Journal: Journal of neurochemistry

Article Title: The de novo Autism Spectrum Disorder RELN R2290C Mutation Reduces Reelin Secretion and Increases Protein Disulfide Isomerase Expression

doi: 10.1111/jnc.14045

Figure Lengend Snippet: (A) A heterozygous R2290C P19 cell line was generated by CRISPR-Cas9 genome editing and verified by sequencing that showed the expected mutation and silent changes indicated below. Silent mutations inactivate the PAM sequence and prevent further Cas9 cleavage in a P19 cell clone. (B) P19 cells grown as neurospheres in MEDII media produce extracellular Reelin by 8 days of differentiation. Restriction digest by PciI demonstrates the heterozygous R2290C mutation. (C) Neurospheres were cryosectioned and stained for Reelin (anti-Reelin, G10 antibody) or Tuj1, a neural-specific marker. (D) Neurosphere lysates and media were collected and analyzed by Western blot for Reelin (G10). (E) The fraction of Reelin in the media in R2290C+/− neurospheres (media/lysate) was significantly decreased compared to wild-type neurospheres (n=4, error bars = SEM, *p value <0.05 by t-test), while the levels of Reelin in the lysates (lysates/actin) trended higher but were not statistically different. (F) Q-PCR showed decreased XBP-1 splicing in R2290C+/− neurospheres compared to WT. (G) Reelin expression in WT and R2290C+/− neurspheres co-localizes with BIP, an ER protein. (H) Western blot of ER stress markers comparing WT and R2290C+/− neurosphere lysates. (I) PDI was significantly increased in R2290C+/− lysates compared to control (n=4, error bars = SEM, *p value <0.05 by t-test).

Article Snippet: RELN null-allele mice were maintained on a C57BL6 background and were from Jackson Laboratories.

Techniques: Generated, CRISPR, Sequencing, Mutagenesis, Staining, Marker, Western Blot, Expressing, Control

Journal: Journal of neurochemistry

Article Title: The de novo Autism Spectrum Disorder RELN R2290C Mutation Reduces Reelin Secretion and Increases Protein Disulfide Isomerase Expression

doi: 10.1111/jnc.14045

Figure Lengend Snippet: (A) Rendering of Reelin repeat 5 and 6 crystal structure (PDB 2E26; Chimera). The positions of the R2290C and R2292C mutations in the RXR motif of 5B are shown facing into the hydrophilic pore of RELN repeat 5. The corresponding locations of the RXR motif in an A repeat are also indicated. The position of the receptor-binding loops in 6A are indicated. (B) Alignment of the conserved region of the Reelin sub-repeat domains (Clustal Omega). The RXR consensus element is boxed and indicated below. (C) Schematic of Reelin structure showing an F-spondin homology domain, an N-terminal domain unique to Reelin, and 8 Reelin repeat domains. Each Reelin repeat domain contains an A and B sub-repeat domain (blue ovals) connected by an EGF domain (green ovals). ASD-associated mutations are indicated above their respective repeat. (D) Less Reelin was detected in media of HeLa cells transfected with full-length mutant (R2290C, R2290H, R1742Q, or R1742W) as compared to wild-type (WT) Reelin by Western blotting (anti-Reelin G10), whereas equal or greater amounts were observed in the cell lysates. One full-length (>400 kD), smeared Reelin band was observed in the cell lysates and three products (full length and two cleavage products: 410, 380, and 180 kD) were observed in the media, as expected (Jossin et al. 2004). Reelin is not expressed in vector-alone transfected HeLa cells (not shown). (E) The ratio of Reelin signal in the media (M) to Reelin lysate signal (L) was significantly greater for WT than mutant Reelin (n=4, error bars ± SEM, and * p value < 0.05 by 1-way ANOVA with Dunnett’s post hoc test). (F) WT Reelin metabolically labeled with 35S-methionine was most abundant in 293 cell lysates at the beginning of a cold chase, but by two hours the majority was detected in the cell media. In contrast, the majority of radiolabeled R2290C and R2290H mutant RELN remained in cell lysates over a 4h time course, as detected by phosphor-imaging of immunoprecipitated Reelin (anti-G10). (G) In the 293 cell lysate the Reelin counts (CL) at times indicated relative to counts at time 0 (C0) decreased more rapidly for WT Reelin than the R2290C and R2290H mutants (n=3, error bars ± SEM, * p<0.05 R2290C, # p<0.05 R2290H by 2-way ANOVA with Dunnett’s post hoc test). (H) In the media the Reelin counts (CM) at indicated times relative to lysate counts at time 0 (C0) increased more rapidly for WT than R2290C or R2290H (n=3, error bars ± SEM, * p<0.05 R2290C, # p<0.05 R2290H by 2-way ANOVA with Dunnett’s post hoc test).

Article Snippet: RELN null-allele mice were maintained on a C57BL6 background and were from Jackson Laboratories.

Techniques: Binding Assay, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Metabolic Labelling, Labeling, Imaging, Immunoprecipitation

Journal: Journal of neurochemistry

Article Title: The de novo Autism Spectrum Disorder RELN R2290C Mutation Reduces Reelin Secretion and Increases Protein Disulfide Isomerase Expression

doi: 10.1111/jnc.14045

Figure Lengend Snippet: ASD Patient RELN mutations by sex. Mutations identified in the RXR consensus sequence in ASD probands are listed along with patient gender, either male (M), female (F), or no report available (N/A). Exome Aggregation Consortium (ExAC) database allele count is listed for each variant ( exac.broadinstitute.org ). This database includes sequence information from 60,706 unrelated individuals from several case-control studies, excluding ASD, but including schizophrenia and bipolar patients.

Article Snippet: RELN null-allele mice were maintained on a C57BL6 background and were from Jackson Laboratories.

Techniques: Sequencing, Variant Assay, Mutagenesis

Journal: Journal of neurochemistry

Article Title: The de novo Autism Spectrum Disorder RELN R2290C Mutation Reduces Reelin Secretion and Increases Protein Disulfide Isomerase Expression

doi: 10.1111/jnc.14045

Figure Lengend Snippet: (A) Schematic of RRfps designed to test all ASD-associated RXR mutations identified in genetic studies thus far. The fusion protein is composed of a nidogen secretion signal, an N-terminal 3xFlag tag, a C-terminal HA tag and either wild-type or mutant versions of Reelin repeat 4, 5, 6, or 7. (B) Representative Western blot of RRfp 5AB for wild-type and mutant constructs. All the RXR mutants are expressed and easily detected in the cell lysates of transfected HeLa cells at comparable levels to the WT counterpart. However, in cell media the mutant proteins are under-represented and barely detectable by Western blotting (anti-Flag tag). (C) All ASD mutant RRfps showed decreased extracellular Reelin ratios (media/lysate) than their respective wild-type controls (n≥3, error bars ± SEM, * p value < 0.05 by 1-way ANOVA with Dunnett’s post hoc test).

Article Snippet: RELN null-allele mice were maintained on a C57BL6 background and were from Jackson Laboratories.

Techniques: Mutagenesis, Western Blot, Construct, Transfection, FLAG-tag

Journal: Journal of neurochemistry

Article Title: The de novo Autism Spectrum Disorder RELN R2290C Mutation Reduces Reelin Secretion and Increases Protein Disulfide Isomerase Expression

doi: 10.1111/jnc.14045

Figure Lengend Snippet: (A) Untagged mutant Reelin (R2290C, H) did not reduce the media accumulation of PA-tagged wild-type Reelin from co-expressing HeLa cells. Western blots show the anti-PA (WT Reelin) and actin levels. (B) Quantification of A (n=3, error bars ± SEM; 1-way ANOVA with Dunnett’s post hoc test, not statistically different, n.s.). (C) Schematic of the canonical Reelin signaling pathway: Reelin interacts with and multimerizes the receptors ApoER2 and VLDLR, recruiting Dab1 to their cytoplasmic domains, and leading to the Dab1-dependent activation of Src, which then phosphorylated Dab1 on tyrosine causing a cascade of downstream events including Dab1 degradation. (D) Dab1 tyrosine phosphorylation (4G10; p-Y) was induced to an equal extent by normalized amounts of WT, R2290C, or R2290H mutant Reelin, in stimulated primary neuronal cultures. (E) Densitometry measurements were quantified by dividing 4G10 signal by total Dab1 for each sample and values reported relative to neurobasal (NB). The stoichiometry of Dab1 tyrosine phosphorylation was augmented by WT and mutant Reelin treatment to approximately the same extent (n=4, error bars ± SEM, * p<0.05, 1-way ANOVA with Dunnett’s post hoc test). There was no significant difference between R2290C, R2290H and wild-type (WT) Reelin-conditioned media (RCM), but all were statistically different from control-conditioned media (CCM) in pairwise comparisons. CCM was not statistically different from NB media.

Article Snippet: RELN null-allele mice were maintained on a C57BL6 background and were from Jackson Laboratories.

Techniques: Mutagenesis, Expressing, Western Blot, Activation Assay, Phospho-proteomics, Control

Journal: Journal of neurochemistry

Article Title: The de novo Autism Spectrum Disorder RELN R2290C Mutation Reduces Reelin Secretion and Increases Protein Disulfide Isomerase Expression

doi: 10.1111/jnc.14045

Figure Lengend Snippet: (A) Western blot of 6-week-old male RELN Orl mice cerebella from wild-type and heterozygous (Orl+/−) mice for Reelin, total Dab1, and ER stress markers. Reelin expression (anti-G10) is reduced and total Dab1 expression (anti-DabH1) is augmented in 6-week-old RELN Orl +/− cerebellum as compared to wild-type (+/+). (B) Quantification of A. Reelin was significantly decreased, while Dab1 was significantly increased in Orl+/− cerebella. PDIA1 was significantly increased in Orl+/− cerebella (n=3, error bars ± SEM, * p< 0.05, t-test). (C) RELN +/− null allele mice cerebellar lysates were analyzed by Western blot for ER stress markers. (D) Quantification of C. Except for reduced Reelin levels in the mutant, no statistically significant differences were seen between the wild-type and RELN +/− null allele cerebella. Dab1 levels tended to be higher in the mutant but this was not significant (n=4-5, error bars ± SEM).

Article Snippet: RELN null-allele mice were maintained on a C57BL6 background and were from Jackson Laboratories.

Techniques: Western Blot, Expressing, Mutagenesis

Journal: Frontiers in Molecular Biosciences

Article Title: Hepatocyte-Specific Knock-Out of Nfib Aggravates Hepatocellular Tumorigenesis via Enhancing Urea Cycle

doi: 10.3389/fmolb.2022.875324

Figure Lengend Snippet: Generation of hepatocyte-specific NFIB-knockout mice (Nfib f/f Alb-Cre). (A) NFIB gene knockout targeting vector strategy design diagram. (B) Genotyping of Nfib loxp/loxp Alb-cre (Nfib f/f Alb-Cre) mice using the tail DNA. The above panel shows the homozygous loxp mice. The wild-type (WT) allele yields an amplicon of 162 bp, while the floxed allele yields an amplicon of 229 bp. The panel below shows the Alb-Cre positive mice, with an amplicon of 390 bp. (C) IHC analysis of NFIB protein in the livers of WT and Nfib f/f Alb-Cre mice. (D) H&E staining showing the histology of the livers of WT and Nfib f/f Alb-Cre mice. All scale bars = 100 μm. Original magnification×20. IHC, immunohistochemistry.

Article Snippet: Mice with homozygous conditional-null alleles of NFIB (Nfib f/f ) were generated with the help of the Cyagen Biosciences (Guangzhou, China).

Techniques: Knock-Out, Gene Knockout, Plasmid Preparation, Amplification, Staining, Immunohistochemistry