene at different stages of development and aging in astrocytes from mouse hippocampus. (C) Representative images of GFAP (green) and TSP‐1 (red) immunostaining in ACSA‐2+ primary astrocytes cultures of the SAMP8 and SAMR1 strains (14 DIV) and in Diff‐Ast SAMP8 and SAMR1 strains (11 DIV). (D, E) Quantification of TSP‐1 protein shows lower levels in SAMP8 ACSA‐2+ astrocytes cultures ( p = 0.021) and in Diff‐Ast SAMP8 ( p = 0.011) than in the control SAMR1 strain. (F) RT‐qPCR of Thbs1 ( p = 0.001) demonstrates lower gene expression levels in Diff‐Ast SAMP8 compared to the control line SAMR1. Three independent experiments per cell type were analyzed ( n = 3). Data are presented as mean ± SEM. Unpaired t ‐test was performed in (D, E). One‐sample t ‐test was done in (F). * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar: 50 μm. Data in (A) and (B) were obtained from databases produced by the Barres lab (Zhang et al. and Clarke et al. available at https://brainrnaseq.org/ ).

Article Snippet: At 11 DIV, neuron medium was totally replaced by ACM from Ast‐Diff or half replaced by ACSA‐2 + ACM during 3 h. Gabapentin (GBP, MedChemExpress, HY‐A0057) and TSP‐1 (MedChemExpress, HY‐P701325) dosages were based on Cheng et al. ( ) procedures.

Techniques: Cell Culture, Derivative Assay, RNA Sequencing, Immunostaining, Control, Quantitative RT-PCR, Gene Expression, Produced

Journal: Aging Cell

Article Title: Astrocyte Senescence Impairs Synaptogenesis due to Thrombospondin‐1 Loss

doi: 10.1111/acel.70382

Figure Lengend Snippet: The hippocampus of 10‐month‐old SAMP8 mice shows deficiencies in TSP‐1 levels. (A) RT‐qPCR of Thbs1 ( p = 0.022) in SAMR1 ( n = 7 independent animals) and SAMP8 ( n = 8 independent animals). (B) TSP‐1 quantification by ELISA test in 10‐m SAMR1 ( n = 4 independent animals) and SAMP8 ( n = 3 independent animals) mice normalized to total protein levels. (C, D) Immunohistochemical analyses and TSP‐1 intensity quantification in 10‐m SAMR1 and SAMP8 hippocampus are shown. Representative astrocytes from the SLM layer in the hippocampus using GFAP (green) as an astroglial marker, and TSP‐1 (red) are depicted magnified. TSP‐1 signal intensity quantification was performed in each layer of the hippocampus and normalized to the quantification area (μm2). Three independent animals of each strain were analyzed for immunohistochemical analysis ( n = 3). Data are presented as mean ± SEM. Unpaired t ‐test was performed. * p < 0.05 and ** p < 0.01. Scale bar: 100 μm; 10 μm (representative astrocyte).

Article Snippet: At 11 DIV, neuron medium was totally replaced by ACM from Ast‐Diff or half replaced by ACSA‐2 + ACM during 3 h. Gabapentin (GBP, MedChemExpress, HY‐A0057) and TSP‐1 (MedChemExpress, HY‐P701325) dosages were based on Cheng et al. ( ) procedures.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Marker

Journal: Aging Cell

Article Title: Astrocyte Senescence Impairs Synaptogenesis due to Thrombospondin‐1 Loss

doi: 10.1111/acel.70382

Figure Lengend Snippet: The competitive TSP‐1 receptor antagonist gabapentin (GBP) blocks the synaptogenic effect of SAMR1 ACMs. (A and C) Immunostaining of MAP2 (gray), VGLUT1 (red) and PSD95 (green), and quantification of excitatory pre‐ (VGLUT1) and postsynaptic (PSD95) vesicles colocalization in hippocampal neurons treated with ACMs from Diff‐Ast SAMR1 and SAMP8 with or without GBP 32 μM. (B and D) Immunostaining of MAP2 (gray), VGLUT1 (red) and PSD95 (green), and quantification of excitatory pre‐ (VGLUT1) and postsynaptic (PSD95) colocalization vesicles in hippocampal neurons treated with ACMs from ACSA‐2 + SAMR1 and SAMP8 of 6‐m mice with or without GBP 32 μM. Three independent experiments were analyzed per cell type and experimental condition. Data are presented as mean ± SEM. One‐way ANOVA Tukey's multiple comparisons test was performed. * p < 0.05, ** p < 0.01, and *** p < 0.001. Scale bar: 50 μm.

Article Snippet: At 11 DIV, neuron medium was totally replaced by ACM from Ast‐Diff or half replaced by ACSA‐2 + ACM during 3 h. Gabapentin (GBP, MedChemExpress, HY‐A0057) and TSP‐1 (MedChemExpress, HY‐P701325) dosages were based on Cheng et al. ( ) procedures.

Techniques: Immunostaining

Journal: Aging Cell

Article Title: Astrocyte Senescence Impairs Synaptogenesis due to Thrombospondin‐1 Loss

doi: 10.1111/acel.70382

Figure Lengend Snippet: TSP‐1 rescues the synaptogenic function of Diff‐Ast SAMP8 ACM. (A, B) Immunostaining of MAP2 (gray), VGLUT1 (red) and PSD95 (green), and quantification of excitatory pre‐ (VGLUT1) and postsynaptic (PSD95) vesicles colocalization in hippocampal neurons treated with ACM from Diff‐Ast SAMP8 with or without TSP‐1 (250 ng/mL) supplementation. (C, D) Immunostaining of MAP2 (gray), VGLUT1 (red) and PSD95 (green), and quantification of excitatory pre‐ (VGLUT1) and postsynaptic (PSD95) vesicles colocalization in hippocampal neurons treated with ACM from transfected Diff‐Ast SAMP8 overexpressing mThbs1 and GFP, or pcDNA3 and GFP as a control. Three independent experiments were analyzed per cell type and experimental condition. Data are presented as mean ± SEM. One‐way ANOVA Tukey's multiple comparisons test was performed. * p < 0.05 and ** p < 0.01. Scale bar: 50 μm.

Article Snippet: At 11 DIV, neuron medium was totally replaced by ACM from Ast‐Diff or half replaced by ACSA‐2 + ACM during 3 h. Gabapentin (GBP, MedChemExpress, HY‐A0057) and TSP‐1 (MedChemExpress, HY‐P701325) dosages were based on Cheng et al. ( ) procedures.

Techniques: Immunostaining, Transfection, Control