Additional file 1 of Astrocyte-specific knockout of YKL-40/Chi3l1 reduces Aβ burden and restores memory functions in 5xFAD mice
Creators: name: Zeng, Xiaoyan
name: Cheung, Stanley K. K.
name: Shi, Mengqi
name: Or, Penelope M. Y.
name: Li, Zhining
name: Liu, Julia Y. H.
name: Ho, Wayne L. H.
name: Liu, Tian
name: Lu, Kun
name: Rudd, John A.
name: Wang, Yubing
name: Chan, Andrew M.
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: Science Policy
subject: Mental Health
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Fig. S1. Age-dependent amyloid-beta plaques deposition in 5xFAD mouse brains and Aβ-induced signaling in primary astrocytes. A. Confocal images (Mag. 10X) of stitched 4m (presymptomatic), 7m (symptomatic), and 9m (advanced) 5xFAD mouse brains immuno-stained with DAPI (blue signal), anti-Aβ (green signal), and anti-GFAP (red signal) antibodies. Scale bars, 2000 μm. B. Higher magnifications of dentate gyrus (DG) region of 7m brains are shown. C. The mean plaque surface area and plaque numbers in DG region were quantified using ImageJ software. 4m (n=8), 7m (n=8), 9m (n=6). Data are mean ± SEM. One-way ANOVA with Tukey’s post hoc comparisons.  *p &lt; 0.05, **p &lt; 0.01,***p &lt; 0.001. D. Parallel immunofluorescence analysis similar to panel (B) was performed with Thioflavin T (ThT, green) and anti-YKL-40 (red). E. Quantification of data from Fig. 1J showing relative phosphorylation levels of AKT, P70S6, mTOR, and S6 following Aβ treatment. Data are mean ± SEM. One-way ANOVA with Tukey’s post hoc comparisons. ns, not significant, *p &lt; 0.05, **p &lt; 0.01. Fig. S2. Neuronal damages induced by Aβ1-42 (Aβ) and YKL-40 treatment. Primary neurons were derived from E17.5 wild type embryos. A. Brightfield photomicrographs taken from DIV14 treated with 10 μM Aβ, 400 ng/ml YKL-40, and 10 μM Aβ+400 ng/ml YKL-40 for 72 hrs. Scale bars, 200 μm. B. Binary figures of MAP2 immuno-stained confocal images, and their reconstructed Simple Neurite Tracing (SNT, Plugin in ImageJ software) tracing images following a 72 hrs exposure to the indicated concentrations of Aβ and YKL-40. C-H. Sholl Analysis (plugin from ImageJ) was performed from tracing images. Quantitative data for soma perimeter, dendritic length, axon length and nodes, dendritic nodes and ends are shown. Data are mean ± SEM of 50 to 70 neurons. One-way ANOVA with Tukey’s post hoc comparisons.  ***p &lt; 0.001 compared to control. Fig. S3. Generation of astrocyte-specific YKL-40/Chi3l1 conditional knockout mice. A. Genotyping of Aldhl11-cre/ERT2. Tails of one month old pups from a typical litter were processed for DNA extraction and PCR analysis was carried out using the following primer pairs: Aldhl1 and Aldhl2; Aldhl3 and Aldhl4. B. Knockout strategy of YKL-40/Chi3l1. C. Mating strategy used to generate the six experimental groups for this study. See Materials and Methods section for detailed descriptions. D. Tails of one month old pups from a typical litter were processed for DNA extraction and PCR analysis was carried out using the following primer pair: mChil1_flox_F and mChil1_flox_R. Wild-type (+/+) and heterozygous (+/- or +/fl) littermates are indicated. Abbreviations, Ma, markers; C, control. E. Primary astrocytes were treated without (-) or with (+) 1 μM tamoxifen solution. The 546-basepair (bp) knockout specific PCR product is indicated. F. Confocal images (Mag. 10X) of dentate gyrus (DG) and (G) thalamus (TH) regions of 3.5-month-old Aldh;Chi3l1+/+, Aldh;Chi3l1+/fl, and Aldh;Chi3l1fl/fl mouse brains immuno-stained with DAPI (blue signal), anti-YKL-40 (green signal), and anti-GFAP (red signal) antibodies. Scale bars, 100 μm. All mice were IP injected with 20 mg/ml tamoxifen in corn oil (100 μl per mice/per day) for 7 consecutive days before brains were harvested. Fig. S4. Knockout of YKL-40 in astrocytes reduces Aβ deposition in 5xFAD mice brain. All mice were IP injected with 20 mg/ml tamoxifen in corn oil (100 µl per mice/per day) for 7 consecutive days before experiments. Experiments were carried out 7 days after last tamoxifen injection. A. Confocal images (Mag. 10X) of motor cortex + metrosplenial cortex (MOC), mortex (CTX), Cornu Ammonis 3 (CA3), and thalamus (TH) regions of 5xFAD;Aldh;Chi3l1+/+; 5xFAD;Aldh;Chi3l1+/fl; 5xFAD;Aldh;Chi3l1fl/fl mouse brain immuno-stained with DAPI (blue signal), anti-Aβ (green signal), and anti-GFAP (red signal) antibodies. Scale bars, 100 μm. B. Quantification of plaque number from Aβ signals and mean surface area. Data are mean ± SEM. One-way ANOVA with Tukey’s post hoc comparisons. *p &lt; 0.05,**p &lt; 0.01, ***p &lt; 0.001 compared to 5xFAD;Aldh;Chi3l1+/+. Fig. S5. YKL-40 depletion in astrocytes reduces glial activation. All mice were IP injected with 20 mg/ml tamoxifen in corn oil (100 μl per mice/per day) for 7 consecutive days before experiments. Experiments were conducted 7 days after last tamoxifen injection. A-B. Confocal images (Mag. 10X) of 7m 5xFAD mice brain immuno-stained with DAPI (blue signal), anti-IBA1 (green signal), and anti-GFAP (red signal) antibodies. Scale bar, 100 μm. C–D. Mean surface area was quantified. n=2 to 4. Data are mean ± SEM. E–F. Western blotting analysis of GFAP, and IBA1 expression from cortex (Cor) and hippocampal (Hip) lysates. GAPDH was used as an internal control. G-H. Quantification results of Western blot analysis are shown. Data are mean ± SEM. One-way ANOVA with Tukey’s post hoc comparisons.*p &lt; 0.05, **p &lt; 0.01,***p &lt; 0.001. Fig. S6. Transcriptomic analysis of YKL-40 knockout astrocytes. (A) The volcano plot shows the 258 differentially expressed genes (294 up-regulated and 64 down-regulated, Foldchange&gt;2, p&lt;0.05) between WT and Chi3l1-/- (KO) in primary astrocytes by RNA-seq.  (B) KEGG enrichment analysis of all DEGs showed that KO-associated with phagosome signaling pathway. GSEA analysis showed that genes associated with (C) phagocytic vesicles and (D) lysosome signaling were significantly up-regulated in KO astrocytes.