Journal: Aging Cell

Article Title: Deficiency of Microglial‐Derived Spp1 Exacerbates Age‐Related Memory Decline by Impairing Mitochondrial Complex I Function

doi: 10.1111/acel.70378

Figure Lengend Snippet: Microglial Spp1 deficiency led to age‐dependent memory deficits. (A) Western blot analysis of Spp1 protein in the hippocampus of young (31–43 years; mean 38.3) and old humans (88–95 years; mean 90.6) ( n = 3 per group). (B) Representative images showing the co‐localization of Spp1 and the microglial marker (IBA1) in brain sections from young and aged humans. Dashed white boxes indicate magnified images of individual cells, and donut chart point to IBA1 + Spp1 +/− cells, Scale bars = 10 μm ( n = 3 per group). (C) Representative images showing the co‐localization of Spp1 mRNA and the microglial marker (IBA1) in brain sections from young (3 M) and aged (22 M) mice. Dashed white boxes indicate magnified images of individual cells, and donut chart indicate the proportion IBA1 + Spp1 mRNA +/− cells, Scale bars = 10 μm ( n = 3 per group). (D) Representative images showing the co‐localization of Spp1 and the microglial marker (IBA1) in brain sections from young (3 M), and aged (22 M) mice. Dashed white boxes indicate magnified images of individual cells, and donut chart point to IBA1 + Spp1 +/− cells, Scale bars = 10 μm ( n = 3 per group). (E) Schematic diagram of the experimental design. (F) Percentage of correct alternations in the Y‐maze test of adult ( n = 12) and aged ( n = 13) Spp1 fl/fl mice and Spp1‐cKO mice. (G/J) Learning curves during Morris Water Maze (MWM) training for adult (G) ( n = 12) and aged (J) ( n = 13) Spp1 fl/fl and Spp1‐cKO mice, measured by latency to find the platform. (H/K) Representative path plots showing the search patterns of adult (H) and aged (K) Spp1 fl/fl and Spp1‐cKO mice during the MWM probe trial. (I) Number of platform crossings during the MWM probe trial of adult ( n = 12) and aged ( n = 13) Spp1 fl/fl mice and Spp1‐cKO mice. (L) Time spent in the target quadrant during the MWM probe trial of adult ( n = 12) and aged ( n = 13) Spp1 fl/fl mice and Spp1‐cKO mice. Data are presented as the mean ± standard error of the mean (SEM). Two‐way repeated measures ANOVA was used to compare the latency to platform acquisition during the learning curves (G/J). Other data were analyzed using unpaired two‐tailed t ‐tests. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = no significant difference. Figure was created using BioRender.

Article Snippet: The Cx3cr1 ‐CreERT2 (Stock No. C001247) and Spp1 ‐flox (Spp1 fl/fl ) (Stock No. S‐CKO‐05240) mice were obtained from Cyagen Biosciences Co. Ltd. (China).

Techniques: Western Blot, Marker, Two Tailed Test

Journal: Aging Cell

Article Title: Deficiency of Microglial‐Derived Spp1 Exacerbates Age‐Related Memory Decline by Impairing Mitochondrial Complex I Function

doi: 10.1111/acel.70378

Figure Lengend Snippet: Deficiency of Spp1 impaired microglial phagocytic function. (A) Heatmap showing differentially expressed genes (DEGs) between two groups of aged MG with high ( n = 3) and low ( n = 4) phagocytic activity. Red indicates upregulated genes, while blue indicates downregulated genes. (B) Representative images of microglial uptake of myelin‐555 in aged mice. Yellow arrows indicate Spp1‐positive microglia phagocytosing myelin‐555; blue arrows indicate Spp1‐negative microglia phagocytosing myelin‐555. Scale bars = 15 μm ( n = 6 mice per group). (C, D) Representative images showing phagocytosis of myelin‐555 by primary microglia isolated from postnatal day 3 (P3) Spp1 fl/fl and Spp1‐cKO mice; white dashed lines indicating magnified images of individual MG. Scale bars = 15 μm. (E) %Myelin + cell (of cell population) = number of phagocytic cells/total cell count ( n = 3 per group). (F) Relative myelin‐555 intensity of phagocytic cells = total fluorescence intensity/total number of phagocytic cells ( n = 3 per group). (G, H) Myelin‐555 was stereotactically injected into the hippocampus of aged Spp1 fl/fl and Spp1‐cKO mice to assess MG phagocytosis. Scale bars, 10 μm (low magnification) and 5 μm (high magnification). (I) Proportion of myelin + MG ( n = 4 per group). (J) Analysis of myelin volume within MG ( n = 4 per group). Data are presented as the mean ± standard error of the mean (SEM). Data were analyzed by unpaired two‐tailed t ‐tests. * p < 0.05, ** p < 0.01, *** p < 0.001. Mouse/Cell pattern was created using BioRender.

Article Snippet: The Cx3cr1 ‐CreERT2 (Stock No. C001247) and Spp1 ‐flox (Spp1 fl/fl ) (Stock No. S‐CKO‐05240) mice were obtained from Cyagen Biosciences Co. Ltd. (China).

Techniques: Activity Assay, Isolation, Cell Characterization, Fluorescence, Injection, Two Tailed Test

Journal: Aging Cell

Article Title: Deficiency of Microglial‐Derived Spp1 Exacerbates Age‐Related Memory Decline by Impairing Mitochondrial Complex I Function

doi: 10.1111/acel.70378

Figure Lengend Snippet: Deficiency of Spp1 disrupted microglia oxidative phosphorylation. (A) Heatmap of GSVA analysis comparing gene set enrichment between primary microglia isolated from postnatal day 3 (P3) Spp1 fl/fl and Spp1‐cKO mice; blue indicates downregulation, and red indicates upregulation. (B) Quantification of ATP levels in primary microglia isolated from postnatal day 3 (P3) Spp1 fl/fl and Spp1‐cKO mice ( n = 6 per group). (C) Measurement of Seahorse XF mitochondrial stress OCR in primary microglia from postnatal day 3 (P3) Spp1 fl/fl and Spp1‐cKO mice. (D) Basal OCR ( n = 3 per group). (E) Maximum OCR ( n = 3 per group). (F) Heatmap of GSVA pathway scores showing significant downregulation of Oxidative Phosphorylation in the microglial cluster of aged (18‐month‐old) Spp1‐cKO mice compared to controls. (G) Subsets of high SPP1‐expressing (SPP1 high MG) and low SPP1‐expressing (SPP1 low MG) in aged human brains ( GSE157827 ). (H) Oxidative phosphorylation pathway scores in SPP1 high and SPP1 low MG in aged human brains. (I) Violin plot showing AUCell scores for SPP1 high MG and SPP1 low MG cells. Data are presented as the mean ± standard error of the mean (SEM). Statistical significance was determined using unpaired two‐tailed t ‐tests for (B, D, E) and Wilcoxon rank‐sum test for (I). FCCP, Carbonyl cyanide 4‐trifluoromethoxyphenylhydrazone; Anti & Rot, Antimycin A and Rotenone; 2‐DG, 2‐Deoxy‐D‐glucose. * p < 0.05, ** p < 0.01, ns = no significant difference. Human/Mouse/Cell pattern was created using BioRender.

Article Snippet: The Cx3cr1 ‐CreERT2 (Stock No. C001247) and Spp1 ‐flox (Spp1 fl/fl ) (Stock No. S‐CKO‐05240) mice were obtained from Cyagen Biosciences Co. Ltd. (China).

Techniques: Phospho-proteomics, Isolation, Expressing, Two Tailed Test

Journal: Aging Cell

Article Title: Deficiency of Microglial‐Derived Spp1 Exacerbates Age‐Related Memory Decline by Impairing Mitochondrial Complex I Function

doi: 10.1111/acel.70378

Figure Lengend Snippet: Deficiency of Spp1 in microglia disrupted mitochondrial complex I‐dependent oxidative phosphorylation. (A) Heatmap showing changes in key OXPHOS‐related genes in primary microglia isolated from postnatal day 3 (P3) Spp1 fl/fl and Spp1‐cKO mice. (B) Workflow diagram of the Oroboros O2k multi‐dimensional energy metabolism analysis system used to measure OCR in BV2 cell homogenates. (C) Basal OCR in BV2 cells ( n = 3 per group). (D‐F) Mitochondrial respiration states in BV2 cells: Complex I respiration (D), complex II respiration (E), and complex IV respiration (F) ( n = 3 per group). (G) Histogram showing the relative mitochondrial complex I enzyme activity ( n = 4 per group). (H) Western blot analysis showing changes in protein expression levels of Spp1 and Ndufs2 after Spp1 knockdown in BV2 cells ( n = 3 per group). (I) Workflow diagram of the Oroboros O2k multi‐dimensional energy metabolism analysis system used to measure OCR in hippocampal tissue from Spp1 fl/fl and Spp1‐cKO mice. (J) Basal OCR in hippocampal tissue from Spp1 fl/fl and Spp1‐cKO mice ( n = 4 per group). (K–M) Mitochondrial respiration states in hippocampal tissue from Spp1 fl/fl and Spp1‐cKO mice: Complex I respiration (K), complex II respiration (L), and complex IV respiration (M) ( n = 4 per group). (N) Schematic diagram showing Spp1 deficiency disrupting OXPHOS via compromised complex I activity. Data are presented as the mean ± standard error of the mean (SEM). Data were analyzed by unpaired two‐tailed t ‐tests. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = no significant difference. Mouse patterns and experimental apparatus were created using BioRender.

Article Snippet: The Cx3cr1 ‐CreERT2 (Stock No. C001247) and Spp1 ‐flox (Spp1 fl/fl ) (Stock No. S‐CKO‐05240) mice were obtained from Cyagen Biosciences Co. Ltd. (China).

Techniques: Phospho-proteomics, Isolation, Activity Assay, Western Blot, Expressing, Knockdown, Two Tailed Test

Journal: Aging Cell

Article Title: Deficiency of Microglial‐Derived Spp1 Exacerbates Age‐Related Memory Decline by Impairing Mitochondrial Complex I Function

doi: 10.1111/acel.70378

Figure Lengend Snippet: Spp1 deficiency inhibited AKT signaling pathway and administration of AKT agonist reversed manifestation. (A) KEGG pathway analysis of differentially expressed genes from sequencing data of primary microglia isolated from postnatal day 3 (P3) Spp1 fl/fl and Spp1‐cKO mice. (B) Heatmap showing GSVA enrichment scores for the “PI3K‐Akt signaling” pathway in primary microglia isolated from postnatal day 3 (P3) Spp1 fl/fl and Spp1‐cKO mice. Each column represents one RNA‐seq sample (red indicating up‐regulation, and blue indicating down‐regulation). (C) scRNA‐seq analysis showing the downregulation of PI3K–Akt signaling pathway in the microglial cluster of 18‐month‐old Spp1‐cKO mice compared to controls. (D) Western blot analysis showing the changes in protein expression levels of p‐AKT, AKT after Spp1 knockdown in BV2 cells ( n = 3 per group). (E) ATP levels ( n = 4 per group). (F) Mitochondrial complex I‐dependent oxygen consumption rate (OCR) ( n = 3 per group). (G) Histogram showing the relative mitochondrial complex I enzyme activity ( n = 4 per group). (H) %Myelin + cell (of cell population) = number of phagocytic cells/total cell count ( n = 4 per group). (I) ATP levels in the hippocampus of Spp1 fl/fl + solvent, Spp1‐cKO + solvent, and Spp1‐cKO + SC79 mice ( n = 3 per group). (J) Western blot analysis showing the changes in protein expression levels of p‐AKT, AKT, and Ndufs2 ( n = 3 per group). (K) Percentage of correct alternations in the Y‐maze test. (L) Learning curves during MWM training, measured by latency to find the platform. (M) Time spent in the target quadrant during the probe phase of the MWM. Data are presented as the mean ± standard error of the mean (SEM). Two‐way repeated measures ANOVA was used to compare the latency to find the platform in the learning curves (L). Figure D was analyzed using two‐tailed t ‐tests. Other data were analyzed using one‐way ANOVA and LSD post hoc test. * p < 0.05, ** p < 0.01, ns = no significant difference. Mouse pattern was created using BioRender.

Article Snippet: The Cx3cr1 ‐CreERT2 (Stock No. C001247) and Spp1 ‐flox (Spp1 fl/fl ) (Stock No. S‐CKO‐05240) mice were obtained from Cyagen Biosciences Co. Ltd. (China).

Techniques: Sequencing, Isolation, RNA Sequencing, Western Blot, Expressing, Knockdown, Activity Assay, Cell Characterization, Solvent, Two Tailed Test

Journal: Aging Cell

Article Title: Deficiency of Microglial‐Derived Spp1 Exacerbates Age‐Related Memory Decline by Impairing Mitochondrial Complex I Function

doi: 10.1111/acel.70378

Figure Lengend Snippet: Microglia‐specific Spp1 overexpression restores ATP production, phagocytosis, and memory in Spp1‐deficient models. (A) Representative confocal images of Spp1 immunofluorescence in BV2 cells: Control (NC), Spp1 knockdown (sh‐Spp1), and sh‐Spp1 rescued by AAV‐Spp1. Scale bars = 10 μm. (B) Quantification of relative Spp1 fluorescence intensity ( n = 3 per group). (C) Representative bright‐field/fluorescence overlays of myelin‐555 uptake (red) by BV2 cells under the three conditions. Scale bars = 10 μm. (D) %Myelin + cell (of cell population) = number of phagocytic cells/total cell count ( n = 3 per group). (E) ATP levels normalized to total protein in control (NC), Spp1 knockdown (sh‐Spp1), and sh‐Spp1 cells rescued by AAV‐Spp1 ( n = 3 per group). (F) Mitochondrial complex I activity in control (NC), Spp1 knockdown (sh‐Spp1), and sh‐Spp1 cells rescued by AAV‐Spp1 ( n = 3 per group). (G) Top: Schematic diagram illustrating the experimental strategy for microglia‐specific Spp1 overexpression. Bottom: Relative fluorescence intensity of Spp1 immunostaining in IBA1 + microglia ( n = 3 mice per group). (H) Representative immunofluorescence images showing the expression of AAV‐driven EGFP in IBA1 + microglia in the hippocampus. Scale bars = 10 μm. (I) Y‐maze spontaneous alternation rate ( n = 8 mice per group). (J) Learning curves during MWM training, measured by latency to find the platform ( n = 8 mice per group). (K) Representative path plots showing the search patterns during the MWM probe trial. (L) Time spent in the target quadrant during the probe phase of the MWM. Data are presented as the mean ± standard error of the mean (SEM). Two‐way repeated measures ANOVA was used to compare the latency to find the platform in the learning curves (J). Other data were analyzed using one‐way ANOVA and LSD post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001. Brain pattern was created using BioRender.

Article Snippet: The Cx3cr1 ‐CreERT2 (Stock No. C001247) and Spp1 ‐flox (Spp1 fl/fl ) (Stock No. S‐CKO‐05240) mice were obtained from Cyagen Biosciences Co. Ltd. (China).

Techniques: Over Expression, Immunofluorescence, Control, Knockdown, Fluorescence, Cell Characterization, Activity Assay, Immunostaining, Expressing

Journal: Aging Medicine

Article Title: Visualization of OPN ‐Targeted Self‐Assembled Micelles Encapsulated With an SIRT6 Activator and Doped Indocyanine Green for Attenuating Vascular Aging

doi: 10.1002/agm2.70045

Figure Lengend Snippet: In vivo antiaging effect and mechanism. (A, D) Pulse wave velocity and quantitative analysis to evaluate the stiffness of arteries. (B, C, E, F) HE and EVG staining and quantitative analysis to evaluate the intima‐media thickness and elastin changes of arteries. (G–I) P16 and p21 expression and quantitative analysis to evaluate senescence of arteries. (J–N) Expression of MMP9, SIRT6, H3K9ac, and H3K56ac after OPN‐MI micelles intervention. Con, control mice injected with PBS; Aged, aged mice injected with PBS; A + MI, aged mice injected with MI micelles; A + OPN‐MI, aged mice injected with OPN‐MI micelles. ( n = 6; * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The primary antibodies used were OPN (88742; CST), SIRT6 (12486; CST), MMP9 (ab283575; Abcam), GAPDH (2118; CST), p21 (37543; CST), p16 (29271; CST), H3K9ac (9649; CST), H3K56ac (MA5‐32138; Invitrogen), and β‐actin (4970; CST).

Techniques: In Vivo, Staining, Expressing, Control, Injection

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: Cellchat cell-cell communication analysis results. a) Outgoing signal heatmap. The intensity of color represents higher communication probability. b) incoming signal heatmap. The intensity of color represents higher communication probability. c) All outgoing signaling network from MGD TAMs. e) Network topology heatmap of SPP1. f) Violin plots of SPP1 transcript abundance across all clusters.

Article Snippet: Recent work showed that using an anti-SPP1 antibody (bioXcell Clone: 100D3) and (clone MPIIIB10) in combination with mIL13Rα2 CAR T cell therapy shows enhanced CAR T cell antitumor response [ ].

Techniques:

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: pySCENIC results highlight that no one TF dominates SPP1 gene expression regulation in MGD TAM. a) TF Regulon activity profile of SPP1 transcription factors, here size of the dot shows regulon activity and color shows gene expression correlation of the corresponding TF with SPP1 gene expression. b) TF gene expression dot plot. The size of the dot represents number of cells expressing corresponding TF in each cell type population, and the color represents average expression of the gene within the cell type population, c) MAFB gene expression feature plot showing expression of this gene across all cell types. d) MAFB violin plot showing expression of this gene across all cell types.

Article Snippet: Recent work showed that using an anti-SPP1 antibody (bioXcell Clone: 100D3) and (clone MPIIIB10) in combination with mIL13Rα2 CAR T cell therapy shows enhanced CAR T cell antitumor response [ ].

Techniques: Gene Expression, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: SPP1 protein structure and sequence analysis results. a) Root mean squared fluctuation of SPP1 protein residues under replica exchange molecular dynamics simulation starting at temperatures 283.15K (black), 303.15K (red), 333.15K (green), and 353.15K (blue). 8, b) Energy landscape-based conformation sampling from four replicate exchange runs. Here y-axis represents radius of gyration values across all 4 replicates and x axis represents RMSD values across all 4 replicates. c) Most stable SPP1 conformation across all 4 replica exchange trajectories. CD44 binding motif (residue 121-140) is shown in orange, 23C3 binding motif is shown in blue, and 2K1 and C2K1 binding motif is shown in cyan. and known SPP1 antibody binding interface shown in blue, and cyan, d) SPP1 protein residue phosphorylation score heatmap. Arrow highlights the top computationally predicted likely phosphorylation site 169 with score > 0.8, e) Sequence alignment of SPP1 across mammals. Orange bar highlights SPP1-CD44 binding motif, blue bar highlights 23C3 binding motif, cyan bar highlights 2K1 and C2K1 binding motif, and red arrow represent phosphorylation site 169 on SPP1 protein sequence. f) Root mean squared fluctuation quadratic mean of normal SPP1 (black) and residue 169 phosphorylated SPP1 (red) across all three independent MD simulation replicates, showing increase in stability in SPP1-CD44 binding interface upon phosphorylation.

Article Snippet: Recent work showed that using an anti-SPP1 antibody (bioXcell Clone: 100D3) and (clone MPIIIB10) in combination with mIL13Rα2 CAR T cell therapy shows enhanced CAR T cell antitumor response [ ].

Techniques: Sequencing, Sampling, Binding Assay, Residue, Phospho-proteomics

Journal: bioRxiv

Article Title: Antibody-Based Targeting of the SPP1-CD44 Axis in Pediatric High-Grade Glioma through Single-Cell and Structural Bioinformatics

doi: 10.1101/2025.05.01.651763

Figure Lengend Snippet: Antibody directed evolution results obtained from RosettaAntibodyDesign tool. a) UMAP showing 2500 new antibodies shown as colored dots and the parental 23C3 antibody shown as blast star. Color of the dots highlight energy required to break interaction between SPP1 and the corresponding antibody variant. UMAP is calculated using cosine distance between amino acid sequence esm2 embeddings of CDR regions of two antibody variants. b) Hex plot showing 2500 antibody variants. Here energy required to break SPP1 binding with the corresponding antibody variant is shown as x axis, and cosine distance of amino acid sequence esm2 embeddings of CDR regions of an antibody variant with parental 23C3 amino acid sequence esm2 embeddings of CDR regions. c) Most stable variant (23C3-v1) protein structure (grey) bonded to SPP1 protein structure (wheat). Blue spheres are the residue changes in 23C3-v1 as compared to 23C3 amino acid sequence. d) 23C3-v1 heavy chain amino acid sequence aligned to 10 humanized antibody heavy chain amino acid sequence. Class I and class II epitope sequence region is highlighted using black line and corresponding residue location, e) 23C3-v1 light chain amino acid sequence aligned to 10 humanized antibody light chain amino acid sequence. Class II epitope sequence region is highlighted using black line and corresponding residue location, f) Heavy chain (light green) mutations (red spheres), and light chain (cyan) mutations (blue spheres) induced in 23C3-v1 to neutralize class I and class II epitope. Heavy chain residues 62-68 are shown as green spheres, g) Three independent MD simulation replicates RMSF quadratic mean of 23C3-v1 (black) and Hu23C3-v1 (red) heavy chain, showing no significant change across SPP1 binding region of heavy chain. A sudden increase in fluctuation was observed in residue 62-68, which is distant and upward from SPP1 binding region (shown as green spheres in ), h) Three independent MD simulation replicates RMSF quadratic mean of 23C3-v1 (black) and humanized 23C3-v1 (Hu23C3-v1) (red) light chain, showing no significant change across SPP1 binding region of light chain, i) Box-whiskers plot of 23C3-v1 and Hu23C3-v1 heavy and light chain binding affinity with SPP1 across all three independent MD simulation replicates calculated using gmx_MMPBSA, shows no significant change in Hu23C3-v1 as compared to 23C3-v1 upon epitope neutralization.

Article Snippet: Recent work showed that using an anti-SPP1 antibody (bioXcell Clone: 100D3) and (clone MPIIIB10) in combination with mIL13Rα2 CAR T cell therapy shows enhanced CAR T cell antitumor response [ ].

Techniques: Variant Assay, Sequencing, Binding Assay, Residue, Neutralization