Additional file 1 of Alzheimer’s disease protective allele of Clusterin modulates neuronal excitability through lipid-droplet-mediated neuron-glia communication

Additional file 1. Figure S1: Bioinformatic and experimental validation of the regulatory effect of rs1532278 on TF-binding and CLU expression, related to Fig. 1. (A) Multiz alignment and phyloP conservation (470 mammals) around rs1532278 (from UCSC hg38 genome browser). (B) JASPAR predicted TF binding sites at rs1532278 and TF expression levels in iGlut (10/23 predicted TFs can be detected by RNA seq). CPM, counts per million reads. (C) Representative images (CD07 line) of iGlut of all three genotypes are also shown, related to Fig. 1D (bottom panel); GFAP and HuNu (human nuclear antigen) staining shows specificity of HuNu and MAP2 staining for iGlut in iGlut-mAst co-cultures. (D) No difference of iGlut differentiation efficiency were found between T/T and CC carriers in iGlut-mAst co-cultures, and none proliferating cells were observed in these neurons indicated by negative staining of Ki67. HuNu +, human cells. n=5 coverslips per group from one differentiation of both CD05 and CD07 lines (2-3 clones per line, one coverslip per clone and 4-5 images per coverslip). (E) DRGX ChIP-qPCR for iGlut-mAst co-cultures of CD07 line on day 30. n=3 biological replicates per group (one clone with 3 biological replicates from the CD07 line) from one independent differentiation. (F-G) ISL2 siRNA knockdown in day-30 pure iGlut (C/C) cultures. Samples of 72 hours post-siRNA transfection were used for qPCR. n=3 biological replicates from one clone per line in one independent differentiation. (H) CLU mRNA levels in iGlut pure cultures. n=6 biological replicates per group (2-3 clones per line and 2-3 biological replicates for each clone) from two independent differentiations of each line (I) sCLU levels detected by ELISA from the supernatant of iGlut pure cultures. n=4 biological replicates per group (2 clones per line and 2 biological replicates for each clone) from two independent differentiations of each line. (J) CLU mRNA levels of mAst in iGlut-mAst co-cultures. n=4 biological replicates per group (2 clones per line and 2 biological replicates for each clone) from two independent differentiations of each line. (K) Three qPCR primer sets that can capture the majority of CLU transcript isoforms (14/17) are used in (L) to detect CLU mRNA levels. Only iGlut-mAst co-cultures of the CD07 line was used. n=6 biological replicates per group (3 biological replicates per clone for 2 clones) from two independent differentiations. (M) rs1532278 did not alter the expression of SCARA3, a CLU-adjacent gene, in iGlut (or when co-cultured with mAst) of the isogenic pairs of both CD05 and CD07 lines. n=10 biological replicates per group (2 biological replicates per clone and 2-3 clones per line) from two independent differentiations of both CD05 and CD07 lines. (N) Representative images (CD05 line) of CLU staining, related to Figure 1K and 1L. Data = mean±SEM. * p<0.05, ** p< 0.01, *** p< 0.001, and **** p< 0.0001. Figure S2. Quality control of the CRISPR/Cas9-edited iPSC lines, related to Fig. 1.(A) Sanger sequencing of top off-target sites (Benchling prediction) with 2 and 3 mismatched sites with gRNA in one clone of the edited alleles in both CD05 and CD07 lines. Note no off-target editing was found. (B) eSNP-karyotyping using RNA-seq data of iGlut of the two isogenic pairs of CRISPR-edited lines (CD05 and CD07; only the iPSC clones used for cellular functional assays were analyzed). Left panel, moving average of the SNP allelic ratio (RNA-seq reads of each allele) along the genome; right panel, stretches of SNP heterozygosity of all common SNPs for each chromosome. Note that no chromosomal abnormalities were found. (C) CLU, ISL2, and DRGX expression in postmortem brain transcriptomic dataset hub ( https://cellxgene.cziscience.com/ ). (D-E) CLU mRNA Levels in iAst differentiated from the isogenic pairs of CRSIPR/Cas9-edited iPSC lines (for donor lines CD05 and CD07). Representative immunofluorescence staining (CD05 line) of S100β and vimentin shows the identity and purity of astrocytes. n=10 biological replicates per group (2-3 clones per line and 2 replicates per clone) from two independent differentiations of both CD05 and CD07 lines. Data, mean±SEM. Figure S3. Synaptic and electrophysiological properties of iGlut carrying TT or C/C alleles of rs1532278 at the CLU locus, related to Fig. 2. (A) Quantification of SYP and PSD-95 puncta density. n=15-17 neurons per group (1-2 neuron per coverslips, 5-6 coverslips per clone, and 2 clones per line) from two independent differentiations of each line. (B-C) Western blotting shows PSD-95 and SYP levels in day-30 iGlut pure cultures. n=4 biological replicates per group (2 clones per line and 2 biological replicates per clone) from two independent differentiations of each line. (D-E) Number of bursts and synchronicity analysis in MEA. AUNCC: Area Under Normalization Cross-Correlation. n=9-12 biological replicates per group (2-3 clones per line and 3-4 biological replicates per clone) from two independent differentiations of each line. Data, mean±SEM. * p<0.05, ** p< 0.01, *** p< 0.001, and **** p< 0.0001. Figure S4. Characterization of neurons carrying the rs1532278-flanking OCR deletion or with exogenous CLU overexpression, related to Fig. 3. (A) Sanger sequencing traces the CRISPR/Cas9-engineered homozygous OCR deletion (representative result of the CD07 line). (B) CLU mRNA levels (n=6 biological replicates per group from two independent differentiations, 2 clones per line, and 3 biological replicates for each clone) and sCLU levels (n=4 biological replicates per group from two independent differentiations, 2 clones per line, and 2 biological replicates for each clone) in iGlut pure co-cultures. (C) Immunofluorescence staining of neuronal CLU in iGlut pure cultures on days 23-25. (D) Quantification of CLU staining shown in panel (C). n=7 coverslips per group (2 clones per line, 3-4 coverslips for each clone, and 3-5 images per coverslip; shown are example images of CD05) from two independent differentiations of each line. (E-F) Western-blot for SYP in day-30 iGlut-mAst co-cultures. n=4 biological replicates per group (2 clones per line, and 2 biological replicates for each clone) from two independent differentiations of each line. (G-H) Number of neuron network bursts and synchronicity analysis in MEA. AUNCC: Area Under Normalization Cross-Correlation. n=5-10 biological replicates per group (2 clones per line and 2-5 biological replicates per clone) from two independent differentiations of each line (I) Immunofluorescence staining (CD07 line) of MAP2 and CLU (Flag tag) to determine AAV-hCLU transduction efficiency (~100%). (J) qPCR to confirm CLU overexpression. n=3 biological replicates per group from one clone of CD07 line in one independent differentiation. (K) Number of network bursts and synchronicity analysis in MEA. AUNCC: Area Under Normalization Cross-Correlation. n=3 biological replicates per group (all from one clone of line CD07) in one independent differentiation. Data, mean±SEM. Scale bars are indicated in corresponding images. * p<0.05, ** p< 0.01, *** p< 0.001, and **** p< 0.0001. Figure S5. Transcriptomic analysis of iGlut-mAst co-cultures and validation, related to Fig. 4. (A) Correlation analysis Salmon sorted iGlut data set to our previous published single-cell data sets iGlut (CD05 and CD07) and others published single cell data sets in postmortem brain with various cell types including ex-neuron, ih-neuron, microglia, astrocyte, oligodendrocytes, and OPC. Ast: astrocyte, ex: excitatory neuron, ih: inhibitory neuron, mic, microglia, Oli: oligodendrocyte, and OPC: oligodendrocyte progenitor cell. (B) MDS (Multidimensional scaling) analysis of all transcripts in iGlut and mAst, which are expressed in ≥75% of samples. n=9 biological replicates per group from two independent differentiations of each line, 2-3 biological replicates per clone, 2 clones per line, and 2 lines in total. (C) ELISA quantification of Aβ in the supernatants of iGlut-mAst co-cultures and iGlut pure cultures. n=4 biological replicates per group (2 clones per line and 2 replicates for each clone) from two independent differentiations of each line. (D) GO Biological process enrichment in significantly downregulated gene sets in iGlut. Blue stars indicate neuron morphology-related pathways. (E) Confirmation of SYP, PSD-95, and CLU expression levels in iGlut in RNA seq analysis. (F) Confirmation of human CLU expression levels by using in-silico qPCR approach to exclude mouse CLU reads interference. Violin plots showed data with median and interquartile range, and all other statistical graphs showed data with mean±SEM. * p<0.05, ** p< 0.01, *** p< 0.001, and **** p< 0.0001. Figure S6. The assessment of the Impact of mouse CLU on human CLU expression in iGlut-mAst co-culture system (related to Figures 1, 3, 4, and 6) and the Analysis Procedure of LipidTox staining in iGlut pure cultures (related to Fig. 5).(A-B) The mRNA expression levels, extracellular (secreted) levels, and intracellular levels of both hCLU and mCLU were analyzed across iGlut-mAst co-cultures and iGlut and mAst mono-cultures in the SNP isogenic pair (A) and CLU overexpression assays (B). To ensure comparability among different cultures, iGlut cells in both mono- and co-cultures were seeded, collected, and processed simultaneously, with the same approach applied to mAst mono- and co-cultures. n=3 biological replicates per group from one clone of CD05 line in one independent differentiation, and C/C of CD05 line was used in CLU overexpression assay. (C) The representative images from CD07 line showing LipidTox and NeuN co-staining analysis. NeuN signals were utilized to create a mask using the CellProfiler program, which excluded any contaminated signals from dead cells in both LipidTox and DAPI staining. The LipidTox signals were then quantified, and cell numbers were determined based on DAPI signals. Data, mean±SEM. Scale bars are indicated in corresponding images. * p<0.05, ** p< 0.01, *** p< 0.001, and **** p< 0.0001. Figure S7. Validation of neuronal CLU as a lipid shuttle in lipid transfer system, related to Fig. 5. (A) A schematic diagram illustrating the experimental setup: Flag-tagged CLU was overexpressed in iGlut using AAV (refer to Fig. 3G), followed by co-culture with mAst in the lipid transfer system (refer to Fig. 5E). Specifically, Day-5 iGlut were seeded on coverslips and infected with AAV-hCLU-Flag at a multiplicity of infection (MOI) of 105 on Day 8. From Day 26 to Day 28, iGlut were co-cultured with mAst pre-seeded on separate coverslips (positioned above with Parafilm separators). GFAP staining was performed to confirm mAst identity, while Flag and MAP2 staining verified successful AAV infection. Representative images from CD05 line. (B) Co-staining of BD493/563 and Flag in mAst was conducted to confirm the co-localization of neuronal CLU and lipid droplets (LDs) in mAst. (C) Validation of 2nd antibody (against Flag antibody) specificity in mAst from experiment (A). (D) Exclusion of gravity effects was tested by reversing the system in (A), placing the mAst coverslip at the bottom and the iGlut coverslip at the top. Representative images from CD05 line. (E) Validation of Flag antibody specificity in only mAst culture. Figure S8. Neuronal CLU facilitates lipid transfer to astrocytes and LD accumulation, related to Fig. 6. (A) The process of cell segmentation in iGlut-mAst co-cultures, related to Fig. 6A. (B) LDs staining and quantification by BD493/503 in day-30 iGlut-mAst co-cultures. n=5-6 coverslips per group (2 clones per line, 2-3 coverslips for each clone with 4-6 images per coverslip, and shown are example images of CD07) from one independent differentiation of each line. Violin plots are shown with data median and interquartile range. Other data, mean±SEM. Scale bars are indicated in corresponding images. * p<0.05, ** p< 0.01, *** p< 0.001, and **** p< 0.0001. Figure S9. Transcriptomic data provide mechanistic support to lipid accumulation and ROS homeostasis in mAst, related to Figs. 6 and 7.(A) Digitonin permeabilization (~0.01%) does not affect CellROX staining (representative images from CD05 co-cultures). (B) iGlut cell numbers in CellRox Staining. n=25-29 images per group (2 clones per line, 2-3 coverslips for each clone with 2-5 images per coverslip) from two independent differentiations of each line. (E) ELISA measurement of CLU level from supernatant after immunodepletion, related to Fig. 7C. n=3 technical replicates per group, all from one supernatant collection of CD07 line. (D) A Sankey plot depicts glutathione metabolism and oxidative stress related pathways in mAst from Wiki pathway analysis (E) Lysosome and autophagy related GO items are enriched in DE gene lists of iGlut and mAst. Violin plots are shown with data median and interquartile range. Other data, mean±SEM. Scale bars are indicated in corresponding images. * p<0.05, ** p< 0.01, *** p< 0.001, and **** p< 0.0001. Figure S10. Raw images for all western blot.

附加文件1. 图S1：rs1532278对转录因子结合（TF-binding）及CLU表达调控作用的生物信息学与实验验证，与图1相关。(A) UCSC hg38基因组浏览器中rs1532278周围的Multiz序列比对及phyloP保守性分析（涵盖470种哺乳动物）。(B) JASPAR预测的rs1532278处转录因子结合位点，以及iGlut中这些转录因子的表达水平（23个预测转录因子中有10个可通过RNA测序（RNA seq）检测到）。CPM，每百万reads计数。(C) 显示所有三种基因型iGlut的代表性图像（CD07系），与图1D（底部面板）相关；GFAP和HuNu（人核抗原）染色显示iGlut-mAst共培养体系中HuNu和MAP2染色对iGlut的特异性。(D) 在iGlut-mAst共培养体系中，T/T与C/C携带者的iGlut分化效率无差异，且Ki67阴性染色表明这些神经元中未观察到增殖细胞。HuNu+，人细胞。每组n=5张盖玻片，来自CD05和CD07系各一次分化实验（每系2-3个克隆，每个克隆1张盖玻片，每张盖玻片4-5张图像）。(E) CD07系第30天iGlut-mAst共培养体系的DRGX染色质免疫沉淀-定量聚合酶链反应（ChIP-qPCR）。每组n=3个生物学重复，来自CD07系一个克隆的一次独立分化实验。(F-G) 第30天纯iGlut（C/C）培养体系中的ISL2小干扰RNA（siRNA）敲低实验。siRNA转染72小时后的样本用于定量聚合酶链反应（qPCR）。每组n=3个生物学重复，来自每系一个克隆的一次独立分化实验。(H) 纯iGlut培养体系中的CLU mRNA水平。每组n=6个生物学重复（每系2-3个克隆，每个克隆2-3个生物学重复），来自每系两次独立分化实验。(I) 酶联免疫吸附测定（ELISA）检测纯iGlut培养上清液中的sCLU水平。每组n=4个生物学重复（每系2个克隆，每个克隆2个生物学重复），来自每系两次独立分化实验。(J) iGlut-mAst共培养体系中mAst的CLU mRNA水平。每组n=4个生物学重复（每系2个克隆，每个克隆2个生物学重复），来自每系两次独立分化实验。(K) 用于（L）中检测CLU mRNA水平的三个qPCR引物组，可捕获大多数CLU转录本异构体（14/17）。仅使用CD07系的iGlut-mAst共培养体系。每组n=6个生物学重复（2个克隆，每个克隆3个生物学重复），来自两次独立分化实验。(M) rs1532278未改变CD05和CD07系同源对iGlut（或与mAst共培养时）中CLU相邻基因SCARA3的表达。每组n=10个生物学重复（每系2-3个克隆，每个克隆2个重复），来自CD05和CD07系各两次独立分化实验。(N) CLU染色的代表性图像（CD05系），与图1K和1L相关。数据表示为平均值±标准误。* p<0.05，** p<0.01，*** p<0.001，**** p<0.0001。图S2：CRISPR/Cas9编辑的诱导多能干细胞（iPSC）系的质量控制，与图1相关。(A) CD05和CD07系编辑等位基因一个克隆中，gRNA存在2-3个错配的潜在脱靶位点（Benchling预测）的Sanger测序结果。未检测到脱靶编辑。(B) 利用CRISPR编辑系同源对（CD05和CD07；仅分析用于细胞功能实验的iPSC克隆）iGlut的RNA seq数据进行eSNP核型分析。左面板：沿基因组的SNP等位基因比率（每个等位基因的RNA seq reads）移动平均值；右面板：每条染色体所有常见SNP的杂合性片段。未发现染色体异常。(C) 尸检脑转录组数据集hub（https://cellxgene.cziscience.com/）中CLU、ISL2和DRGX的表达。(D-E) CRISPR/Cas9编辑iPSC系同源对（供体系CD05和CD07）分化的iAst中CLU mRNA水平。S100β和波形蛋白的代表性免疫荧光染色（CD05系）显示星形胶质细胞的身份和纯度。每组n=10个生物学重复（每系2-3个克隆，每个克隆2个重复），来自CD05和CD07系各两次独立分化实验。数据表示为平均值±标准误。图S3：CLU基因座rs1532278位点携带TT或C/C等位基因的iGlut的突触与电生理特性，与图2相关。(A) SYP和PSD-95 puncta密度定量。每组n=15-17个神经元（每张盖玻片1-2个神经元，每个克隆5-6张盖玻片，每系2个克隆），来自每系两次独立分化实验。(B-C) 蛋白质印迹（Western blotting）显示第30天纯iGlut培养体系中PSD-95和SYP水平。每组n=4个生物学重复（每系2个克隆，每个克隆2个生物学重复），来自每系两次独立分化实验。(D-E) 微电极阵列（MEA）中的爆发次数和同步性分析。AUNCC：归一化互相关下面积。每组n=9-12个生物学重复（每系2-3个克隆，每个克隆3-4个生物学重复），来自每系两次独立分化实验。数据表示为平均值±标准误。* p<0.05，** p<0.01，*** p<0.001，**** p<0.0001。图S4：携带rs1532278侧翼开放染色质区域缺失（OCR deletion）或外源性CLU过表达的神经元表征，与图3相关。(A) Sanger测序追踪CRISPR/Cas9工程化的纯合OCR缺失（CD07系代表性结果）。(B) 纯iGlut培养体系中CLU mRNA水平（每组n=6个生物学重复，来自两次独立分化实验，每系2个克隆，每个克隆3个生物学重复）和sCLU水平（每组n=4个生物学重复，来自两次独立分化实验，每系2个克隆，每个克隆2个生物学重复）。(C) 第23-25天纯iGlut培养体系中神经元CLU的免疫荧光染色。(D) 面板（C）中CLU染色的定量。每组n=7张盖玻片（每系2个克隆，每个克隆3-4张盖玻片，每张盖玻片3-5张图像；显示CD05系示例图像），来自每系两次独立分化实验。(E-F) 第30天iGlut-mAst共培养体系中SYP的蛋白质印迹。每组n=4个生物学重复（每系2个克隆，每个克隆2个生物学重复），来自每系两次独立分化实验。(G-H) MEA中的神经元网络爆发次数和同步性分析。AUNCC：归一化互相关下面积。每组n=5-10个生物学重复（每系2个克隆，每个克隆2-5个生物学重复），来自每系两次独立分化实验。(I) MAP2和CLU（Flag标签）的免疫荧光染色（CD07系）以确定AAV-hCLU转导效率（~100%）。(J) qPCR验证CLU过表达。每组n=3个生物学重复，来自CD07系一个克隆的一次独立分化实验。(K) MEA中的网络爆发次数和同步性分析。AUNCC：归一化互相关下面积。每组n=3个生物学重复（均来自CD07系一个克隆），来自一次独立分化实验。数据表示为平均值±标准误。比例尺见对应图像。* p<0.05，** p<0.01，*** p<0.001，**** p<0.0001。图S5：iGlut-mAst共培养体系的转录组分析及验证，与图4相关。(A) Salmon处理的iGlut数据集与我们先前发表的iGlut（CD05和CD07）单细胞数据集及其他发表的尸检脑不同细胞类型（包括兴奋性神经元、抑制性神经元、小胶质细胞、星形胶质细胞、少突胶质细胞和少突胶质前体细胞）单细胞数据集的相关性分析。Ast：星形胶质细胞，ex：兴奋性神经元，ih：抑制性神经元，mic：小胶质细胞，Oli：少突胶质细胞，OPC：少突胶质前体细胞。(B) 在≥75%样本中表达的iGlut和mAst所有转录本的多维尺度分析（MDS）。每组n=9个生物学重复，来自每系两次独立分化实验，每个克隆2-3个生物学重复，每系2个克隆，共2个系。(C) ELISA定量iGlut-mAst共培养体系和纯iGlut培养上清液中的Aβ水平。每组n=4个生物学重复（每系2个克隆，每个克隆2个重复），来自每系两次独立分化实验。(D) iGlut中显著下调基因集的基因本体论（GO）生物学过程富集分析。蓝色星号表示神经元形态相关通路。(E) RNA seq分析中iGlut中SYP、PSD-95和CLU表达水平的验证。(F) 通过硅基qPCR方法排除小鼠CLU reads干扰，验证人CLU表达水平。小提琴图显示数据的中位数和四分位距，其他统计图显示平均值±标准误。* p<0.05，** p<0.01，*** p<0.001，**** p<0.0001。图S6：iGlut-mAst共培养体系中小鼠CLU对人CLU表达的影响评估（与图1、3、4、6相关）及纯iGlut培养体系中LipidTox染色的分析流程（与图5相关）。(A-B) 在SNP同源对（A）和CLU过表达实验（B）中，分析iGlut-mAst共培养体系及iGlut、mAst单培养体系中人CLU（hCLU）和小鼠CLU（mCLU）的mRNA表达水平、细胞外（分泌）水平和细胞内水平。为确保不同培养体系间的可比性，单培养和共培养中的iGlut细胞同时接种、收集和处理，mAst单培养和共培养也采用相同方法。每组n=3个生物学重复，来自CD05系一个克隆的一次独立分化实验，CLU过表达实验使用CD05系的C/C基因型。(C) CD07系的代表性图像显示LipidTox和NeuN共染色分析。利用CellProfiler程序将NeuN信号转化为掩模，排除LipidTox和DAPI染色中死细胞的污染信号。随后定量LipidTox信号，并基于DAPI信号确定细胞数量。数据表示为平均值±标准误。比例尺见对应图像。* p<0.05，** p<0.01，*** p<0.001，**** p<0.0001。图S7：神经元CLU作为脂质转运系统中脂质穿梭体的验证，与图5相关。(A) 实验设置示意图：利用AAV在iGlut中过表达Flag标签的CLU（参考图3G），随后与脂质转运系统中的mAst共培养（参考图5E）。具体而言，第5天iGlut接种于盖玻片，第8天以105的感染复数（MOI）感染AAV-hCLU-Flag。第26-28天，iGlut与预先接种于另一盖玻片的mAst共培养（用Parafilm分隔层置于上方）。GFAP染色确认mAst身份，Flag和MAP2染色验证AAV感染成功。CD05系代表性图像。(B) BD493/563与Flag在mAst中的共染色，以确认神经元CLU与mAst中脂滴（LDs）的共定位。(C) 实验（A）中mAst的二抗（抗Flag抗体）特异性验证。(D) 通过反转（A）中的系统（将mAst盖玻片置于底部，iGlut盖玻片置于顶部）测试重力效应排除。CD05系代表性图像。(E) 仅mAst培养体系中Flag抗体的特异性验证。图S8：神经元CLU促进脂质向星形胶质细胞转运及脂滴积累，与图6相关。(A) iGlut-mAst共培养体系中的细胞分割流程，与图6A相关。(B) BD493/503对第30天iGlut-mAst共培养体系中脂滴的染色及定量。每组n=5-6张盖玻片（每系2个克隆，每个克隆2-3张盖玻片，每张盖玻片4-6张图像；显示CD07系示例图像），来自每系一次独立分化实验。小提琴图显示数据的中位数和四分位距。其他数据表示为平均值±标准误。比例尺见对应图像。* p<0.05，** p<0.01，*** p<0.001，**** p<0.0001。图S9：转录组数据为mAst中脂滴积累和ROS稳态提供机制支持，与图6和7相关。(A) 洋地黄皂苷透化（~0.01%）不影响CellROX染色（CD05共培养体系代表性图像）。(B) CellRox染色中的iGlut细胞数量。每组n=25-29张图像（每系2个克隆，每个克隆2-3张盖玻片，每张盖玻片2-5张图像），来自每系两次独立分化实验。(C) 免疫耗竭后上清液中CLU水平的ELISA测定，与图7C相关。每组n=3个技术重复，均来自CD07系一次上清液收集。(D) Sankey图描绘Wiki通路分析中mAst的谷胱甘肽代谢和氧化应激相关通路。(E) iGlut和mAst差异表达基因列表中溶酶体与自噬相关的GO条目富集。小提琴图显示数据的中位数和四分位距。其他数据表示为平均值±标准误。比例尺见对应图像。* p<0.05，** p<0.01，*** p<0.001，**** p<0.0001。图S10：所有蛋白质印迹实验的原始图像。