Fig 1: DEG in the V321L DG can be largely accounted for by a loss of nuclear back signaling. A, Top, Schematic of the experimental paradigm. P4–5 hippocampal cultures were maintained for 8–9 d after which they were treated with DMSO (vehicle control) or 20 µM DAPT overnight. On DIV9–10, cultures were stimulated with 20 nM soluble ErbB4 (sErbB4) or equal volume 1× PBS in culture media (vehicle control) for 4 h prior to RNA extraction. The four treatment conditions are shown: vehicle alone, sErbB4 alone, DAPT alone, DAPT + sErbB4. N = 3 platings from 6 to 8 mice from three litters per genotype for each condition. Bottom, Venn diagram showing overlap in the DEGs (FC >1.25 in either direction; post hoc corrected, p < 0.05). Local back signaling did not result in significant changes in gene expression. High overlap in the baseline-adjusted ErbB4 effect and the nuclear back signaling effect indicates a high level of baseline back signaling at DIV9–10 consistent with data shown in Figure 3B. Also see Extended Data Tables 6-1 and 6-2. B, Left, Heatmaps showing changes in gene expression in response to sErbB4 treatment compared with expression of the same genes in the WT DG tissue relative to V321L DG tissue for genes whose expression is changed in the same direction in vitro and in vivo. All genes detected in both datasets irrespective of the degree of FC are displayed. Each row represents a gene, and every 10th row is labeled with the gene name on either side of the heatmap. The genes are ordered from most downregulated (blue) to most upregulated (red) genes from the in vitro experiment. Color of the heatmap represents the FC in gene expression given by the legend displayed alongside. (Right) Heatmaps showing changes in gene expression in response to sErbB4 treatment compared with expression of the same genes in WT DG tissue relative to V321L DG tissue for genes whose expression is oppositely regulated in vitro compared with in vivo. Each row represents a gene, and every 10th row is labeled with the gene name on either side of the heatmap. The genes are ordered from most downregulated (blue) to most upregulated (red) genes from the in vitro experiment. Color of the heatmap represents the FC in gene expression given by the legend displayed alongside. C, DEGs were subjected to pathway enrichment analysis using IPA, which identified significantly enriched pathways represented by the DEGs as well as a z-score of direction in which these pathways are predicted to be altered based on the directions of changes in expression of the genes comprising each pathway. Each pathway is indicated by a circle with the size of the circle indicating the proportion of genes comprising that pathway in the IPA database that are represented in the DEG set. The color of the circles is according to the heatmaps displayed alongside indicating the adjusted p values. Red pathways are predicted to become upregulated, and blue pathways are predicted to become downregulated based on the aggregate gene expression changes effected by Nrg1 nuclear back signaling.
Fig 2: Generation of Nrg1 V321L knock-in mice. A, A schematic of the different modes of signaling engaged by Type III Nrg1. Cleavage by Bace1 is an essential step to generate a substrate for γ-secretase. The extracellular EGF-like domain of Nrg1 can interact with ErbB4 on neighboring cells to engage the canonical forward signaling by activating ErbB4. ErbB4 interaction can also result in activation of back signaling by Nrg1. Local back signaling engages PI3K signaling at the membrane, whereas cleavage by γ-secretase results in liberation of the C-terminal ICD, which can translocate to the nucleus–nuclear back signaling. The V321L mutation blocks cleavage by γ-secretase and is predicted to disrupt nuclear back signaling. B, A schematic diagram of the Nrg1 genomic structure. The Nrg1 gene encodes six families of isoforms as a result of alternative promoter usage. Types 1, 2, 4, and 5 contain Ig-like domains (encoded by Exons 3 and 4). Exon 7 is the unique 5′ coding exon for Type III Nrg1, encoding an N-terminal, cysteine-rich transmembrane domain. Exon 8, in combination with Exon 9 or 10, and various combinations of Exons 11, 12, and 13 encode an EGF-like domain common to all Nrg1 isoforms. Exon 13 encodes a common C-terminal transmembrane domain. The C-terminal ICD is encoded by Exons 14–17. The missense SNP that results in a valine-to-leucine substitution in the C-terminal transmembrane domain is shown above the gene. b′, Schematic diagram of the BAC clone used for generating the target construct corresponding to a region of the Nrg1 gene that comprises the entire Type III coding region. Below the BAC clone is a diagram of the targeting construct, including a 5.8 kbp left homology arm, a neoR cassette in the antisense orientation flanked by Frt sites, and a 2.5 kbp right homology arm. b″, Diagram of WT and mutant alleles (the mutant allele following flippase removal of the neo cassette). The C-terminal transmembrane domain sequence is underlined. Black arrows labeled “NDEL1” and “NDEL2” indicate approximate genomic locations of genotyping primers. C, An example genotyping gel of offspring from a het × het cross illustrating a WT, heterozygote and homozygote. D, The mutant mouse line was maintained by breeding heterozygotes. The genotype of pups from >100 litters was analyzed for deviation from the expected 1:2:1 ratio. No deviation was found. Mice with the genotype V/V are referred to as WT in the manuscript and L/L are referred to as V321L as denoted in parenthesis.
Fig 3: The V321L substitution decreases nuclear back signaling. A, Immunoblot of triplicate nuclear fractions isolated from pooled cortical and hippocampal lysates. Nrg1 ICD was detected using Santa Cruz Biotechnology antibody sc-348. Histone H3 served as a nuclear loading control and Na+/K+ ATPase as a marker for the membrane fraction (N = 3 mice/genotype). B, Immunoblot of two replicates of membrane fractions isolated from pooled cortical and hippocampal lysates. NRG1 ICD was detected using Santa Cruz Biotechnology antibody sc-348. Na+/K+ ATPase served as a marker for the membrane fraction; note the lack of the nuclear marker Histone H3 or cytoplasmic marker CyclophilinA indicating clean membrane preps. FL Nrg1 is indicated with a yellow arrowhead as “Nrg1-FL,” and the membrane-bound C-terminal fragment not cleaved by γ-secretase is indicated as “Nrg1 TM-CTF.” A positive control consisting of total lysate from N2A cells transfected with a Type III Nrg1 plasmid is shown in the lane labeled “C.” (N = 2 mice/genotype.) C, Left, Hippocampal neurons from WT (dark blue) and V321L (light blue) neonatal pups (P4) were cultured for 17 d in vitro and were stimulated with either vehicle (Veh), 20 nM sERBB4 (sB4), or 20 nM sErbB4 after a 24 h pretreatment with 20 µM of the γ-secretase inhibitor DAPT (DAPT). Neurons were fixed and stained using an antibody directed against the Nrg1 ICD and counterstained with DAPI. Scale bar, 10 µm. Right, Quantification of nuclear clusters of Nrg1-ICD. Neurons from WT mice show increased nuclear ICD clusters in response to sB4 stimulation, which is counteracted by pretreatment with DAPT (DAPT). Neurons from V321L mice do not respond to sB4 stimulation (N = 6–13 neurons, 3 platings/mouse, 3 mice/genotype; one-way ANOVA p values corrected for multiple comparisons using Tukey’s post hoc test; WT Veh vs WT sB4, p < 0.0001 (****); WT sB4 vs WT sB4 + DAPT, ****p < 0.0001; WT sB4 vs V321L Veh, ****p < 0.0001; WT sB4 vs V321L B4, ****p < 0.0001). All other comparisons are statistically not statistically significant. D, Cortical neurons from embryonic WT (dark blue) and V321L mice (light blue; E18.5) were cultured for DIV3 and were stimulated with soluble ErbB4 (sB4), PI3K inhibitor WM, γ-secretase inhibitor L-685,458 (L6), WM + B4, or L6 + B4. Neurons that underwent no drug treatments/sB4 stimulation are indicated as the control group (C). Neurons were fixed and axonal length was quantified. (Two-way ANOVA with Tukey’s post hoc correction, WT C vs WT B4, ****p = 0.0002; WT L6 vs WT L6 + B4, **p = 0.0047; V321L C vs V321L B4, ***p = 0.001; V321L WM vs V321L WM + B4, p = 0.1; V321L L6 vs V321L L6 + B4, *p = 0.03.) N = 20–37 neurons per genotype per condition. ns, not significant. E, Treatment and conditions same as in D. Quantification is for dendritic length. (two-way ANOVA w/ Tukey’s post hoc correction: WT C vs WT B4, **p = 0.002; WT WM vs WT WM + B4, ****p < 0.0001). N = 20–37 neurons per genotype per condition. ns, not significant.
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