REST and Neural Gene Network Dysregulation in iPSC Models of Alzheimer's Disease

Abstract

The molecular basis of the earliest neuronal changes that lead to Alzheimer's disease (AD) is unclear. Here, we analyze neural cells derived from sporadic AD (SAD), APOE4 gene-edited and control induced pluripotent stem cells (iPSCs). We observe major differences in iPSC-derived neural progenitor (NP) cells and neurons in gene networks related to neuronal differentiation, neurogenesis, and synaptic transmission. The iPSC-derived neural cells from SAD patients exhibit accelerated neural differentiation and reduced progenitor cell renewal. Moreover, a similar phenotype appears in NP cells and cerebral organoids derived from APOE4 iPSCs. Impaired function of the transcriptional repressor REST is strongly implicated in the altered transcriptome and differentiation state. SAD and APOE4 expression result in reduced REST nuclear translocation and chromatin binding, and disruption of the nuclear lamina. Thus, dysregulation of neural gene networks may set in motion the pathologic cascade that leads to AD.

Keywords: Alzheimer’s disease; REST; apolipoprotein E; epigenetic; induced pluripotent stem cell; neural differentiation; neural progenitor; neurogenesis; organoid; polycomb.

Figure 1.. Differentiation and Characterization of SAD Neural Progenitors

(A) Schematic of iPSC differentiation to neural progenitor (NP) cells. (B) Representative NP cells expressing the neural stem cell markers Nestin, Musashi, and SOX2. NL1: NP cells generated from an iPSC line derived from a normal control. SAD1: NP cells generated from an iPSC line derived from a patient with sporadic Alzheimer’s disease. Scale bar, 50 mm. (C) Quantification of neural stem cell markers Nestin, Musashi, and SOX2 shown in (B) using MetaMorph software. Data represent the mean ± SEM from 5 fields per cell line. Each point represents one cell line with 3 replicates (Table S2). (D) Unsupervised hierarchical clustering of genes differentially expressed between sporadic Alzheimer’s disease (SAD) and NL NP cells. Differentially expressedgenes (rows) and cells (columns) were clustered, and gene expression, transformed to a z-score per gene, is represented in a heatmap. NL, n = 5; SAD, n = 5. (E) Gene Ontology (GO) biological process groups enriched in genes upregulated in SAD NP cells. (F) Schematic of neurogenesis and neuronal differentiation based on the adult subgranular zone.

Figure 2.. Accelerated Differentiation of SAD Neural Progenitors

(A) Elevated expression of neurogenesis genes in NP cells derived from SAD iPSCs. Shown is mRNA expression determined by qRT-PCR. Data represent themean fold change ± SEM relative to the mean expression level of the NL NP cell lines after normalizing to GAPDH expression. Each dot represents one cell line with 3 replicates (Table S2). (B) Decreased mRNA expression of APOE and CCND2 in SAD NP cells as determined by qRT-PCR. Data represent the mean ± SEM relative to the mean expression level of the SAD lines normalized to GAPDH expression. Each dot represents one cell line with 3 replicates (Table S2). (C) Light scatterplots from FACS analysis using the neural lineage differentiation cell surface marker CD24 in NP cells. (D) Percentage of NP cells with high CD24 levels (inner boxed area in C) in NL and SAD NP cell lines (Table S2). Data represent the mean ± SEM; n = 3. (E) Confocal fluorescence microscopy of representative NL3 and SAD3 NP cells after labeling for doublecortin (DCX) (red), Nestin (green), and DAPI (blue). Scalebar, 100 μm. (F) Representative immunofluorescence image of SAD3 showing neuronal morphology in a subset of cells in SAD NP cell cultures. The top panel is double-labeledfor the NP cell marker Nestin (green) and the early neuronal marker DCX (red). The bottom panel is double-labeled for DCX (red) and another early neuronal marker b-tubulin III (green). Both panels are also labeled with DAPI (DNA, blue). Scale bar, 50 μm. (G) Quantification of western blot analysis using DCX and tau antibodies in NL and SAD NP cells (Table S2). Protein levels were normalized to the loading controls actin and GAPDH. Data represent the mean ± SEM; n = 3.. (H) Levels of Aβ40 in 5-day conditioned cell culture medium from NL and SAD NP cells (Table S2) determined by ELISA and normalized to total protein. Values represent mean ± SEM of n = 3. (I) Decreased Aβ40 production in SAD NP cells after treatment with the γ-secretase inhibitors DAPT (2 μM) or Compound E (CE, 20 nM), or the BACE1 inhibitor IV (0.2 μM). (J) Fold change in mRNA expression of DCX, CD24, and ASCL1 in SAD versus NL NP cells following inhibition of Aβ production by DAPT (2 μM) or Compound E (CE) (20 nM), or the BACE1 inhibitor IV (0.2 μM). Values represent the mean ± SEM; n = 3 independent replications from one NL and one SAD NP cell line. *p < 0.05, **p < 0.01, and ***p < 0.001 by the MannWhitney U test.

Figure 3.. Accelerated Maturation and Increased Excitability of SAD Neurons.

(A) Increased synapsin-1-positive puncta in SAD neurons (days in vitro [DIV], 6 weeks). Representative NL4 and SAD2 neurons were labeled for synapsin 1 (red) or MAP2 (green). Data represent the mean ± SEM from 5–6 fields per cell line. (B) Assessment of timing of action potential (AP) induction in NL and SAD neurons. Representative profiles of induced APs from SAD and NL neurons (top) andmeasured APs after 4.5–15 weeks of differentiation (bottom). (C) AP spike number in SAD and control neurons after 4.5–12 weeks of differentiation. Some SAD and NL NP cells were co-cultured with rat astrocytes and thenassessed after 10–12 weeks of neuronal differentiation (indicated by red and gray bars). (D) Maximum AP amplitude of NL and SAD neurons determined relative to either resting membrane potential (RMP) or threshold after 10–12 weeks ofdifferentiation. (E) Representative current profiles following cell depolarization by voltage steps from −50 to +55 mV. (F and G) Minimum inward (Sodium Current) (F) and maximum outward (Potassium Current) (G) currents were measured in NL and SAD neurons after the indicated duration of differentiation. (C–G) n = 4–43 neurons; p values: *p < 0.05, **p < 0.01, and ***p < 0.001 by the Mann-Whitney U test except for (D) (Student’s t test). Refer to Table S2 for experimental details.

Figure 4.. Altered Regulation of REST and Accelerated Neuronal Differentiation in SAD NP Cells

(A and B) Transcription factor prediction based on enrichment in genes that are differentially expressed in SAD versus NL NP cells (A) and neurons (B) using the ENCODE ChIP-seq database. Predictions are stratified for genes that were upregulated or downregulated, and all genes in the SAD versus NL comparison. (C) REST-RE1 site binding is markedly reduced in SAD NP cells. REST ChIP-PCR (REST antibody; Millipore; #17–641) was performed for RE1 sites in SNAP25, SCN3B, CALB1, DCX, and ASCL1. N = 2 biological replicates per line for 10 NL and 7 SAD NP cell lines. (D) FACS analysis and quantification of nuclear REST levels in NL and SAD NP cells. Data represent the mean ± SEM from 3–5 replicates per line for 10 NL and 7 SAD NP cell lines. (E) Quantification of REST nuclear intensity (left) and fraction of total cellular REST signal in the nucleus (right) by confocal immunolabeling of REST (anti-REST;Millipore; #07–579). Data represent the mean ± SEM from 4 replicates in 3 SAD and 3 control NP cell lines. (F) Immunolabeling of representative SAD4 NP cells for REST (Millipore; #07–579), nestin, and DCX. The DCX-positive soma is delineated by a dotted circle. Scalebar, 50 μm. (G) Quantification of REST nuclear intensity in DCX-positive and DCX-negative cells. Data represent the mean ± SEM from 3 SAD NP cell lines. (H) REST knockdown by two previously described REST shRNAs (sh-RESTa and sh-RESTb; Lu et al., 2014) increases DCX mRNA levels in the control NP cell line NL3. DCX expression levels were normalized to expression in the control shRNA-treated culture (sh-Scramble). Values represent the mean ± SEM from 3 experiments. (I) Quantification of DCX mRNA expression in NL and SAD NP cell lines after overexpression of REST. Cells were transduced with lentiviral vectors encoding GFP (Control) or REST (REST-OE) and analyzed by qRT-PCR. Data represent fold change relative to the mean of the DCX mRNA levels in the NL lines transduced with the GFP control lentivirus and normalized to GAPDH. Values represent the mean ± SEM from 3 experiments. (J) Quantification of gene expression in SAD NP cells after overexpression of REST. Cells were transduced with lentiviral vectors encoding GFP (GFP) or REST(REST-OE) and analyzed by qRT-PCR. Data represent the mean from 3 experiments ± SEM. (K) Representative confocal immunofluorescence images of DCX-positive neuronal cells and nestin (green) in SAD3 NP cell cultures transduced with RESTlentivirus (SAD+REST) or GFP control lentiviral particles (SAD). Scale bar, 50 μm. (L) Quantification of DCX-positive cells in the presence (+) or absence () of REST overexpression. Data represent the mean ± SEM from four experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 by Student’s t test.

Figure 5.. Altered Regulation of REST and Accelerated Neuronal Differentiation in Gene-Edited NP Cells Expressing APOE4

(A) Expression of DCX, ASCL1, and MAPT in APOE4 isogenic (E4 ISO) versus parental APOE3 (E3) NP cells. Data represent the mean ± SEM from 3 biological replicates of 1 APOE3 parental line and 2 individual APOE4 isogenic clones. Expression was determined by droplet digital PCR (ddPCR) and normalized to TBP. Isogenic lines were derived by gene editing from the APOE3/E3 and APOE4/E4 lines AG09173 and AG10788, respectively (see Table S1). (B) Expression of DCX, ASCL1, and MAPT in APOE4 parental (E4) versus isogenic APOE3 (E3 ISO) NP cells. Data represent the mean ± SEM from 3 biological replicates of 1 APOE4 parental line and 2 individual APOE3 isogenic clones, and was normalized to TBP. (C) Immunolabeling for nestin (red) and DCX (green) in isogenic APOE NP cells. Shown are E3 parental NP cells (E3), E4 isogenic NP cells (E4 ISO), E4 parental NPcells (E4), and E3 isogenic NP cells (E3-ISO). Scale bar, 100 μm. (D) Quantification of the immunolabeling shows a marked increase in DCX-positive cells in both parental APOE4 and isogenic APOE4 cell lines relative to APOE3. Data represent the mean ± SEM from 5 fields from 3 independent biological replicates. (E and F) Quantification (E) of western blot analysis (F) of DCX protein levels in 2 E3-ISO lines compared to parental E4 and 2 E4-ISO lines compared to E3. (G) Individual cell analysis of REST in the nucleus and cytosol, as well as the fraction of total cellular REST in the nucleus (Nuclear Fraction/Total) after REST immunolabeling (REST Antibody; Millipore; #07–579). *p < 0.05, **p < 0.01 and ***p < 0.001 by Student’s t test.

Figure 6.. Altered Gene Regulation and Neuronal Differentiation in APOE4 Cerebral Organoids

(A) Unsupervised hierarchical clustering of genes differentially expressed between parental APOE4 and isogenic APOE3 cerebral organoids after 46 days of maturation. Differentially expressed genes (rows) and organoids (columns) were clustered, and gene expression was transformed to a z-score and represented in the heatmap. Parental APOE4 (E4), n = 3; isogenic APOE3 (E3 ISO), n = 3. (B) GO biological process groups enriched in genes upregulated in parental APOE4 compared to isogenic APOE3 organoids. (C) Expression of DCX, ASCL1, MAPT and APOE mRNA in parental APOE4 (E4) versus isogenic APOE3 (E3 ISO) organoids. Data represent the mean ± SEM from 3 experiments using a total of 45 individual organoids (DIV, 46 days). (D) Western blot analysis of REST (Millipore; #07–579), DCX, total tau (Tau5), p-tau(Ser202), and APOE in parental APOE4 and isogenic APOE3 organoids. Eachlane was loaded with protein lysate from 3 organoids (left). Normalization to tubulin or actin shown in adjacent western blots was used for quantification (right). Data represent the mean ± SEM from 3 individual lysates. (E) Transcription factor prediction based on enrichment in APOE4 differentially expressed genes using the ENCODE ChIP-seq database. Predictions are stratifiedfor genes that were upregulated or downregulated, and all genes in the APOE4 versus APOE3 comparison. (F) Venn diagram illustrating the overlap of APOE4-upregulated genes and REST targets based on RE1 motif analysis (see STAR Methods). Overlap significance was determined by Fisher’s exact test. For (C) and (D): *p < 0.05, **p < 0.01, and ***p < 0.001 by unpaired Student’s t test.

Figure 7.. Disruption of the Nuclear Lamina in SAD and APOE4 NP Cells

(A) Representative confocal immunofluorescence microscopy of NL1 and SAD4 NP cell lines for lamin B. Dotted circles delineate nuclei represented in the highermagnification images. Arrowheads indicate nuclei with abnormal morphology. Scale bar, 50 μm. (B) Quantification of cells showing abnormal nuclear lamina morphology. Data represent the mean ± SEM from 3 biological replicates using 3 different NL and SAD NP cell lines. (C) Representative images from APOE isogenic NP cell lines after lamin B immunolabeling. Scale bar, 50 μm. (D) Quantification of cells with abnormal nuclear morphology. Data represent the mean ± SEM from 3 biological replicates using 2 different isogenic lines. (E) Representative images from NL3 and SAD2 neurons differentiated by doxycycline induced NGN2 overexpression after lamin B immunolabeling (neurons DIV,13 days). Scale bar, 50 μm. (F) Quantification of cells with abnormal nuclear morphology. Data represent the mean ± SEM from 3 NL and 4 SAD lines; *p < 0.05, **p < 0.01 and ***p < 0.001 by unpaired Student’s t test.