Journal: Nature Communications

Article Title: Targeting osteoblastic 11β-HSD1 to combat high-fat diet-induced bone loss and obesity

doi: 10.1038/s41467-024-52965-4

Figure Lengend Snippet: a , b Osteogenic activity of MC3T3-E1 osteoblastic cells with 11β-HSD1 overexpression (MC3T3-HSD1 cells) and transfected control cells (MC3T3-GFP cells). a Alkaline phosphatase (ALP) and alizarin red staining. b The mRNA expression of RUNX family transcription factor 2 ( Runx2 ) and bone gamma-carboxyglutamate protein ( Bglap ). c , d Combination analysis of RNA sequencing (RNA-seq) and assays for transposase-accessible chromatin sequencing (ATAC-seq) in MC3T3-GFP cells and MC3T3-HSD1 cells. c Intersection of differential genes from RNA-seq and ATAC-seq. d Biological processes associated with Egr2. e The mRNA and protein expression of Egr2 in MC3T3-GFP cells and MC3T3-HSD1 cells. f The mRNA expression of Egr2 during osteogenic differentiation. g , h The osteogenic activity of MC3T3-HSD1 cells with Egr2 overexpressing. g Alizarin red staining. h Bglap mRNA expression. i The glucose uptake test of MC3T3-GFP cells and MC3T3-HSD1 cells. j , k The mRNA expression of key glucose transporter proteins and components of insulin-dependent glucose uptake pathway in MC3T3-GFP cells and MC3T3-HSD1 cells. j Glucose transporter type 1 (Glut1) , 3 (Glut3) and 4 (Glut4) . k: Insulin receptor (Ir), Insulin receptor substrate 1 (Irs1), Phosphatidylinositol-3-kinase catalytic subunit regulatory subunit 1 (Pik3r), alpha (Pik3ca) and subunit beta (Pik3cb) . l , m The glucose uptake test and mRNA expression of Glut4 and Pik3cb in MC3T3-HSD1 cells with Egr2 overexpressing. l Glucose uptake test. m Glut4 and Pik3cb . n Dual-luciferase reporter analysis and ChIP quantitative polymerase chain reaction (ChIP-qPCR) analysis of Ege2 target interactions for Pik3cb and Glut4 gene promotors. n Pik3cb and Glut4 gene promotors. o ChIP-qPCR analysis. p Pik3cb and Glut4 gene promotors with different mutations. Note: Data were presented as mean value ± SEM for ( a , b , e – m , p ). n = 3 biologically independent samples for RNA-seq, ATAC-seq, western blot analysis, glucose uptake test and osteogenic staining; n = 6 biologically independent samples for RT-qPCR analysis. Statistical significance was calculated using two-tailed Student’s t-test ( e ), one-way ANOVA followed by Tukey’s post-hoc test ( n , p ), and two-way ANOVA followed by a two-stage step-up method by Benjamini, Krieger and Yekutieli ( a , b , f – m ) to adjust for multiple comparisons. All tests were two-sided.

Article Snippet: Following blocking with 5% nonfat milk in TBST, the membrane was incubated overnight at 4 °C with primary antibodies (1:500) against Actin (4967, Cell Signaling, USA), p110β (ab151549, abcam, UK), AKT (4691 T, Cell Signaling, USA), phosphorylated AKT (4060 T, pAKT, Cell Signaling, USA), Glut4 (21619, SAB, USA), 11β-HSD1 (AF3397, R&D system, USA), Egr2 (EPR4004, Abcam, UK), H6PDH (EPR12338, Abcam, UK).

Techniques: Activity Assay, Over Expression, Transfection, Control, Staining, Expressing, RNA Sequencing Assay, Sequencing, Luciferase, Real-time Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting osteoblastic 11β-HSD1 to combat high-fat diet-induced bone loss and obesity

doi: 10.1038/s41467-024-52965-4

Figure Lengend Snippet: a The mRNA expression of glucocorticoid target gene Glucocorticoid-induced leucine zipper ( Gilz ) in MC3T3-GFP cells and MC3T3-HSD1 cells after treating with 11β-HSD1 inhibitor (AZD8329). b , c The mRNA and protein expression of early growth response 2 ( Egr2 ) in MC3T3-GFP cells and MC3T3-HSD1 cells after treatment of AZD8329. b The mRNA expression. c The protein expression. d The mRNA expression of Egr2 in MC3T3-GFP cells and MC3T3-HSD1 cells after treating with AZD8329 during osteogenic differentiation. e – h The osteogenic activity of MC3T3-GFP cells and MC3T3-HSD1 cells after treatment of AZD8329. e Alizarin red staining. f Bone gamma-carboxyglutamate protein (Bglap) mRNA expression. g Runt-related transcription factor 2 (Runx2) mRNA expression. h Osterix mRNA expression. i – j The glucose uptake test and mRNA expression levels of Glucose transporter type 4 (Glut4) and Phosphatidylinositol-3-kinase catalytic subunit beta (Pik3cb) in MC3T3-GFP cells and MC3T3-HSD1 cells after treatment of AZD8329. g: Glucose uptake test. h Glut4 and Pik3cb mRNA expression. Note: Data were presented as mean value ± SEM for ( a , b , d – j ). n = 3 biologically independent samples for osteogenic staining, western blot analysis and LC-MS/MS analysis, n = 6 biologically independent samples for RT-qPCR analysis. Statistical significance was calculated using one-way ANOVA followed by Tukey’s post-hoc test ( a – c ), and two-way ANOVA followed by a two-stage step-up method by Benjamini, Krieger and Yekutieli ( d – j ) to adjust for multiple comparisons. All tests were two-sided.

Article Snippet: Following blocking with 5% nonfat milk in TBST, the membrane was incubated overnight at 4 °C with primary antibodies (1:500) against Actin (4967, Cell Signaling, USA), p110β (ab151549, abcam, UK), AKT (4691 T, Cell Signaling, USA), phosphorylated AKT (4060 T, pAKT, Cell Signaling, USA), Glut4 (21619, SAB, USA), 11β-HSD1 (AF3397, R&D system, USA), Egr2 (EPR4004, Abcam, UK), H6PDH (EPR12338, Abcam, UK).

Techniques: Expressing, Activity Assay, Staining, Western Blot, Liquid Chromatography with Mass Spectroscopy, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Targeting osteoblastic 11β-HSD1 to combat high-fat diet-induced bone loss and obesity

doi: 10.1038/s41467-024-52965-4

Figure Lengend Snippet: a Design of the bone-targeted 11β-HSD1 inhibitor, (DSS) 6 -AZD8329. b , c Organ distribution and skeletal distribution of (DSS) 6 -AZD8329 ( n = 3) and (RKK) 6 -AZD8329 ( n = 3). b The organ distribution. c The skeletal distribution, scale bar=20 μm. d Experimental design of wild-type mice treated with (DSS) 6 -AZD8329 and (RKK) 6 -AZD8329. e – g The micro-CT analysis and bone histometric analysis. e The trabecular bone and cortical bone microstructure. f The percentage change of trabecular bone volume/total volume (Tb. BV/TV) and trabecular bone density (Tb. v. BMD). g The percentage change of cortical bone density (Ct. v. BMD). h The calcein double labeling, scale bar=20 μm. i The percentage change of mineral apposition rate (MAR), bone formation rate per bone surface (BFR/BS) and the number of osteoblasts per bone surface (N.Ob/BS). j – k Weight gain and energy intake during HFD. * P < 0.05, ** P < 0.01, *** P < 0.001 when other groups vs. HFD+vehicle group. j Weight gain. k Energy intake. l Weights and representative photographs of gonadal white adipose tissues (gWAT). m – o Glucose handling tests ( n = 8 for two vehicle-treated groups, n = 12 for two drug-treated groups). * P < 0.05, ** P < 0.01, *** P < 0.001 when other groups vs. HFD + vehicle group. m Fasting blood glucose. n: Insulin tolerance test (ITT). o Oral glucose tolerance test (oGTT). p Glucose uptake tests ( n = 8 for two vehicle-treated groups, n = 12 for two drug-treated groups). Left: Representative images of glucose uptake. Right: Quantitative analysis of glucose uptake into liver, WAT, muscle and bone. q Skeletal mRNA expression of Hsd11b1 , Gilz , Egr2 , Pik3cb and Glut4 . Note: Data were presented as mean value ± SEM for ( f , g , i – q ). Chow+vehicle ( n = 8), mice with Chow feeding and start administration of vehicle since week 8; HFD+vehicle ( n = 8), HFD + (RKK) 6 -AZD8329 ( n = 13), HFD + (DSS) 6 -AZD8329 ( n = 13), mice with HFD feeding and start administration of vehicle, (RKK) 6 -AZD8329, or (DSS) 6 -AZD8329, respectively, since week 8. All samples are biologically independent samples. Statistical significance was calculated using one-way ANOVA followed by Tukey’s post-hoc test ( f – g , i , l , m – q ) and two-way ANOVA followed by Sidak’s multiple comparisons test ( j – k , n – o ). All tests were two-sided.

Article Snippet: Following blocking with 5% nonfat milk in TBST, the membrane was incubated overnight at 4 °C with primary antibodies (1:500) against Actin (4967, Cell Signaling, USA), p110β (ab151549, abcam, UK), AKT (4691 T, Cell Signaling, USA), phosphorylated AKT (4060 T, pAKT, Cell Signaling, USA), Glut4 (21619, SAB, USA), 11β-HSD1 (AF3397, R&D system, USA), Egr2 (EPR4004, Abcam, UK), H6PDH (EPR12338, Abcam, UK).

Techniques: Micro-CT, Labeling, Expressing

Journal: Nature

Article Title: CRISPR–Cas9 screens reveal regulators of ageing in neural stem cells

doi: 10.1038/s41586-024-07972-2

Figure Lengend Snippet: a , In vivo screen platform in old mice. Old (20–22 months) Cas9 mice were injected with lentivirus expressing sgRNAs into the lateral ventricles (in proximity to the SVZ NSC niche). Five weeks after injection, genomic DNA was collected from the olfactory bulb (OB), and sgRNAs were sequenced to determine enrichment or depletion. b , Immunofluorescence images of SVZ niche sections from old (21 months) Cas9 mice, 48 h after injection of lentiviruses expressing mCherry and 100 control sgRNAs. One experiment. mCherry (lentivirus-infected cells, red), GFAP (NSCs and astrocytes, magenta), Ki67 (proliferative cells, green), DAPI (nuclei, blue). Right, high-magnification images. Scale bars, 100 µm (left); 10 µm (right). c , d , Normalized sgRNA counts of the top 10 gene pool (50 sgRNAs) and control (100 sgRNAs) libraries in various brain regions, 1 day ( c ) or 5 weeks ( d ) after virus injection. Top 10 gene pool, gene knockouts that boosted old NSC activation in in vitro screens 1 and 2. sgRNA counts were normalized to total counts between brain regions to account for sequencing depth variance. e , CasTLE gene scores for 50 gene knockouts tested in groups of 10 in old (19–22 months) female Cas9 mice, analysing for enrichment or depletion in the OB. Each sgRNA library of 10 genes was injected into n = 4 old Cas9 mice, n = 1 mouse for 1 day after injection SVZ sequencing of starting sgRNA pool, and n = 3 mice for 5 weeks after injection sequencing of OB sgRNAs. OB sgRNA enrichment computed with CasTLE and comparing with the 24-h SVZ sequenced sample. Mean ± s.e.m. of CasTLE score in n = 3 mice. Each dot represents the gene score from one mouse. Asterisks indicate gene hits with a 95% confidence interval that did not contain 0 by CasTLE analysis. Colour indicates screen number. f , g , Relative enrichment and frequency of each sgRNA targeting Slc2a4 (purple), Vmn1r107 (green) or control (grey) sgRNA pool in the OB. Hashed line: CasTLE-computed relative enrichment effect size for sgRNAs of interest relative to control sgRNA pool. Vertical dashed lines indicate relative enrichment of the five sgRNAs targeting Slc2a4 or Vmn1r107 .

Article Snippet: The GLUT4 antibody we used for immunostaining of brain sections (R&D Systems, MAB1262, clone 1F8) was validated in vivo by the Slc2a4 knockout (see above).

Techniques: In Vivo, Injection, Expressing, Immunofluorescence, Control, Infection, Virus, Activation Assay, In Vitro, Sequencing

Journal: Nature

Article Title: CRISPR–Cas9 screens reveal regulators of ageing in neural stem cells

doi: 10.1038/s41586-024-07972-2

Figure Lengend Snippet: a , b , Normalized count matrices of all sgRNA counts across samples at Day 4 ( a ) or Day 14 ( b ). c , Growth curves with number of cells at each passage (p3-p12) for primary cultures of NSCs from young (3-4 months) and old (18–21 months) mice used for the genome-wide screens. Dots represent cell counts for the sample at each passage (x10 6 ). Dotted line at 1.4×10 9 represents the required number of cells for each biological replicate. d - f , Pairwise comparison of CasTLE scores across young screen samples at Day 14. Correlation plots between Screen 1 and Screen 2, Screen 2 and Screen 3; Screen 1 and Screen 3. Spearman ρ is indicated. g - i , Principal Component Analysis (PCA) performed on all gene scores of the three independent screens at Day 4 (g,h) and Day 14 (i): Young 1, 2, 3 (blue) and Old 1, 2, 3 (red), with Principal Components 1 vs. 2 ( g ) and Principal Components 3 vs. 4 ( h , i ). j , k , Volcano plots of example screen results (screen 1) at Day 14 for young ( j ) or old NSCs ( k ), showing gene scores as a function of effect size. Each dot represents one gene. Labelled dots are top ranking gene knockouts in at least 2 of the 3 independent screens (FDR < 0.1). Selecting genes that intersect screen 1 (day 4 or 14) with screen 2 (day 4 or 14) that boost NSC activation (purple, corresponding to enriched sgRNAs) or impede NSC activation (green, corresponding to depleted sgRNAs) in an age-dependent manner or gene knockouts that boost activation regardless of age (grey, corresponding to enriched sgRNAs). See Supplementary Table for complete list of gene scores. l , Comparison of significantly depleted genes (FDR < 0.1) in genome-wide screen (Day 14) and essential genes identified from Online GEne Essentiality database (OGEE) . m , Comparison of significantly depleted genes (FDR < 0.1) in genome-wide screen (Day 14) and essential genes identified from Core Essential Genes 2 (CEG2) . n , Validation of gene knockout efficiency at the genomic level. qNSCs were infected with lentivirus expressing sgRNAs targeting individual genes (5 sgRNAs per gene) and genomic DNA was extracted. Percentage of knockout was quantified by sequencing PCR products followed by DECODRv3.0. Top: Experiment 1. Dot plot of the percentage of knockout for n = 2 independent primary NSC cultures, each derived from 2 mice (one young, one old). Connecting line indicates range. Bottom: Experiment 2. Dot plot of the percentage of knockout for n = 1 independent primary NSC culture derived from 1 mouse (young). Each dot represents the percentage knockout for one sgRNA. #: knockout detected by DECODRv3.0, but with low confidence (r 2 < 0.6) (see Source Data). No data point: knockout not detected by DECODRv3.0 (see Source Data). Dotted red line: sum of knockout percentages for high confidence and detected knockouts. See Extended Data Fig. for genomic knockout examples and knockout efficiency by western blot and FACS for Slc2a4 (GLUT4).

Article Snippet: The GLUT4 antibody we used for immunostaining of brain sections (R&D Systems, MAB1262, clone 1F8) was validated in vivo by the Slc2a4 knockout (see above).

Techniques: Genome Wide, Comparison, Activation Assay, Gene Knockout, Infection, Expressing, Knock-Out, Sequencing, Derivative Assay, Western Blot

Journal: Nature

Article Title: CRISPR–Cas9 screens reveal regulators of ageing in neural stem cells

doi: 10.1038/s41586-024-07972-2

Figure Lengend Snippet: a , b , Immunofluorescence images of SVZ niche sections from old (21 months) female ( a ) or OB sections from old (22 months) male ( b ) Cas9 mice 5 weeks after injection of lentivirus expressing Scl2a4 sgRNAs into the lateral ventricles (LVs). One experiment. mCherry (lentivirus-infected cells, red), GLUT4 (green), DAPI (blue). Dashed rectangles highlight examples of mCherry-infected cells. Insets, zoomed-in images. c , d , Quantification of GLUT4 mean fluorescence intensity in the SVZ ( c ) and the OB ( d ). Mean ± s.e.m. of GLUT4 fluorescence intensity for n = 285 mCherry – and n = 285 mCherry + GFAP + cells ( c ) and n = 1,333 mCherry – and n = 897 mCherry + cells ( d ) in 1 old (22 month) Cas9 male mouse injected with Slc2a4 sgRNAs. Each dot represents one cell. Two-tailed Mann–Whitney test. e , f , z stack confocal ( e ) and immunofluorescence ( f ) images of OB sections from old (20 months) male Cas9 mice 5 weeks after injection of lentivirus expressing control or Slc2a4 sgRNAs into the LVs. Mice were injected with EdU once per week for 4 weeks, starting 1 week after virus injection. One experiment. e , NeuN (mature neurons, green), mCherry (lentivirus-infected cells, red), GFAP (astrocytes, blue), Dcx (neuroblasts, magenta). Arrowheads indicate lentivirus-infected NeuN + cells. z stack positions indicated on the bottom right. f , EdU (newborn cells, green), mCherry (lentivirus-infected cells, magenta), NeuN (mature neurons, red), DAPI (blue). Arrows indicate lentivirus-infected NeuN + cells. Right, zoomed-in images. g , Newborn neuron quantification in the OB. Mean ± s.e.m. of newborn neurons (NeuN + mCherry + EdU + ) average number over total EdU + cell number from 3 OB sections (normalized to control) for n = 6 (control), n = 6 ( Slc2a4 ), n = 3 ( Vnm1r107 ) old (18–21 months) male Cas9 mice, over 3 experiments. Each dot represents one mouse. Two-tailed Mann–Whitney test. h , Immunofluorescence images of SVZ sections from old (21 months) male Cas9 mice 5 weeks after injection of lentivirus expressing Slc2a4 sgRNAs into the LVs. One experiment. Ki67 (green), GFAP (magenta), S100a6 (red), DAPI (blue). qNSCs (qN; GFAP + S100a6 + Ki67 – ), aNSCs (aN; GFAP + S00a6 + Ki67 + ), neuroblasts (Nb; GFAP – Ki67 + ), astrocytes (As; GFAP + S100a6 – ). i , Quantification of qNSC, aNSC, neuroblast and astrocyte number in the SVZ. Mean ± s.e.m. of average cell number from about 8 SVZ sections for n = 8 (control) and n = 8 ( Slc2a4 ) old (18–21 months) male Cas9 mice, over 3 experiments. Each dot represents one mouse. Two-tailed Mann–Whitney test. j , Immunofluorescence images of GLUT4 levels in SVZ sections from young (3–4 months) and old (18–21 months) male Cas9 mice. One experiment. GLUT4 (red), Ki67 (green), GFAP (magenta), DAPI (blue). qNSC and astrocytes (GFAP + Ki67 – ), aNSCs (GFAP + Ki67 + ), neuroblasts (GFAP – Ki67 + ), other cells (GFAP – Ki67 – ). Right, zoomed-in images. k , Quantification of GLUT4 mean fluorescence intensity in the SVZ niche. Cell types as in j . Mean ± s.e.m. of GLUT4 mean fluorescence intensity for n = 7 (young) and n = 7 (old) male Cas9 mice, over 2 experiments. Each dot represents one mouse. Two tailed Mann–Whitney test. Scale bars, 50 µm ( a , b , e , f (middle and right), j ) or 100 µm ( f (left), h ).

Article Snippet: The GLUT4 antibody we used for immunostaining of brain sections (R&D Systems, MAB1262, clone 1F8) was validated in vivo by the Slc2a4 knockout (see above).

Techniques: Immunofluorescence, Injection, Expressing, Infection, Fluorescence, Two Tailed Test, MANN-WHITNEY, Control, Virus

Journal: Nature

Article Title: CRISPR–Cas9 screens reveal regulators of ageing in neural stem cells

doi: 10.1038/s41586-024-07972-2

Figure Lengend Snippet: a , Top panel: immunofluorescence images of SVZ sections from old (22 months old) female Cas9 mice, 5 weeks after injection with lentivirus expressing Slc2a4 (GLUT4) sgRNAs directly into the lateral ventricles. One experiment; representative of 3 independent experiments. DAPI (nuclei, blue), GFAP (qNSCs/astrocytes, magenta), Ki67 (proliferative cells, green), S100a6 (NSCs, red), merge image. Bottom panel: immunofluorescent images of sections of the SVZ from the same mice as in top panel. DAPI (nuclei, blue), mCherry (lentivirus-infected cells, red), and GFAP (qNSCs/astrocytes, green). b , Immunofluorescence images of olfactory bulb (OB) sections from old (~20 months old) male Cas9 mice, 5 weeks after injection of lentivirus expressing control (unannotated genomic regions), Slc2a4 (GLUT4) or Vmn1r107 sgRNAs directly into the lateral ventricles. One experiment; representative of 3 independent experiments. Mice were injected with EdU once per week, starting one week after virus injection, for 4 weeks. EdU (newborn cells, green) and nuclei (DAPI, blue). Zoomed-out images with the dashed white squares representing the insets (top) and zoomed-in images as insets (bottom). c , Example image of mCherry (lentivirus-infected cells, magenta) and EdU (newborn cells, green) staining (left panel) and QuPath image quantification of the average number of EdU + and mCherry + cells in the olfactory bulbs, normalized to total EdU + cells (right panel) in old Cas9 mice (18–23 months old) (right panel). Mean +/- SEM of the average number of cell counts for one mouse, from 3 serial sections taken at 100 µm intervals across the olfactory bulb pair (normalized to average of control), for n = 9 old mice (4 males, 5 females, 20–23 months) for control sgRNAs, n = 6 old mice (3 males, 3 females, 20 – 23 months) for Slc2a4 (GLUT4) sgRNAs, and n = 5 old Cas9 mice (3 males, 2 females, 18–22 months) for Vmn1r107 sgRNAs, over 3 independent experiments. Each dot represents one mouse. P -values: two-tailed Mann-Whitney test. d , Immunofluorescence images of olfactory bulb sections from old (20 months) male Cas9 mouse, 5 weeks after injection of lentivirus expressing control (unannotated genomic regions) or Vmn1r107 sgRNAs directly into the lateral ventricles. One experiment; representative of 3 independent experiments. Mice were injected with EdU once per week, starting one week after virus injection, for 4 weeks. EdU (newborn cells, green), mCherry (lentivirus-infected cells, magenta), NeuN (mature neurons, red), and DAPI (nuclei, blue). Dashed white squares: NeuN + cells that are infected with lentivirus (expressing one of the sgRNAs). Insets: zoomed-in images. e , QuPath image quantification of NeuN mean fluorescent intensity in infected (mCherry + ) newborn cells (EdU + ) in the olfactory bulb of old Cas9 mice (18–23 months old) (mix of males and females, see Source Data), 5 weeks after injection of lentivirus expressing control (unannotated genomic regions), Slc2a4 (GLUT4), or Vmn1r107 sgRNAs directly into the lateral ventricles. Mice were injected with EdU once per week, starting one week after virus injection, for 4 weeks. Mean +/- SEM of the average NeuN fluorescence intensity of all enumerated cells in one mouse, from 3 serial sections taken at 100 µm intervals across the olfactory bulb pair, for n = 9 old Cas9 mice (4 males, 5 females, 20–23 months) for control sgRNAs, n = 6 old Cas9 mice (3 males, 3 females, 20–23 months) for Slc2a4 (GLUT4) sgRNAs, and n = 5 old Cas9 mice (3 males, 2 females, 18–22 months) for Vmn1r107 sgRNAs, over 3 independent experiments. Each lavender dot represents one mouse. P -values: two tailed Mann-Whitney test. Each grey dot represents a single cell NeuN fluorescence intensity, showing all cells across all samples for each respective treatment. f , Image quantification of the percentage of newborn cells (EdU + ) that are also NeuN + , comparing cells with (mCherry + ) or without (mCherry − ), in control, Slc2a4 , or Vmn1r107 sgRNA expression conditions. Mean +/− SEM of the average (%NeuN + ) of all cell quantifications from a single mouse for n = 5 old Cas9 mice for each condition, over 3 independent experiments. Each dot represents one mouse. P -values: two-tailed Mann-Whitney test. g , Immunofluorescence images of olfactory bulb sections from old (20 months old) male Cas9 mouse, 5 weeks after injection of lentivirus expressing Slc2a4 (GLUT4) sgRNAs directly into the lateral ventricles. Mice were injected with EdU once per week, starting one week after virus injection, for 4 weeks. mCherry (lentivirus-infected cells, red), Tuj1 (neuron marker, green), and DAPI (nuclei, blue). Insets and white ovals: Tuj1 + cells that are infected with lentivirus (expressing one of the sgRNAs targeting Slc2a4 (GLUT4)). One experiment; representative of 2 independent experiments. h , QuPath image quantification of Tuj1 mean fluorescent intensity in infected (mCherry + ) newborn cells (EdU + ) in the olfactory bulb of old Cas9 mice (18–21 months old) (mix of males and females), 5 weeks after injection of lentivirus expressing control (unannotated genomic regions), or Slc2a4 (GLUT4) sgRNAs directly into the lateral ventricles. Mean +/− SEM of the average Tuj1 fluorescence intensity of all enumerated cells in one mouse, sections taken at 100 µm intervals across the olfactory bulb pair for n = 4 old Cas9 mice for each condition, over 2 independent experiments. Each lavender dot represents one mouse. P -values: two tailed Mann-Whitney test. Each grey dot represents a single cell Tuj1 fluorescence intensity, showing all cells across all samples for each respective treatment. i , Immunofluorescence images of olfactory bulb sections from old (20 months old) male Cas9 mouse, 5 weeks after injection of lentivirus expressing Slc2a4 (GLUT4) sgRNAs directly into the lateral ventricles. One experiment. Mice were injected with EdU once per week, starting one week after virus injection, for 4 weeks. mCherry (lentivirus-infected cells, red), Calretinin (mature neuron marker, green), and DAPI (nuclei, blue). j , Immunofluorescence images of olfactory bulb sections from old (20 months old) male Cas9 mouse, 5 weeks after injection of lentivirus expressing Slc2a4 (GLUT4) sgRNAs directly into the lateral ventricles. Mice were injected with EdU once per week, starting one week after virus injection, for 4 weeks. Left panels: mCherry (lentivirus-infected cells, red), GFAP (astrocytes, blue, top panels), Dcx (immature neuroblast, blue, bottom panels), NeuN (neurons, green). Right panels: mCherry (lentivirus-infected cells, red), DAPI (nuclei, blue), Sox10 (oligodendrocytes, green, top panels), Olig2 (oligodendrocytes, magenta, lower panels). Note that images are the exact same in the top and lower panels to show the different channels, except for the Slc2a4 (GLUT4) sgRNA condition for Dcx (lower panel), as there were no Dcx + cells (which are very rare) in the same image. One experiment. k , QuPath image quantification of the mean fluorescence intensity with an isotype antibody control (mouse IgG) in sections from the SVZ neural stem cell niche from 4 young (3-4 months old) and 3 old (18–21 months old) male Cas9 mice (control for Fig. ). Cell types were identified as follows: qNSC/astrocyte (GFAP + Ki67 − ), aNSC (GFAP + Ki67 + ), Neuroblast (GFAP − Ki67 + ) and other cells (ependymal, microglia; GFAP − Ki67 − ). Mean +/− SEM of control average fluorescent intensities of each cell type, as identified above, from each mouse, for n = 4 (young qNSCs/astrocytes, young aNSCs, young other cells), n = 3 (young neuroblasts, old qNSCs/astrocytes, old neuroblasts, old other cells), or n = 2 (old aNSCs) Cas9 mice, over 2 experiments. Each dot represents one mouse. P -values determined by two tailed Mann-Whitney test. l , Violin plots comparing the log-normalized expression of Slc2a4 (GLUT4) transcripts between young (blue) and old (red) in single cell RNA-seq of qNSCs/astrocytes, aNSCs, neuroblasts, and all other cells from the neurogenic niche . P -values determined by two-sided two-sample Welch’s t-test. m , Representative immunofluorescence images of coronal sections of the SVZ neural stem cell niche from young (3-4 months old) and old (18–21 months old) male Cas9 mice. GLUT4 (red), S100a6 (NSC marker, green), GFAP (NSC and astrocyte marker, magenta), and DAPI (nuclei, blue). Dotted squares: regions with NSCs shown in the insets. Insets: zoomed-in images. n , QuPath image quantification of the mean fluorescence intensity of the GLUT4 antibody in S100a6 + /GFAP + cells quantified from sections of the SVZ neural stem cell niche. Mean +/− SEM of the average fluorescent intensities of cells from one mouse, for n = 4 young (3-4 months old) and n = 4 old (18–21 months old) male Cas9 mice, over 2 independent experiments. Each dot represents a mouse. P -values: two tailed Mann-Whitney test.

Article Snippet: The GLUT4 antibody we used for immunostaining of brain sections (R&D Systems, MAB1262, clone 1F8) was validated in vivo by the Slc2a4 knockout (see above).

Techniques: Immunofluorescence, Injection, Expressing, Infection, Control, Virus, Staining, Two Tailed Test, MANN-WHITNEY, Fluorescence, Marker, RNA Sequencing Assay

Journal: Nature

Article Title: CRISPR–Cas9 screens reveal regulators of ageing in neural stem cells

doi: 10.1038/s41586-024-07972-2

Figure Lengend Snippet: a , Glucose uptake pathway. b , Heatmap plotting glucose and insulin pathway median gene scores from genome-wide in vitro screens (day 4). Gene knockouts that boost (green) or impede (red) NSC activation. c , Immunofluorescence image of GLUT4 staining in cultured qNSCs and aNSCs from young (3–4 months) and old (18–21 months) Cas9 mice (mixed sex). GLUT4 (red), DAPI (blue). One experiment. Scale bar, 10 µm. d , e , Quantification of GLUT4 ( d ) and STX4A ( e ) mean fluorescence. Mean ± s.e.m. of mean fluorescence for n = 4 (young qNSCs, old qNSCs, young aNSCs) and n = 3 (old aNSCs) cultures, each from six pooled Cas9 mice (three males, three females). One experiment. Each dot represents one NSC culture. Two-tailed Mann–Whitney test. f , Schematic (top) and quantification (bottom) of bioluminescent glucose uptake in primary qNSCs and aNSCs from young and old mice. Mean ± s.e.m. of results for n = 6 (young qNSCs, aNSCs) and n = 8 (old qNSCs, aNSCs) cultures, each from six pooled Cas9 mice (three males, three females), over two experiments. Each dot represents one NSC culture. Two-tailed Mann–Whitney test. g , ECAR and OCR in qNSCs from young and old mice. qNSCs infected with control or Slc2a4 sgRNAs lentiviruses. ECAR, mean ± s.d. of results for n = 16 (young and old, control), n = 12 (young, Slc2a4 ) and n = 15 (young, Slc2a4 ) NSC cultures, each from two pooled mice (one male, one female), over two experiments. OCR, mean ± s.d. of results for n = 9 (young, control), n = 15 (young and old, Slc2a4 ) and n = 12 (old, control) NSC cultures, each from two pooled Cas9 mice (one male, one female), over two experiments. Each dot represents one NSC culture. One-tailed Wilcoxon signed-rank test (within age groups), one-tailed Mann–Whitney test (across age groups). h , Schematic (top) and quantification (bottom) of bioluminescent glucose uptake in primary qNSC cultures from old mice. qNSCs infected with Slc2a4 or control sgRNAs lentiviruses, or uninfected, were assessed for glucose uptake at day 4 or day 8. Mean ± s.d. of results for n = 3 (uninfected), n = 4 (day 4) and n = 5 (day 8) NSC cultures, each from six pooled Cas9 mice (three males, three females), over two experiments. Each dot represents one NSC culture. Two-tailed Mann–Whitney test. i , Schematic (top) and quantification (bottom) of NSC activation efficiency for young and old qNSCs (FACS). qNSCs infected with control or Slc2a4 sgRNAs lentiviruses were placed in quiescence medium with (grey) or without (pink) glucose for 2 days, then in activation medium (with glucose) for 4 days. Mean ± s.d. of Ki67 + cell percentage for n = 6 NSC cultures (each condition), each from six pooled Cas9 mice (three males, three females), over two experiments. Each dot represents one NSC culture. Two-tailed Mann–Whitney test. P values in source data. j , Summary of gene knockout interventions that boost old NSC activation in both in vitro and in vivo screens. The models in a and j were created using BioRender ( https://biorender.com ).

Article Snippet: The GLUT4 antibody we used for immunostaining of brain sections (R&D Systems, MAB1262, clone 1F8) was validated in vivo by the Slc2a4 knockout (see above).

Techniques: Genome Wide, In Vitro, Activation Assay, Immunofluorescence, Staining, Cell Culture, Fluorescence, Two Tailed Test, MANN-WHITNEY, Infection, Control, One-tailed Test, Gene Knockout, In Vivo

Journal: Nature

Article Title: CRISPR–Cas9 screens reveal regulators of ageing in neural stem cells

doi: 10.1038/s41586-024-07972-2

Figure Lengend Snippet: a , Bar plots showing the log2 fold change in average expression of various Slc2 transcripts expressed in qNSCs/astrocytes from young and old mice in single cell RNA-seq data , where log2 fold changes greater than 0 indicate higher expression in cells of old animals than in young animals. b , Immunofluorescence image of STX4A in primary NSC cultures from young (3-4 months old) or old (18–21 months old) mice. NSCs were plated in quiescence NSC media (qNSCs) for 7 days prior to imaging, and NSCs were plated in activated NSC media (aNSCs) 2 days prior to imaging. STX4A (green) and DAPI (nuclei, blue). One experiment; quantification in Fig. . c , Representative immunofluorescence images of coronal sections from SVZ NSC niche sections from young (3-4 months old) and old (18–21 months old) Cas9 mice. Ki67 (proliferation maker, green), GFAP (NSC and astrocyte marker, magenta), DAPI (nuclei, blue), and STX4A (red). Cell types were identified as follows: qNSC/astrocytes (GFAP + Ki67 − ), aNSCs (GFAP + Ki67 + ), Neuroblasts (GFAP − Ki67 + ), and other cells (ependymal, microglia; GFAP − Ki67 − ). d , QuPath image quantification of STX4A mean fluorescence in cells of the neural stem cell niche. Cell types were identified as follows: qNSC/astrocyte (GFAP + Ki67 − ), aNSC (GFAP + Ki67 + ), Neuroblast (GFAP − Ki67 + ) and other cells (ependymal, microglia; GFAP − Ki67). Mean +/− SEM of STX4A mean fluorescence intensity for n = 4 young (~4 months) and n = 4 old (~19 months) Cas9 mice, over 2 independent experiments. Each dot represents one mouse. P -values: two tailed Mann-Whitney test. e , Expression of Scl2a4 (GLUT4) transcripts in young (6–8 weeks old) qNSCs and aNSCs in culture from a published RNA-seq dataset . Mean +/− SD from n = 4 qNSC and aNSC cultures. P -values: two-tailed Mann-Whitney test. f , FACS-based glucose uptake assay with 2NBDG (2-( N -(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) (Sigma, 72987) on primary NSC cultures (qNSCs and aNSCs) from young (3-4-months-old) or old (18–21-months-old) mice. Mean +/− SEM of FACS results from n = 4 (young and old qNSCs, young aNSCs) and n = 3 (old aNSCs) independent NSC cultures, each from a pool of 6 Cas9 mice, 3 males and 3 females. Each dot represents one independent NSC culture. P -values: two-tailed Mann-Whitney test. g , Violin plots showing the log-normalized expression of all genes in the fatty acid oxidation gene signature across young and old qNSC/astrocyte cells in single cell RNA-seq data . P -value: two-sample Welch’s t-test. h , Top panel: scheme of the Slc2a4 (GLUT4) locus with the location of sgRNAs 1–5. Bottom panel: genomic sequences for Slc2a4 (GLUT4) sgRNAs 1–5 from DECODRv3.0 analysis tool indicating the sgRNA target as well as cut site and indel distribution. i , Guide sequences used for Slc2a4 (GLUT4) sgRNAs 1–5 and the DECODRv3.0 knockout percentage for each sgRNA. j , GLUT4 knockout efficiency in young (3-4 months) qNSC culture by western blot. Western blot analysis of GLUT4 levels in qNSCs infected with control sgRNA (targeting unannotated regions of the genome) or sgRNA targeting Slc2a4 (GLUT4), 10 days after infection by lentivirus and 3 days of selection with puromycin. β-actin is used as a loading control. Data from n = 1 (control sgRNAs) and n = 1 ( Slc2a4 (GLUT4) sgRNAs) NSC culture, each derived from one young Cas9 mouse. The western blot was repeated 3 times with similar results. For western blot source data, see Supplementary Fig. . k , Quantification of western blot: GLUT4 intensity, normalized to β-actin intensity. l , GLUT4 knockout efficiency in primary qNSC cultures by FACS. Intracellular FACS analysis of GLUT4 levels in fixed qNSCs treated with control sgRNA or sgRNA targeting Slc2a4 (GLUT4), 10 days after lentivirus infection to express sgRNA. No antibody control panel is on the left. Plots show mCherry + gated cells, GLUT4 fluorescence. m , Quantification of FACS data, normalized to control. n , Data from Fig. , presented as the boost in qNSC activation ability with Slc2a4 (GLUT4) knockout, with or without glucose starvation. Mean +/− SEM of NSC activation ability of Slc2a4 (GLUT4) knockout relative to control, for n = 6 young (3-4 months) or n = 6 old (18–21 months) independent NSC cultures, each derived from a pool of 6 Cas9 mice (3 males, 3 females), over 2 experiments. Each dot represents one independent NSC culture. P -values: two-tailed Mann-Whitney test. o , Quantification of NSC activation efficiency in cultured NSC from young (3-4 months old) or old (18–21 months old) mice 4 days after transition to aNSC media, as assessed by Ki67 intracellular FACS analysis. NSCs were placed in qNSC media for 4 days. Then the cell media was replaced with qNSC media with or without 2-Deoxy-D-glucose (2-DG) (2 mM) for 48 h, at which point the media was replaced with aNSC media and the cells were allowed to activate for 4 days prior to intracellular FACS analysis with Ki67. Mean +/− SEM of the percentage of Ki67 + cells for n = 4 (young) or n = 4 (old) independent NSC cultures, each derived from a pool of 2 Cas9 mice (1 male, 1 female). Each dot represents one independent NSC culture. P -values: two-tailed Mann-Whitney test.

Article Snippet: The GLUT4 antibody we used for immunostaining of brain sections (R&D Systems, MAB1262, clone 1F8) was validated in vivo by the Slc2a4 knockout (see above).

Techniques: Expressing, RNA Sequencing Assay, Immunofluorescence, Imaging, Marker, Fluorescence, Two Tailed Test, MANN-WHITNEY, Genomic Sequencing, Knock-Out, Western Blot, Infection, Control, Selection, Derivative Assay, Activation Assay, Cell Culture