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: Science Advances

Article Title: Emilin 2 promotes the mechanical gradient of the cochlear basilar membrane and resolution of frequencies in sound

doi: 10.1126/sciadv.aba2634

Figure Lengend Snippet: ( A ) In the Emlin2 reporter allele, lacZ replaces the first two coding exons of the gene. Gray box, 5′-untranslated region; arrowhead, promoter. ( B ) Western blot of cochlea (P8) from +/+ and Emilin2 −/− mice and for control 293T cells transfected with Emilin2 expression vector (Em2) or empty vector (vect). The blot was reprobed for actin, as control. ( C ) Histochemical detection of β-galactosidase [encoded by lacZ (blue)] in the BM in intact cochlea in an Emilin2 +/− pup. BM spiral and “unwound” spiral diagrams depict gradients of width and frequency sensitivity along the BM. ( D ) Diagram of the organ of Corti. ( E ) Immunofluorescence analysis of emilin 2 protein (red) and β-galactosidase protein (blue) in cryosections of +/+, +/−, and −/− mice. Whitish color, double-positive cells in +/− mice. ( F and G ) In the organ of Corti flat mounts, emilin 2 (red) was in the acellular BM (F) and in tympanic border cell (tbc) and spiral vessel (spv) (G). Cell nuclei, blue [4′,6-diamidino-2-phenylindole (DAPI)]. cc, Claudius cells; he, Hensen cells; ih, inner hair cell; ip, inner pillar cell; oh, outer hair cell; op, outer pillar cell; OSL, osseous spiral lamina; SL, spiral ligament; tc, tunnel of Corti.

Article Snippet: To generate an Emilin2 null allele, lacZ was fused in-frame at the ATG start codon of the gene by homologous recombination in embryonic stem (ES) cells derived from C57BL/6J mice (Ozgene Pty Ltd., Western Australia).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Immunofluorescence

Journal: Science Advances

Article Title: Emilin 2 promotes the mechanical gradient of the cochlear basilar membrane and resolution of frequencies in sound

doi: 10.1126/sciadv.aba2634

Figure Lengend Snippet: ( A ) Intact organ of Corti in Emilin2 −/− mice (6 months old; mid-turn histological sections). The spiral vessel structure is diminished. Arrows d, f, and g indicate views in (D), (F), and (G), respectively. ( B ) Compacted BM in Emilin2 −/− mice [cross-sectional area of pectinate zone relative to width of arcuate-pectinate zone; n = 6 mice, 1 month old; 8 to 10 cochleae; views: +/+ basal 30, apical 14; Emilin2 −/− basal 20, apical 17 (au, arbitrary units)]. ( C ) BM width and thickness [means ± SEM; asterisk (*), apical: t = 5.227, df = 29, P = 0.00001348; basal: t = 6.351, df = 50, P = 0.000000062, two-tailed t test; from data in (B)]. ( D and E ) Transmission electron micrographs [pectinate zone (mid-basal turn)]. Filaments (f) are normally interspersed in ground substance (gs). Emilin2 −/− mice have denser filaments and less gs (3 months old). ( F ) Arcuate zone below inner pillar cell (ipc) shows loss of gs in Emilin2 −/− mice. ( G ) The spiral vessel normally fills with gs, but gs is lacking in Emilin2 −/− mice. bt, basement membrane; sc, support cell of sensory epithelium; ST, scala tympani.

Article Snippet: To generate an Emilin2 null allele, lacZ was fused in-frame at the ATG start codon of the gene by homologous recombination in embryonic stem (ES) cells derived from C57BL/6J mice (Ozgene Pty Ltd., Western Australia).

Techniques: Two Tailed Test, Transmission Assay

Journal: Science Advances

Article Title: Emilin 2 promotes the mechanical gradient of the cochlear basilar membrane and resolution of frequencies in sound

doi: 10.1126/sciadv.aba2634

Figure Lengend Snippet: ( A ) LC-PolScope images of fibers and color wheel for quantification. Similar fiber colors for +/+ indicate uniform alignment; varied colors for Emilin2 −/− indicate nonuniformity (10 μm by 10 μm views, mid-turn examples). ( B ) Range of relative fiber orientations in apical, middle, and basal turns. Fiber measurements were assigned to 10° bins (for fiber numbers, see Materials and Methods). Mean orientation in each preparation was normalized (to ~101°). Histograms were fitted with normal distribution curves. SD from the mean: apical, 2.2 (+/+) and 11.9 (−/−); middle, 4.7 (+/+) and 28.1 (−/−); and basal 4.7 (+/+) and 26.2 (−/−). Groups, three mice (six cochleae); differences in SD, ** P < 0.0001, F test statistics. ( C to F ) Fiber and fiber bundle thickness and spacing. Bundles were not observed in apical turns. Groups, three mice, means ± SD, * P < 0.001 and ** P < 0.0001, two-tailed unpaired t test. ( G ) Filament array images reconstructed from stacks of pectinate zone optical sections (0.5 μm apart; 8 apical and 18 middle optical sections; total thicknesses are 4 and 9 μm, respectively). ( H ) Interpretative diagram showing an orderly array in +/+ mice and braided appearance in Emilin2 −/− mice.

Article Snippet: To generate an Emilin2 null allele, lacZ was fused in-frame at the ATG start codon of the gene by homologous recombination in embryonic stem (ES) cells derived from C57BL/6J mice (Ozgene Pty Ltd., Western Australia).

Techniques: Two Tailed Test

Journal: Science Advances

Article Title: Emilin 2 promotes the mechanical gradient of the cochlear basilar membrane and resolution of frequencies in sound

doi: 10.1126/sciadv.aba2634

Figure Lengend Snippet: ( A to C ) BM frequency tuning curves at 56-kHz equivalent location (vertical dashed line). BM threshold displacements (0.3-nm criterion) as functions of stimulus frequencies. Emilin2 −/− mice are less sensitive to a 56-kHz tone and have broader, multipeaked responses (arrows). ( D to F ) BM/malleus displacement ratios [from data in (A) to (C)] as functions of stimulus level in 5-dB steps. Dashed lines, 56-kHz reference; arrows, additional peaks coincident with additional tuning curve minima for −/− mice. Red numbers, cochlear amplification (in dB) at peaks, calculated as the difference in BM/malleus displacement ratio for active (+/+, 20 dB SPL; −/−, 30 dB SPL) and passive (80 dB SPL) responses. ( G ) Mean tuning curves for five +/+ (black) and five −/− (blue) mice from three litters (mean and SD) showing more sensitive low-frequency tails for −/− mice (* P < 0.05, each point in range). At 56 kHz, +/+ mice were more sensitive ( t = 6.0867, df = 8, P = 0.0033). ( H and I ) Irregular tuning curve shapes for five Emilin2 −/− mice compared to five +/+ mice. Curves i, ii, iii, and iv are each incremented up (arrow) by 15 dB SPL from curve below.

Article Snippet: To generate an Emilin2 null allele, lacZ was fused in-frame at the ATG start codon of the gene by homologous recombination in embryonic stem (ES) cells derived from C57BL/6J mice (Ozgene Pty Ltd., Western Australia).

Techniques: Amplification

Journal: Science Advances

Article Title: Emilin 2 promotes the mechanical gradient of the cochlear basilar membrane and resolution of frequencies in sound

doi: 10.1126/sciadv.aba2634

Figure Lengend Snippet: ( A and B ) Mean displacement thresholds for active (open symbols) and passive (postmortem; solid symbols) responses ( n = 5, two different litters from groups in ). Mean and SD; BM threshold displacement (0.3-nm criterion) at 56-kHz equivalent place. The 56-kHz peak is lost postmortem in +/+ mice; irregular peaks persist in Emilin2 −/− mice. ( C and D ) Expanded 35- to 70-kHz region from (A and B). For +/+, pre- and postmortem responses differed over 45 to 58 kHz ( P < 0.001, each point). For Emilin2 −/− , peaks remained at 56 and ~50 kHz; large variations between mice yielded significant differences (*) in mean curves only at 40 kHz ( t = 4.0468, df = 8, P = 0.0038) and 53 kHz ( t = 3.9450, df = 8, P = 0.0042). ( E and F ) BM displacement thresholds ( y axis, left) and phase ( y axis, right; 50–dB SPL stimulus) as functions of stimulus frequency. Low-frequency responses (10 to 45 kHz) were significantly less sensitive for +/+ than Emilin2 −/− mice. Displacement phase for Emilin2 −/− mice significantly lagged +/+ mice at <47 kHz and led at >58 kHz, indicating reduced BM stiffness in Emilin2 −/− mice. * P ≤ 0.005, two-tailed unpaired t test.

Article Snippet: To generate an Emilin2 null allele, lacZ was fused in-frame at the ATG start codon of the gene by homologous recombination in embryonic stem (ES) cells derived from C57BL/6J mice (Ozgene Pty Ltd., Western Australia).

Techniques: Two Tailed Test

Journal: Science Advances

Article Title: Emilin 2 promotes the mechanical gradient of the cochlear basilar membrane and resolution of frequencies in sound

doi: 10.1126/sciadv.aba2634

Figure Lengend Snippet: ( A and B ) Masker tone thresholds required to suppress CAP threshold response to probe tones as functions of masker tone frequency: +/+ [(A), black] and Emilin2 −/− [(B), blue] for 12 kHz (open symbols) and 20 kHz (solid symbols) probe tones. Each point: means ± SD of 10 measurements. Gray symbols, CAP threshold audiograms. ( C and D ) Mean curves (±SD) for five mice. Dashed horizontal line, CAP threshold at probe tone frequency for +/+ (N1 threshold). Responses are significantly less sensitive for Emilin2 −/− than +/+ mice. Minima for 12-kHz probe tone (C): +/+, 5.7 ± 4.2 dB SPL and −/−, 37.7 ± 14.4 dB SPL ( t = 4.7703, df = 8, P = 0.0014). Minima for 20-kHz probe tone (D): +/+, −4.8 ± 6.3 dB SPL and −/−, 22.9 ± 9.2 dB SPL ( t = 5.5549, df = 8, P = 0.0005). Emilin2 −/− 20-kHz curves are broader with minima on high- and low-frequency slopes. Q 10dB (probe tone frequency/bandwidth of 10 dB from tip) for 12-kHz probe tone: +/+, 5.4 and −/−, 2.4; for 20-kHz probe tone: +/+, 14.3 and −/−, 11.1. Red “+” (+/+) and “x” (−/−) symbols are probe tone frequency and SPL, respectively.

Article Snippet: To generate an Emilin2 null allele, lacZ was fused in-frame at the ATG start codon of the gene by homologous recombination in embryonic stem (ES) cells derived from C57BL/6J mice (Ozgene Pty Ltd., Western Australia).

Techniques: