Impairment of DET1 causes neurological defects and lethality in mice and humans

Abstract

COP1 and DET1 are components of an E3 ubiquitin ligase that is conserved from plants to humans. Mammalian COP1 binds to DET1 and is a substrate adaptor for the CUL4A-DDB1-RBX1 RING E3 ligase. Transcription factor substrates, including c-Jun, ETV4, and ETV5, are targeted for proteasomal degradation to effect rapid transcriptional changes in response to cues such as growth factor deprivation. Here, we link a homozygous DET1^R26W mutation to lethal developmental abnormalities in humans. Experimental cryo-electron microscopy of the DET1 complex with DDB1 and DDA1, as well as co-immunoprecipitation experiments, revealed that DET1^R26W impairs binding to DDB1, thereby compromising E3 ligase function. Accordingly, human-induced pluripotent stem cells homozygous for DET1^R26W expressed ETV4 and ETV5 highly, and exhibited defective mitochondrial homeostasis and aberrant caspase-dependent cell death when differentiated into neurons. Neuronal cell death was increased further in the presence of Det1-deficient microglia as compared to WT microglia, indicating that the deleterious effects of the DET1 p.R26W mutation may stem from the dysregulation of multiple cell types. Mice lacking Det1 died during embryogenesis, while Det1 deletion just in neural stem cells elicited hydrocephalus, cerebellar dysplasia, and neonatal lethality. Our findings highlight an important role for DET1 in the neurological development of mice and humans.

Keywords: COP1; DET1; E3 ligase; neurodevelopment; ubiquitin.

Fig. 1.

Identification of human patients bearing mutant DET1 and the generation of mice lacking Det1 in neural stem and progenitor cells. (A) Patient pedigree chart. Mutation screening was done for the patients as well as their parents and healthy siblings. Patients are indicated by red filled symbols. (B) DNA sequence of the DET1^R26W missense patient mutation. (C) Littermates aged 4 wk. (D) Hematoxylin and eosin-stained brain sections from mice aged 4 wk. Results representative of 5 Det1^{fl/fl or fl/−} Nestin.cre and 4 Det1^{fl/+ or +/+} Nestin.cre mice aged 3 to 4 wk. (Scale bars: cortex, 800 μm; cerebellum, 200 μm.) (E) Immunolabeling of c-JUN (brown) in brain sections from mice aged 4 wk. (Scale bars: cortex, 100 μm; cerebellum, 200 μm.) Results representative of 3 Det1^{fl/fl or fl/−} Nestin.cre and 3 control (Det1^fl/+Nestin.cre or Det1^+/+) mice aged 4 wk.

Fig. 2.

Differentiation of WT and DET1^R26W iPSCs into neurons. (A) Representative WT and DET1^R26W cells during NGN2-driven iPSC-to-neuron differentiation. (Scale bar, 100 μm.) (B) Western blots of two clones of DET1^R26W iPSCs. (C) ETV4 and ETV5 mRNA expression in iPSCs. Symbols represent biological replicates. Lines indicate median values. (D) Cell confluency during NGN2-driven iPSC-to-neuron differentiation. Initial seeding density = 4,000 cells/well. Data are the mean ± SEM (n = 4 biological replicates). ****P < 0.0001 by the unpaired t test. (E) LDH detected in the media of differentiating iPSCs. Data are the mean ± SEM. n = 3 biological replicates. ****P < 0.0001 by the unpaired t test. (F) Percentage of LDH released from differentiating iPSCs at day 7. Data are the mean ± SEM. n = 4 biological replicates for WT and DET1^R26W. (G) Number of dead cells staining with Cytotox Red (4,632, Sartorius) at day 7. Data are the mean ± SEM. n = 6 to 18 biological replicates. Emr, emricasan. (H) Cell viability determined by CellTiter-Glo assay (G7570, Promega) at day 14. Initial seeding density = 6,000 cells/well. Data are normalized to confluency and are mean ± SEM (n = 6 to 18 biological replicates).

Fig. 3.

Enhanced neurotoxicity of Det1-deficient microglia in vitro. (A) Immunolabeling of MAP2 and TUBB2A in day 14 iPSC-derived neurons. (Scale bars, 50 μm.) Data are the mean ± SEM. n = 6 to 16 biological replicates. **P < 0.01 by the unpaired t test. (B) Cytokines and chemokines secreted by mouse microglia. Cells were treated with 100 ng/mL LPS overnight. Circles represent independent experiments. WT untreated, n = 2; WT LPS treated, n = 3; KO untreated, n = 3; KO LPS treated, n = 4. Bars, mean ± SEM. **P-value < 0.01, ***P-value < 0.005, ****P-value < 0.001 by the t test. (C) Representative cocultures of iPSC-derived neurons (red) and mouse microglia (green). (Scale bars, 100 μm.) MAP2 neuronal staining (red) is graphed. Bars indicate the mean ± SEM (n = 2 to 6 biological replicates). *P < 0.05, **P < 0.01 and ***P < 0.005 by the unpaired t test.

Fig. 4.

RNA sequencing and proteomics of iPSC-to-neuron differentiation. (A) Volcano plot comparing the transcriptomes of WT and DET1^R26W iPSCs. The FDR-based top 50 significantly up- or down-regulated genes are highlighted. Data collected from three biological replicates. (B) Normalized abundances of the proteins in cluster 1. Plot displays the median (line), and the lower (0.25) and upper (0.75) quartiles (gray area). Data collected from three biological replicates. (C) Overrepresentation analysis of the proteins in cluster 1. Proteins were annotated with GO terms for Biological Process, Molecular Function, and Cellular Component. −log10 FDR and enrichment scores calculated using Fisher’s exact test. The top five most significant terms for each category are shown. (D) Oxygen consumption rates (OCR) in neurons at baseline and under stress. Data are the mean ± SD (n = 4 biological replicates per genotype). **P < 0.01 by the unpaired t test. (E and F) Overrepresentation analysis of transcripts and proteins of WT and DET1^R26W neurons. Proteins were annotated with GO Biological Process terms. −log10 Benjamini–Hochberg adjusted P-values (FDR) and scores determined using the 1D enrichment test. (G) Transcription factor enrichment analysis of the FDR-based top 100, 200, and 500 differentially expressed genes in DET1^R26W versus WT iPSCs. Heatmap illustrates the enrichment scores of the top 10 overlapped transcription factors.

Fig. 5.

DET1^R26W fails to degrade substrates and interact with DDB1. (A) Western blots of A375 DET1 KO cells stably reconstituted with DET1 variants, treated with cobimetinib (1 µM, 1 h). Results representative of two independent experiments. (B) Workflow for the identification of DET1-associated proteins. (C) Affinity-enrichment MS analysis quantifying proteins associated with WT DET1 versus DET1^R26W. Data collected from three biological replicates. (D) Western blots of DET1-containing complexes immunoprecipitated from reconstituted A375 DET1 KO cells. Cells treated with DMSO or cobimetinib (1 µM, 1 h). Results representative of two independent experiments. (E) Cryo-EM structure of the DDB1:DDA1:DET1 ternary complex at an average resolution of 2.9 Å. DDB (ivory). DDA1 (teal). DET1 (magenta). DET1 forms a partial beta propeller while two amino terminal helices project into the DDB1 cleft between BPA and BPC domains. (F) The Hbox motif of human and mouse DET1 with the patient variant highlighted red. (G) A cutaway view of the DET1 Hbox helix (residues 17 to 27) highlights interactions between key residues of DET1 and DDB1. Salient hydrogen bonds are indicated with dashed lines. Some foreground sections of DDB1 cartoons have been hidden for clarity. (H) An example of the density map quality at the region of interest where local resolution of the maps approaches 2.5 Å. Here, key DET1:DDB1 interaction is mediated in part by the patient variant position R26.