Journal: bioRxiv

Article Title: Cell surface markers identify astrocyte subpopulations in the adult hippocampus with a heterogeneous response to aging

doi: 10.1101/2025.11.07.685575

Figure Lengend Snippet: (a) Diagram of the procedure followed for the isolation of astrocytes. Hippocampi (and forebrain as a control) were dissected from C57BL/6JRcc mice at 2 and 18 months of age. Cell suspensions were obtained by enzymatic digestion and mechanic dissociation. Astrocytes were labeled with GLAST-ACSA-1-PE and ACSA-2-FITC antibodies, sorted by FACS and RNA was sequenced using Illumina. (b) Quantification of astrocyte subpopulations in hippocampus of young (Y, 2m) and old wild-type mice (O, 18m) and forebrain of young mice by FACS. Frequency of single- and double-positive astrocytes (A + and A + /G + ) in the two brain regions are shown as percentages (n≥3). Bars indicate mean ± SEM. Unpaired t-test was performed. * p < 0.05, ** p < 0.01. (c) Representative FACS dot plots of ACSA-2 and GLAST-ACSA-1 expression in young and old hippocampus and in young forebrain. For complete gating strategies, see Supplementary Figure 2. (d) Gene expression heatmap of the different central nervous system cell type markers. Orange, high expression and white, no expression. (e-f) Overlap of top 500 expressed genes in A + /G + and A + population, respectively, with published mouse astrocyte gene sets ( ; Borggrewe et al., 2020; ) visualized in a Venn diagram. (g) Mean expression of genes involved in lactate metabolism, myelination, blood brain barrier, and cholesterol synthesis of published dataset (Borggrewe et al., 2020), illustrated as z-scores per group (n≥3). For lists of genes see Supplementary Table 4. (h) Mean expression of genes associated with astrocyte subtypes of published dataset illustrated as z-score per group (n≥3). For list of genes see Supplementary Table 5. Representation shown in (a) was created with BioRender.com.

Article Snippet: Primary antibodies used for the staining were as follows: ATP1B2 (1:300, Alomone Labs, ANP-012), GLAST (ACSA-1) biotin (1:50, Miltenyi, 130-119-161), S100β (1:250, Synaptic Systems, 287004), GFAP (1:1000, Synaptic Systems, 173004), VGLUT1 (1:500, GeneTex, GTX133148), PSD95 (1:200, Fisher, MA1045), Anti-Pan-gamma-protocadherin (10μg, NeuroMab, clone N159/5) and Anti-gamma-protocadherin-C3 (10μg, NeuroMab, clone N174B/27).

Techniques: Isolation, Control, Labeling, Expressing, Gene Expression

Journal: bioRxiv

Article Title: Cell surface markers identify astrocyte subpopulations in the adult hippocampus with a heterogeneous response to aging

doi: 10.1101/2025.11.07.685575

Figure Lengend Snippet: (a) Box and whiskers plot of RNAseq data showing VST normalized counts of Pcdhga3 , Pcdhga7, Pcdhga8 and Pcdhgc3 genes in Y-A + /G + and O-A + /G + astrocytes. Values were compared with the DESeq2 package. (b) Bar plots of RNAseq data showing FPKM expression of four protocadherin-related genes ( Pcdhga3 , Pcdhga7 , Pcdhga8 , and Pcdhgc3 ) in cortical astrocytes and neurons. FPKM, fragments per kilobase of exon per million mapped fragments. (c) Schematic diagram showing the experimental strategy for isolation of astrocytes (ACSA-2 + ) from the hippocampus of young (Y, 2m) and old (O, 18-24m) wild-type mice by MACS and subsequent culture in vitro . (d) Representative immunostaining of GLAST (green), ATP1B2 (red) and S100β (white) in ACSA-2 + astrocytes from young (2m) adult mice, cultured during 14 DIV. Scale bar = 25 µm. (e) Quantification of A + /G + and A + astrocytes in young hippocampal astrocyte primary cultures at 14DIV. Percentage of S100β + /ATP1B2 + /GLAST + (A + /G +) and S100β + /ATP1B2 + /GLAST - (A + ) astrocytes is shown as mean ± SEM. (f) Representative immunofluorescence images of GFAP (red) and γ-Pcdh-C3 (green) in Y-A + /G + (2m) and O-A + /G + (24m) astrocytes at 14 DIV. Scale bar = 15 µm. (g) γ-Pcdh-C3 mean fluorescence quantification in Y-A + /G + and O-A + /G + astrocytes. Data is shown as mean ± SEM. Paired t-test was performed. (h) Schematic diagram showing the experimental procedure followed to obtained neurons (E18) and astrocytes (2 and 18m) for the heterochronic co-culture. (i) Immunostaining of GFAP (white), VGLUT1 (green) and PSD95 (red) in neurons co-cultured with young or old astrocytes at 8 DIV. Scale bar = 25 µm. (j) Quantification of excitatory pre-(VGLUT1) and postsynaptic (PSD95) vesicles colocalization in hippocampal neurons co-cultured with hippocampal young (2m) and old (18m) astrocytes. Three independent experiments were performed (n=3). Data are presented as mean ± SEM. One-sample t-test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Data in (b) were obtained from the database produced by the Barres lab ( available at https://brainrnaseq.org/ ). Representations shown in (c) and (h) were created with BioRender.com.

Article Snippet: Primary antibodies used for the staining were as follows: ATP1B2 (1:300, Alomone Labs, ANP-012), GLAST (ACSA-1) biotin (1:50, Miltenyi, 130-119-161), S100β (1:250, Synaptic Systems, 287004), GFAP (1:1000, Synaptic Systems, 173004), VGLUT1 (1:500, GeneTex, GTX133148), PSD95 (1:200, Fisher, MA1045), Anti-Pan-gamma-protocadherin (10μg, NeuroMab, clone N159/5) and Anti-gamma-protocadherin-C3 (10μg, NeuroMab, clone N174B/27).

Techniques: Expressing, Isolation, In Vitro, Immunostaining, Cell Culture, Immunofluorescence, Fluorescence, Co-Culture Assay, Produced

Journal: Communications Biology

Article Title: 1,1-Diethoxyethane enhances aerobic respiration in human mitochondria via activation of AMP-activated protein kinase

doi: 10.1038/s42003-026-09797-3

Figure Lengend Snippet: a Exposure of AC16 cells to 1-DEE (15 mM) for 30 min ( n = 3). b Exposure of WT and AMPK DKO MEF cells to 1,1-DEE (15 mM) for 30 min ( n = 3). c Pretreatment of AC16 cells with compound C (10 µM) for 4 h, followed by incubation with 1,1-DEE (15 mM) for 60 min ( n = 3). Western blot and quantitative analyses of AMPK, ACC, and PFKFB2 phosphorylation were performed. d , e Exposure of AC16 cells to 1,1-DEE (15 mM) for 8 h. The mRNA expression levels of SREBF1 and FASN were assessed by semiquantitative PCR ( d ) and qRT-PCR ( n = 3) ( e ). Data are presented as the mean ± SEM. Statistical significance was determined using one-way ANOVA ( a, e ) and two-way ANOVA ( c ): ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

Article Snippet: The following antibodies were used: AMPK (1:1000; #2532), phospho-AMPK (Thr172) (1:1000; #2535), ACC (1:1000; #3676), phospho-ACC (Ser79) (1:1000; #11818), PFKFB2 (1:1000; #13029), phospho-PFKFB2 (Ser483) (1:1000; #13064), PGC-1α (1:1000; #2178), NRF1 (1:1000; #46743), NRF2 (1:1000; #12721), β-actin (1:1000; #4970), anti-rabit IgG (1:5000; #7074) from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Incubation, Western Blot, Phospho-proteomics, Expressing, Quantitative RT-PCR

Journal: Communications Biology

Article Title: 1,1-Diethoxyethane enhances aerobic respiration in human mitochondria via activation of AMP-activated protein kinase

doi: 10.1038/s42003-026-09797-3

Figure Lengend Snippet: 1,1-DEE treatment can reversibly activate AMPK. Acute activation of AMPK is caused by the docking of 1,1-DEE at the ubiquinone site of hRCI, which transiently suppresses mitochondrial respiration, reduces ATP production, and increases ROS generation. AMPK activation is responsible for both the acute response (within 30 min) via post-translational phosphorylation of ACC and PFKFB2, promoting fatty acid oxidation and glycolytic activity, and the prolonged effect (within 8 h) via upregulation of the PGC-1α/TFAM signaling pathway for enhancing mitochondrial biogenesis and respiration, and via increase NRF2 expression and nuclear accumulation for upregulating antioxidant defense. Created in BioRender. Lee, S. (2026) https://BioRender.com/qo0brn4 .

Article Snippet: The following antibodies were used: AMPK (1:1000; #2532), phospho-AMPK (Thr172) (1:1000; #2535), ACC (1:1000; #3676), phospho-ACC (Ser79) (1:1000; #11818), PFKFB2 (1:1000; #13029), phospho-PFKFB2 (Ser483) (1:1000; #13064), PGC-1α (1:1000; #2178), NRF1 (1:1000; #46743), NRF2 (1:1000; #12721), β-actin (1:1000; #4970), anti-rabit IgG (1:5000; #7074) from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Phospho-proteomics, Activity Assay, Expressing