Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Molecular modeling-based predictions of protein stability and surface charge distribution in SPN-ARR domains of the Shank3 protein harboring Q321R and other ASD-risk mutations. (A) Structure of the SPN and ARR domains of the Shank3 protein (PDB ID: 5G4X), with the locations of amino acid residues mutated in autistic individuals (R12C, L68P, A198G, R300C, and Q321R) indicated. The SPN domain, the linker connecting the SPN and ARR domains, and the ARR domain of Shank3 are indicated in gray, green, and orange colors, respectively, as a ribbon diagram. The ASD-risk residues are indicated by ball-and-stick models. (B) Stability predictions of SPN and ARR domains of the Shank3 protein containing the indicated ASD-risk mutations, obtained using I-Mutant 2.0 software. A negative predicted free energy change (ΔΔG, in kcal/mol) indicates a decrease in the stability of the mutant protein. (C) Electrostatic surface charge distribution patterns in WT and mutant (Q321R) SPN and ARR domains of the Shank3 protein. Negative and positive surface charges are indicated in red and blue, respectively. Hydrophobic surfaces are indicated in white. Surface areas with the Shank3 Q321R mutation are indicated by dotted orange circles.

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: Mutagenesis, Software

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Generation and characterization of Shank3 Q321R mice. (A) A schematic diagram showing the location of the Shank3 Q321R mutation in the Shank3 protein. Ank, ankyrin repeat region; SH3, Src homology 3 domain; PDZ, PSD-95/Dlg/ZO-1 domain; Proline-rich, proline-rich region; SAM, sterile alpha motif domain. (B) A schematic diagram showing the gene-targeting strategy used to generate Shank3 Q321R mice. Note that the mutated codon corresponding to the Q321R mutation is located in exon 8 of the Shank3 gene. Primers (forward and reverse) for PCR genotyping are indicated. Neo, neomycin resistance; DTA, diphtheria toxin. (C) Confirmation of the Shank3 Q321R mutation by genomic DNA sequencing of Shank3 Q321R/Q321R mice. Results from a wild-type (WT) mouse are shown for comparison. (D) PCR genotyping of Shank3 Q321R/Q321R (HM/homozygous) and Shank3 +/Q321R (HT/heterozygous) mice. (E) Known Shank3 protein variants. The target region (aa 1431–1590) of the Shank3 antibody used in the immunoblot analysis (F) is indicated. (F) The levels of Shank3a, but not Shank3c/d or Shank3e, protein variants, are substantially decreased in whole-brain lysates from WT and Shank3 Q321R/Q321R and Shank3 +/Q321R mice (2 months; female), as determined by immunoblot analysis using Shank3-specific antibodies targeting amino acids 1431–1590 in the proline-rich region of the protein that can detect all three protein splice variants. n = 3 mice (WT, HT, and HM), ∗ P < 0.05, ∗∗ P < 0.01, ns, not significant, One sample t -test. (G) Normal gross morphology of the brains of WT and homozygous Shank3 Q321R/Q321R mice (2 months; female), as shown by immunostaining of coronal sections for the neuronal marker, NeuN. Scale bar, 1 mm.

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: Mutagenesis, Sterility, DNA Sequencing, Comparison, Western Blot, Immunostaining, Marker

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Suppressed neuronal excitability but normal mEPSCs and mIPSCs in hippocampal CA1 pyramidal neurons from male Shank3 Q321R/Q321R mice. (A) Normal mEPSC frequency and amplitude in CA1 pyramidal neurons in the hippocampus of male Shank3 Q321R/Q321R mice (P21–25). The indicated values represent means ± SEM. n = 14 neurons from 3 mice (WT) and 15 neurons from 3 mice (Q321R), ns, not significant, Mann–Whitney U -test (for frequency) and Student’s t -test (for amplitude). (B) Normal mIPSC frequency and amplitude in CA1 pyramidal neurons in the hippocampus of male Shank3 Q321R/Q321R mice (P21–25). n = 12 neurons from 5 mice (WT) and 13 neurons from 3 mice (Q321R), ns, not significant, Student’s t -test (for frequency) and Student’s t -test (for amplitude). (C–F) Suppressed neuronal excitability in hippocampal CA1 pyramidal neurons from male Shank3 Q321R/Q321R mice (P22–26), as shown by the number of action potential fired plotted against injected currents (F) . Note that the current-voltage relationship (C) , input resistance (D) , and Sag ratio (E) were not changed. n = 14 neurons from 5 mice (WT) and 16 neurons from 4 mice (Q321R), ∗ P < 0.05, ∗∗∗ P < 0.001, ns, not significant, repeated measures two-way ANOVA (for current-membrane potential and current-firing curves) and Mann–Whitney U -test (for input resistance and sag ratio). (G) Normal mEPSC frequency and amplitude in dorsolateral striatal neurons from male Shank3 Q321R/Q321R mice (P28–43). n = 16 neurons from 4 mice (WT) and 16 neurons from 5 mice (Q321R), ns, not significant, Mann–Whitney U -test (for frequency) and Student’s t -test (for amplitude). (H) Normal mIPSC frequency and amplitude in dorsolateral striatal neurons from male Shank3 Q321R/Q321R mice (P28–43). n = 16 neurons from 6 mice (WT) and 17 neurons from 7 mice (Q321R), ns, not significant, Student’s t -test (for frequency) and Mann–Whitney U -test (for amplitude).

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: MANN-WHITNEY, Injection, Membrane

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Normal locomotor activity and moderate anxiolytic-like behavior in heterozygous Shank3 +/Q321R mice. (A,B) Normal locomotor activity in Shank3 +/Q321R mice (2–3 months; male) in Laboras cages, where locomotor activity was measured together with other movements for four consecutive days in the absence of habituation. OFF/ON, light-off/on. n = 13 mice (WT) and 13 mice (HT), ns, not significant, two-way ANOVA (genotype main effect p -value = 0.1813 in A ), and Student’s t -test (B) . (C,D) Normal locomotor activity in Shank3 +/Q321R mice (2–3 months; male) in the open-field test, as shown by the distance moved. n = 13 mice (WT) and 13 mice (HT), ns, not significant, repeated measures two-way ANOVA (C) and Student’s t -test (D) . (E) Normal anxiety-like behavior in Shank3 +/Q321R mice (2–3 months; male) in the open-field test, as shown by the time spent in the center region of the open-field arena. n = 13 mice (WT) and 13 mice (HT), ns, not significant, Mann–Whitney U -test. (F) Normal anxiety-like behavior in Shank3 +/Q321R mice (2–3 months; male) in the elevated plus-maze test, as shown by the time spent in the open arms of the maze. n = 13 mice (WT) and 13 mice (HT), ns, not significant, Welch’s t -test. (G) Anxiolytic-like behavior in Shank3 +/Q321R mice (2–3 months; male) in the light-dark test, as shown by the time spent in the light chamber of the light-dark apparatus. n = 13 mice (WT) and 12 mice (HT), ∗∗ P < 0.01, Student’s t -test.

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: Activity Assay, MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Normal social interaction, moderately enhanced social communication and self-grooming, and suppressed digging in heterozygous Shank3 +/Q321R mice. (A) Normal social approach in Shank3 +/Q321R mice (2–3 months; male) in the three-chamber test, as shown by time spent sniffing. S1, a stranger; O, object; S2, new stranger. Social novelty recognition, measured by the preference for new stranger (S2) over old stranger (S1), could not be determined due the lack of normal social novelty recognition in WT mice. n = 10 mice (WT), 13 mice (HT), ∗∗∗ P < 0.001, ns, not significant, Welch’s t -test, Mann–Whitney U -test, and Student’s t -test. (B) Moderately increased courtship USVs emitted by Shank3 +/Q321R mice (2–3 months; male) upon encounter with a novel female stranger, as shown by the normal number of USVs but increased mean duration of each USV calls. n = 12 mice (WT), 12 mice (HT), ∗ P < 0.05, ns, not significant, Student’s t -test. (C) Enhanced self-grooming in Shank3 +/Q321R mice (2–3 months; male) in home cages with bedding (10 min), as shown by total self-grooming time. n = 12 mice (WT), 13 mice (HT), ∗ P < 0.05, Student’s t -test. (D) Suppressed digging in Shank3 +/Q321R mice (2–3 months; male) in home cages with bedding (10 min), as shown by total digging time. n = 12 mice (WT), 13 mice (HT), ∗ P < 0.05, Welch’s t -test. (E–I) Normal self-grooming in Shank3 +/Q321R mice (2–3 months; male) in Laboras cages, as shown by total self-grooming duration. n = 13 mice (WT), 13 mice (HT), ns, not significant, two-way ANOVA (genotype main effect p -value = 0.4087 in E ), Mann-Whitney test (F,H,I) , Student’s t -test (G) .

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Normal locomotor activity and moderate anxiolytic-like behavior in homozygous Shank3 Q321R/Q321R mice. (A,B) Normal locomotor activity in Shank3 Q321R/Q321R mice (2–4 months; male), in Laboras cages, where locomotor activity was measured together with other movements for four consecutive days in the absence of habituation. Note that the WT data in this panel and other panels in this figure are identical to those shown in because WT, heterozygous, and homozygous mice were tested together in the same behavioral tests. OFF/ON, light-off/on. n = 13 mice (WT) and 14 mice (Q321R), ns, not significant, two-way ANOVA ( A , genotype main effect p -value = 0.9356) and Student’s t -test (B) . (C,D) Normal locomotor activity in Shank3 Q321R/Q321R mice (2 months; male) in the open-field test, as shown by the distance moved. Note that Shank3 Q321R mice spent a normal amount of time in the center region of the open-field arena, suggestive of normal anxiety-like behavior. n = 13 mice (WT) and 14 mice (Q321R), ∗∗∗ P < 0.001, ns, not significant, repeated measures two-way ANOVA (C) and Student’s t -test (D) . (E) Normal anxiety-like behavior in Shank3 Q321R/Q321R mice (2 months; male) in the open-field test, as shown by the time spent in the center region of the open-field arena. n = 13 mice (WT) and 14 mice (Q321R), ns, not significant, Student’s t -test. (F) Anxiolytic-like behavior in Shank3 Q321R/Q321R mice (2 months; male) in the elevated plus-maze test, as shown by the increased time spent in the open arms of the maze. n = 13 mice (WT) and 14 mice (Q321R), ∗ P < 0.05, Mann–Whitney U -test. (G) Anxiolytic-like behavior in Shank3 Q321R/Q321R mice (2 months; male) in the light-dark test, as shown by the increased time spent in the light chamber of the light-dark apparatus. n = 13 mice (WT) and 14 mice (Q321R), ∗ P < 0.05, Welch’s t -test.

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: Activity Assay, MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Normal social interaction and communication, moderately enhanced stress-induced self-grooming, and suppressed digging in homozygous Shank3 Q321R/Q321R mice. (A) Normal social approach in Shank3 Q321R/Q321R mice (3 months; male) in the three-chamber test, as shown by time spent sniffing social (S1) or object (O) target. Social novelty recognition, measured by the preference for new stranger (S2) over old stranger (S1), could not be determined due the lack of normal social novelty recognition in WT mice. Note that the WT data in this panel and other panels in this figure are identical to those shown in because WT, heterozygous, and homozygous mice were tested together in the same behavioral tests. n = 10 mice (WT), 14 mice (Q321R), ∗∗∗ P < 0.001, ns, not significant, Welch’s t -test (for S1 vs. O) and Student’s t -test (for S1 vs. S2). (B) Normal social interaction in Shank3 Q321R/Q321R mice (3 months; male) in bidirectional direct social-interaction tests, as shown by total time spent in social interaction. n = 10 pairs (WT), 13 pairs (Q321R), ns, not significant, Student’s t -test. (C) Normal USVs emitted by Shank3 Q321R/Q321R mice (4 months; male) upon encounter with a novel female stranger (courtship USVs), as shown by the total number of USVs and the mean duration of each USV calls. n = 12 mice (WT), 14 mice (Q321R), ns, not significant, Student’s t -test. (D) Enhanced self-grooming in Shank3 Q321R/Q321R mice (2 months; male) in home cages with bedding (10 min), as shown by total self-grooming time. n = 12 mice (WT), 13 mice (Q321R), ∗∗∗ P < 0.001, Student’s t -test. (E) Suppressed digging in Shank3 Q321R/Q321R mice (2 months; male) in home cages with bedding (10 min), as shown by total digging time. n = 12 mice (WT), 13 mice (Q321R), ∗ P < 0.05, Welch’s t -test. (F–J) Normal self-grooming in Shank3 Q321R/Q321R mice (2 months; male) in Laboras cages, where self-grooming was measured together with other movements for four consecutive days in the absence of habituation. OFF/ON, light-off/on. Note that there are no genotype differences during the first 10 min, 1 or 12 h, the entire session (96 h), or during light-off and light-on periods (48 h each). n = 13 mice (WT) and 14 mice (Q321R), ns, not significant, two-way ANOVA (genotype main effect p -value = 0.9754 in F ), Student’s t -test (H) and Mann–Whitney U -test (G,I,J) .

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Normal object-recognition and fear memory in homozygous male Shank3 Q321R/Q321R mice. (A) Normal levels of object recognition memory in Shank3 Q321R/Q321R mice (2–4 months; male) in the novel object recognition test, as shown by the discrimination index over a familiar object and a novel object (see Materials and Methods for details) presented 24 h after exploring two identical objects on the first day. n = 11 mice (WT), 13 mice (Q321R), ns, not significant, Student’s t -test. (B) Normal acquisition, retrieval, and extinction of contextual fear memory Shank3 Q321R/Q321R mice (3–6 months; male), as shown by freezing levels. Mice were given 5 foot shocks (2-min intervals) during the 12-min fear memory acquisition phase (day 1), and were consecutively exposed to the same context 24 h after the training (day 2) and also during days 3–9 for fear extinction. n = 13 mice (WT), 17 mice (Q321R), repeated measures two-way ANOVA. (C) Normal somatosensory function in Shank3 Q321R/Q321R mice (6 months; male) in the hot plate test, as shown by latency to first licking/jumping. n = 17 mice (WT) and 20 mice (Q321R), ns, not significant, Student’s t -test (for latency to first licking), and Mann–Whitney U -test (for latency to first jumping). (D) Normal somatosensory function in Shank3 Q321R/Q321R mice (2 months; male) in the von Frey test, as shown by threshold for response to stimulation. n = 10 mice (WT) and 11 mice (Q321R), ns, not significant, Mann–Whitney U -test.

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: Hot Plate Test, MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Abnormal EEG patterns in Shank3 Q321R/Q321R mice. (A,B) Abnormal EEG patterns in frontal and parietal lobes of the Shank3 Q321R/Q321R brain (3 months; male). EEG power was calculated by combining data from both left and right hemispheres in the frontal or parietal lobe. Note that the EEG power in the delta frequency range is decreased, whereas that in the alpha frequency range is increased. The vertical dotted lines in the power spectral density (PSD) diagrams indicate the boundaries between different frequency ranges (delta, 0–4 Hz; theta, 4–12 Hz; alpha, 12–30 Hz; low gamma, 30–80 Hz; high gamma, 80–130 Hz). n = 10 mice (WT) and 10 mice (Q321R) for frontal lobe and 9 mice (WT) and 10 mice (Q321R) for parietal lobe, ∗ P < 0.05, ns, not significant, Student’s t -test, Mann–Whitney U -test, Welch’s t -test.

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Decreased susceptibility to PTZ-induced seizures in Shank3 Q321R/Q321R mice. (A) Percentage of animals that can reach stage 1 (behavioral arrest) seizures. Seizures were induced in WT and Shank3 Q321R/Q321R mice (3–4 months; male) by intraperitoneal injection of PTZ (50 mg/kg). n = 10 mice (WT), 13 mice (Q321R), ∗∗ P < 0.01, log-rank (Mantel-Cox) test ( p = 0.0047). (B) Latency to reach stage 1 (behavioral arrest) seizures. ∗ P < 0.05, Mann–Whitney U -test. (C) Percentage of animals that can reach stage 2 (myoclonic) seizures. ns, not significant, log-rank (Mantel-Cox) test ( p = 0.1881). (D) Latency to reach stage 2 (myoclonic) seizures. ns, not significant, Mann–Whitney U -test. (E) Seizure susceptibility based on the latencies to reach stage 1, 2, or 3 seizure (see Materials and Methods for details on the definition of seizure susceptibility). ∗∗ P < 0.01, Student’s t -test. (F) Duration of seizure stages 1, 2, and 3. ∗∗ P < 0.01, ∗∗∗ P < 0.001, ns, not significant, one-way ANOVA with Kruskal–Wallis/Holm–Sidak test (within-genotype stage 1/2/3 comparison) and Mann–Whitney U -test (between-genotype stage 1/2/3 comparison). (G) Final seizure stages that animals reached, expressed as a percentage of animals. ∗ P < 0.05, Chi-square test.

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: Injection, MANN-WHITNEY, Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: Shank3 Mice Carrying the Human Q321R Mutation Display Enhanced Self-Grooming, Abnormal Electroencephalogram Patterns, and Suppressed Neuronal Excitability and Seizure Susceptibility

doi: 10.3389/fnmol.2019.00155

Figure Lengend Snippet: Summary of electrophysiological, behavioral, and brain function phenotypes in Shank3 Q321R/Q321R mice.

Article Snippet: A mouse embryonic stem (ES) cell line harboring the Shank3 Q321R mutation was generated by Cyagen.

Techniques: Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: The small GTPase RAB28 is required for phagocytosis of cone outer segments by the murine retinal pigmented epithelium

doi: 10.1074/jbc.RA118.005484

Figure Lengend Snippet: Generation of Rab28−/− mice. A, schematic representation of the mouse Rab28 gene and three splice variants, V1–V3. The mouse Rab28 gene consists of nine exons (yellow rectangles). Dashed gray, red, and green lines indicate three types of mRNA splicing. V1, V2, and V3 share exons 1–6 but differ in the C terminus, which is encoded by exons 8, 7, and 9, respectively. aa, amino acids. B, Western blotting of HEK293 lysates expressing mCherry-RAB28V2 (lane 1) and EGFP-RAB28V1 (lane 2). The polyclonal antibody used in this study recognizes both variants (red arrow); the faster moving polypeptide is nonspecific. C, mCherry-RAB28V2 and EGFP-RAB28V1 expression pattern in HEK293 cells. V1 and V2 colocalize in the cytoplasm, and V2 is also present in the nucleus. Top and right small panels, orthogonal views of y and z planes at the lines indicated. Scale bar, 20 μm. D, schematic representation of the Rab28 gene, the location of the gene trap in intron 2, the floxed allele, and the knockout allele. The En2SA-IRES-LacZ-pGK-Neo GT cassette is flanked by two FRT sites (green triangle) and is inserted into intron 2 by homologous recombination. Exon 3 is flanked by two LoxP sites (red triangle). Small arrows represent the approximate position of primers for genotyping. E, PCR genotyping of the 3′ and 5′ recombination arm, third LoxP, second FRT/LoxP, and first FRT sites in ES cells. D1 and A5, two ES cell clones and the derived mice with identical genotyping results. The right-most panel represents a PCR genotyping result of one litter of Rab28+/− × Rab28+/− pups. Primer sequences are listed in Table S1. F, retinal lysate Western blotting. Left lane, RAB28 with a mobility of 25 kDa; right lane, RAB28 is absent in Rab28−/− retina lysate. The identity of the slower moving species is unknown. G, immunohistochemistry of WT (left) and knockout retina cryosections (right) with anti-RAB28 antibody. RAB28 is present in WT photoreceptors and RPE and absent in Rab28−/− sections (right). H, expression of mCherry-RAB28V2 in rod photoreceptors by neonatal electroporation. Scale bars, 20 μm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Generation of Rab28 −/− mouse Rab28 embryonic stem (ES) cells (clones EPD0688_3_D01 and HEPD0677_7_A05), in C57BL/6N genetic background, JM8A3.N1 subline) containing a gene trap in intron 2 were acquired from EUCOMM (Helmholtz Zentrum, Munich, Germany).

Techniques: Western Blot, Expressing, Knock-Out, Homologous Recombination, Clone Assay, Derivative Assay, Immunohistochemistry, Electroporation

Journal: The Journal of Biological Chemistry

Article Title: The small GTPase RAB28 is required for phagocytosis of cone outer segments by the murine retinal pigmented epithelium

doi: 10.1074/jbc.RA118.005484

Figure Lengend Snippet: Reduced function of Rab28−/− photoreceptors. A and B, representative scotopic ERG (A) traces at −1.6, 0.4, and 2.4 log cd s/m2 and photopic ERG traces (B) at 0.4, 0.9, and 1.4 photopic log cd s/m2 from 3-month-old (3m) Rab28+/+ and Rab28−/− mice. C and D, scotopic a-wave amplitude (C) and photopic b-wave amplitude (D) as a function of light intensity. Significant differences were observed for the scotopic a-wave amplitude at 0.4, 1.4, and 2.4 log cd s/m2 and for the photopic b-wave amplitude at all light intensities of −0.01, 0.4, 0.9, 1.4, and 1.9 log cd s/m2. Shown are mean ± S.D. (error bars), n ≥ 5 for each group, one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Generation of Rab28 −/− mouse Rab28 embryonic stem (ES) cells (clones EPD0688_3_D01 and HEPD0677_7_A05), in C57BL/6N genetic background, JM8A3.N1 subline) containing a gene trap in intron 2 were acquired from EUCOMM (Helmholtz Zentrum, Munich, Germany).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: The small GTPase RAB28 is required for phagocytosis of cone outer segments by the murine retinal pigmented epithelium

doi: 10.1074/jbc.RA118.005484

Figure Lengend Snippet: Progressive retina degeneration in Rab28−/− mice. A, immunostaining of retina frozen sections with anti-GC1 antibody (top panels) and with anti-cone arrestin (bottom panels) at 1, 3, 6, and 13 months of age. Note the gradual reduction of ROS length and ONL thickness and the more rapid cone degeneration. Scale bar, 20 μm. B and C, decline of scotopic a-wave (B) and photopic b-wave amplitudes (C) at 1, 3, 6, and 13 months of age. D–F, average ONL thickness (D), ROS length (E), and cone density (F) of WT and Rab28−/− retina near optic nerve at 1, 3, 6, and 13 months of age. All are significantly reduced at 3, 6, and 13 months. Shown are mean ± S.D. (error bars), n = 4, one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Generation of Rab28 −/− mouse Rab28 embryonic stem (ES) cells (clones EPD0688_3_D01 and HEPD0677_7_A05), in C57BL/6N genetic background, JM8A3.N1 subline) containing a gene trap in intron 2 were acquired from EUCOMM (Helmholtz Zentrum, Munich, Germany).

Techniques: Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: The small GTPase RAB28 is required for phagocytosis of cone outer segments by the murine retinal pigmented epithelium

doi: 10.1074/jbc.RA118.005484

Figure Lengend Snippet: Cone outer segment elongation and tip enlargement in Rab28−/− retina. A, immunohistochemistry of 1-month-old WT (top panels) and Rab28−/− (bottom panels) retina frozen sections with antibodies against rhodopsin, CNGA1/A3, PDE6, rod Tα, and ROM1, as indicated. The immunostaining showed correct localization of ROS proteins in Rab28−/− rod photoreceptors. B–D, immunostaining of 1-month-old WT (top panels) and Rab28−/− (bottom panels) retina sections with anti-CNGA3 (B), ML-opsin (C), and S-opsin (D). White arrows point to the bulged COS tips at the apical RPE (B–D, bottom panels). Red arrows, cone opsin mislocalization in the IS, ONL, and OPL (C and D, bottom panels). E, ML-opsin staining of P14 WT (top) and Rab28−/− (bottom) retina sections. Mutant COS are slightly elongated, and enlarged COS tips are occasionally observed (dashed arrow in E). F–H, high-magnification confocal images of two WT COS (F) and two mutant COS (G and H). WT COS taper toward the RPE, but mutant COS have a balloon-like enlargement at the distal tip (red arrows). Scale bars, 20 μm (A–D), 10 μm (E), and 5 μm (F–H). I, average length of 1-month-old mutant COS (21.7 ± 4.9 μm) is significantly longer than seen in WT COS (15.1 ± 1.6 μm). Shown are mean ± S.D. (error bars). More than 80 cones from four animals were measured; one-way ANOVA. ***, p < 0.001.

Article Snippet: Generation of Rab28 −/− mouse Rab28 embryonic stem (ES) cells (clones EPD0688_3_D01 and HEPD0677_7_A05), in C57BL/6N genetic background, JM8A3.N1 subline) containing a gene trap in intron 2 were acquired from EUCOMM (Helmholtz Zentrum, Munich, Germany).

Techniques: Immunohistochemistry, Immunostaining, Staining, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The small GTPase RAB28 is required for phagocytosis of cone outer segments by the murine retinal pigmented epithelium

doi: 10.1074/jbc.RA118.005484

Figure Lengend Snippet: Reduction of prenylated protein levels in Rab28−/− cone OS. A–C, immunostaining of WT and GRK1 (A), cPDEα′ (B), and cTγ (C) in 1-month WT (top) and Rab28−/− (bottom) retinas. All three cone OS prenylated proteins showed a reduction in intensity in Rab28−/− compared with WT, whereas GRK1 in rod OS is not reduced. D, GRK1 (red) and cone arrestin (green) staining in P14 WT (top) and Rab28−/− (bottom) retina. GRK1 reduction in cone OS is already obvious at this stage. Scale bars, 20 μm. E, Western blotting of 7-week-old Rab28+/− and Rab28−/− retina lysate with anti-cPDEγ, anti-cTγ, anti-cPDEα′, anti-cArrestin, anti-CNGA3, anti-GRK1, and anti-M- and S-opsin antibodies. α-Tubulin was used as loading control. F, quantification of Western blotting signal intensity (mean ± S.D. (error bars)), normalized to α-tubulin. Only the reduction of cPDEγ and cTγ is statistically significant. *, p < 0.05, n = 3, one-way ANOVA.

Article Snippet: Generation of Rab28 −/− mouse Rab28 embryonic stem (ES) cells (clones EPD0688_3_D01 and HEPD0677_7_A05), in C57BL/6N genetic background, JM8A3.N1 subline) containing a gene trap in intron 2 were acquired from EUCOMM (Helmholtz Zentrum, Munich, Germany).

Techniques: Immunostaining, Staining, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: The small GTPase RAB28 is required for phagocytosis of cone outer segments by the murine retinal pigmented epithelium

doi: 10.1074/jbc.RA118.005484

Figure Lengend Snippet: Reduced COS phagosomes in P24 Rab28−/− retina. A and B, immunohistochemistry of P24 WT (A) and Rab28−/− (B) retina cryosections with anti-ML- and anti-S-opsin antibodies (mixed 1:1) (green). Retina samples were collected at 1.5 h after light onset. Cone opsin-positive COS phagosomes (arrows) in mutant (bottom) were diminished compared with WT control (top). At the right of each panel is an overlay with PNA (red) and the nuclear marker, DAPI (blue). C, quantitative evaluation of COS phagosomes at P24. Mutant COS phagosome count is significantly reduced compared with WT (Rab28−/− 7.5 ± 2.6 versus WT 39.8 ± 6.0 per 2-mm retina). Phagosomes of 12 sections from three animals (four sections for each retina) were counted. Shown are mean ± S.D. (error bars), one-way ANOVA. ***, p < 0.001. D and E, rhodopsin immunostaining of 1-month WT (D) and Rab28−/− (E) retina sections collected 1.5 h after light onset. Both the density and size of rhodopsin-positive phagosomes (green dots at RPE) are comparable between WT and Rab28−/−. F, quantitative evaluation of ROS phagosomes at P24. Mutant ROS phagosomes are comparable with WT (Rab28−/− 51.4 ± 5.5 versus WT 56.3 ± 7.1 per 200-μm RPE). Phagosomes of 12 sections from three animals (four sections for each retina) were counted. ns, not significant. G and H, S-opsin immunostaining of P24 Nrl−/− (G) and Rab28−/−; Nrl−/− (H) retina sections collected at 1.5 h after light onset. S-opsin-positive phagosomes are barely detected in the RPE of both genotypes. However, the OS tips are larger in Rab28−/−; Nrl−/− cones compared with Nrl−/− cones (arrows). Scale bars, 20 μm (A, B, D, and E) and 10 μm (G and H).

Article Snippet: Generation of Rab28 −/− mouse Rab28 embryonic stem (ES) cells (clones EPD0688_3_D01 and HEPD0677_7_A05), in C57BL/6N genetic background, JM8A3.N1 subline) containing a gene trap in intron 2 were acquired from EUCOMM (Helmholtz Zentrum, Munich, Germany).

Techniques: Immunohistochemistry, Mutagenesis, Control, Marker, Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: The small GTPase RAB28 is required for phagocytosis of cone outer segments by the murine retinal pigmented epithelium

doi: 10.1074/jbc.RA118.005484

Figure Lengend Snippet: Ultrastructure. A, longitudinal image of 1-month-old Rab28−/− outer segments, showing an elongated COS (arrow) wedged between two ROS and reaching the apical RPE. A swollen, degenerating COS tip (red asterisk), belonging to the same cone, is membrane-bound and filled with vacuoles. B, enlargement of yellow boxed area in A, showing the partially disorganized distal COS (yellow arrows). C and D, representative electron micrographs of 1-month-old (1m) Rab28+/− (C) and Rab28−/− retinas (D). The asterisk in C marks a normal phagosome. Arrows in D indicate three enlarged mutant COS tips filled with membrane stacks or vacuoles at the RPE/photoreceptor interface. Note the increased pigment density in the mutant RPE (arrows). E–H, magnified images of three enlarged tips, ∼2 μm in the shortest dimension, in mutants with variable-size vacuoles (dashed arrows) and distorted membranes (arrowheads). Scale bars, 5 μm (A), 1 μm (B), 5 μm (C and D), 2 μm (E–G), and 100 nm (H).

Article Snippet: Generation of Rab28 −/− mouse Rab28 embryonic stem (ES) cells (clones EPD0688_3_D01 and HEPD0677_7_A05), in C57BL/6N genetic background, JM8A3.N1 subline) containing a gene trap in intron 2 were acquired from EUCOMM (Helmholtz Zentrum, Munich, Germany).

Techniques: Membrane, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The small GTPase RAB28 is required for phagocytosis of cone outer segments by the murine retinal pigmented epithelium

doi: 10.1074/jbc.RA118.005484

Figure Lengend Snippet: Rab28-interacting proteins. A, immunoblot verification of co-IP (Table S2) between RAB28 and select candidate proteins. Verified interactants include PDE6D, KCNJ13, MEF2D, GLUT1, and VAC14. GRK1 and CAND1 failed verification. Lane 1, input; lane 2, normal goat IgG control IP; lane 3, RAB28 antibody IP. Correctly verified proteins (red arrows) and two proteins that failed verification (yellow arrows) are indicated. B and C, representative immunohistochemistry using anti-KCNJ13 antibody (green) and PNA (red) in P24 WT (B) and Rab28−/− (C) retinas. KCNJ13 localization at RPE apical microvilli is comparable between WT and Rab28−/−. D and E, immunohistochemistry with anti-KCNJ13 antibody (red) to show relative delivery of KCNJ13 in P21 Nrl−/− (D) and Rab28−/−; Nrl−/− (E) double-knockout retinas/RPE. KCNJ13 is partially trapped at the base of RPE apical microvilli in double-knockout mice (arrows in E). Scale bars, 20 μm (B and C) and 10 μm (C and D).

Article Snippet: Generation of Rab28 −/− mouse Rab28 embryonic stem (ES) cells (clones EPD0688_3_D01 and HEPD0677_7_A05), in C57BL/6N genetic background, JM8A3.N1 subline) containing a gene trap in intron 2 were acquired from EUCOMM (Helmholtz Zentrum, Munich, Germany).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Control, Immunohistochemistry, Double Knockout

Journal: The Journal of Biological Chemistry

Article Title: The small GTPase RAB28 is required for phagocytosis of cone outer segments by the murine retinal pigmented epithelium

doi: 10.1074/jbc.RA118.005484

Figure Lengend Snippet: Rab28 interaction with PDE6D is Rab28 prenylation–dependent. A, co-IP and Western blotting of PDE6D and RAB28 interaction in vitro. PDE6D-EGFP and mCherry-RAB28V2 or mCherry-RAB28V2-C217A were co-expressed in HEK293 cells. Co-IP was performed using monoclonal anti-mCherry antibody or normal mouse IgG, and the blot was probed with anti-EGFP antibody. mCherry-RAB28V2, but not nonfarnesylated mCherry-RAB28V2-C217A, pulls down PDE6D-EGFP. mCherry was used as the co-IP control. B, confocal images of proteins expressed in vitro. mCherry-RAB28V2 (red) and PDE6D-EGFP (green) colocalize in the ER and cytoplasm when co-expressed (top panels); mCherry-RAB28V2-C217A distributes uniformly throughout the cytoplasm due to the loss of membrane association (bottom panels). C, WT (left) and PDE6D−/− (right) retina cryosections probed with anti-RAB28 antibody. RAB28 fails to enter the Pde6d−/− OS and accumulates primarily in the IS. D, immunoblot of Rab28+/− and Rab28−/− retina homogenates probed with antibodies directed against PDE6D and α-tubulin; PDE6D is not down-regulated in Rab28−/− retinas. Scale bars, 40 μm (B) and 20 μm (C).

Article Snippet: Generation of Rab28 −/− mouse Rab28 embryonic stem (ES) cells (clones EPD0688_3_D01 and HEPD0677_7_A05), in C57BL/6N genetic background, JM8A3.N1 subline) containing a gene trap in intron 2 were acquired from EUCOMM (Helmholtz Zentrum, Munich, Germany).

Techniques: Co-Immunoprecipitation Assay, Western Blot, In Vitro, Control, Membrane

Journal: The Journal of Biological Chemistry

Article Title: The small GTPase RAB28 is required for phagocytosis of cone outer segments by the murine retinal pigmented epithelium

doi: 10.1074/jbc.RA118.005484

Figure Lengend Snippet: Model of COS disc shedding failure in Rab28−/− mouse retina. In WT retina (left), COS length is maintained by the daily addition of new disc membranes at the proximal COS and shedding of aged discs at the distal tip. Shed discs are phagocytized by RPE processes. In Rab28−/− (right), COS shedding at the distal tip fails, elongating COS toward the RPE and accumulating membrane material. Membrane incorporation at the base continues, jamming the ciliary gate and blocking the entry of prenylated proteins. CC, connecting cilium; BB, basal body; CIS, cone inner segment.

Article Snippet: Generation of Rab28 −/− mouse Rab28 embryonic stem (ES) cells (clones EPD0688_3_D01 and HEPD0677_7_A05), in C57BL/6N genetic background, JM8A3.N1 subline) containing a gene trap in intron 2 were acquired from EUCOMM (Helmholtz Zentrum, Munich, Germany).

Techniques: Membrane, Blocking Assay

Journal: The Journal of Biological Chemistry

Article Title: Deletion of both centrin 2 (CETN2) and CETN3 destabilizes the distal connecting cilium of mouse photoreceptors

doi: 10.1074/jbc.RA118.006371

Figure Lengend Snippet: Generation of Cetn3GT/GT and Cetn3−/− mice. A, schematic representation of the mouse Cetn3 gene trap and conditional and knockout alleles. The mouse Cetn3 gene consists of 5 exons (yellow rectangles). The En2SA-IRES-LacZ-pGK-Neo GT cassette, which is flanked by two FRT sites (green triangles), is inserted into intron 2 by homologous recombination. Exon 3 is flanked by two LoxP sites (red triangles). B and C, PCR genotyping of 3′ and 5′ recombination arms (B) and 1st FRT, 2nd LoxP, and 3rd LoxP sites in two ES cell clones, B09 and E12 (C). D and E, PCR genotyping result of one litter each of Cetn3GT/+ × Cetn3GT/+ (D) and Cetn3+/− × Cetn3+/− (E) pups. Primer sequences are listed in Table 2. F and G, immunohistochemistry of WT (F) and Cetn3GT/GT (G) retina cryosections incubated with anti-CETN3 antibody. Signal attributable to CETN3 is present in CC and basal bodies of WT photoreceptors (left) but absent from Cetn3GT/GT photoreceptors (right). Scale bar: 5 μm.

Article Snippet: Cetn3 embryonic stem (ES) cells (clones EPD0630_2_E12 and_ EPD0630_2_B09) on a C57BL/6N genetic background, JM8A3.N1 subline) and containing a gene trap (GT) in intron 2, were acquired from EUCOMM (Helmholtz Zentrum, Munich, Germany).

Techniques: Knock-Out, Homologous Recombination, Clone Assay, Immunohistochemistry, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Deletion of both centrin 2 (CETN2) and CETN3 destabilizes the distal connecting cilium of mouse photoreceptors

doi: 10.1074/jbc.RA118.006371

Figure Lengend Snippet: Pup counts for centrin KO lines Total litters counted are 17, 47, and 43 for Cetn3, Cetn2, and Cetn2/3 lines, respectively. *For Cetn2 and Cetn2/3 lines, the ratio represents male KO ( Cetn2 −/Y and Cetn2 −/Y ; Cetn3 GT/GT ) of total male pups.

Article Snippet: Cetn3 embryonic stem (ES) cells (clones EPD0630_2_E12 and_ EPD0630_2_B09) on a C57BL/6N genetic background, JM8A3.N1 subline) and containing a gene trap (GT) in intron 2, were acquired from EUCOMM (Helmholtz Zentrum, Munich, Germany).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Deletion of both centrin 2 (CETN2) and CETN3 destabilizes the distal connecting cilium of mouse photoreceptors

doi: 10.1074/jbc.RA118.006371

Figure Lengend Snippet: Cetn3 mutant photoreceptors display normal ERGs and ciliary trafficking. A–C, scotopic ERG a-wave amplitude (A) and b-wave amplitude (B) at −1.6, −0.6, 0.4, 1.4, and 2.4 log cd s/m2 light intensity and photopic b-wave amplitude (C) at −0.01, 0.4, 0.9, 1.4, and 1.9 log cd s/m2 from 8-month-old (8m) Cetn3+/+, Cetn3GT/+, and Cetn3GT/GT mice. Scotopic a-wave amplitudes are (in μV) 34, 140, 205, 271, and 326 for +/+; 31, 154, 196, 276, and 347 for GT/+; 31, 134, 184, 256, and 320 for GT/GT; and scotopic b-wave amplitudes are (in μV) 326, 373, 485, 559, and 658 for +/+; 314, 375, 489, 566, and 664 for GT/+; 321, 350, 492, 594, and 653 for GT/GT at −1.6, −0.6, 0.4, 1.4, and 2.4 log cd s/m2, respectively. Photopic b-wave amplitudes are: 22, 70, 105, 121, and 131 for +/+; 25, 73, 108, 136, and 139 for GT/+; 20, 67, 103, 127, and 137 for GT/GT at −0.01, 0.4, 0.9, 1.4, and 1.9 log cd s/m2, respectively, No significant difference was detected among the three genotypes. Shown are mean ± S.D., n ≥ 5 for each group; one-way ANOVA; p > 0.5. D, localization of OS markers, rhodopsin, GC1, PDE6, CNGA1/A3, ROM1, and S- and ML-opsin in 12-month-old Cetn3GT/+ (top row) and Cetn3GT/GT (bottom row) retina sections. E, cone opsin (combined S- and ML-antibodies) immunostaining of 26-month-old Cetn3+/+ and Cetn3−/− retina sections, contrasted with PNA (red, for cone sheaths) and DAPI (blue, for nuclei). No reduction of ONL thickness or cone density was noticed in the Cetn3−/− mutant. Scale bars: 20 μm.

Article Snippet: Cetn3 embryonic stem (ES) cells (clones EPD0630_2_E12 and_ EPD0630_2_B09) on a C57BL/6N genetic background, JM8A3.N1 subline) and containing a gene trap (GT) in intron 2, were acquired from EUCOMM (Helmholtz Zentrum, Munich, Germany).

Techniques: Mutagenesis, Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: Deletion of both centrin 2 (CETN2) and CETN3 destabilizes the distal connecting cilium of mouse photoreceptors

doi: 10.1074/jbc.RA118.006371

Figure Lengend Snippet: Model of CETN2 and CETN3 function in mouse photoreceptors. In WT photoreceptors, CETN1–3 are localized to the CC and the centriole lumen formed by the inner microtubule wall. SPATA7 is located outside along the outer CC microtubule wall. The axoneme (diameter of ∼250 nm) extends into the OS where discs are formed. In Cetn2−/−;Cetn3GT/GT photoreceptors, CETN1 accumulates at the CC center and SPATA7 is gradually depleted from the CC outside wall. As a consequence, distal CC and basal OS axonemes are destabilized, the microtubule structure disintegrates, and OS discs misalign; BB, basal body; DC, daughter centriole.

Article Snippet: Cetn3 embryonic stem (ES) cells (clones EPD0630_2_E12 and_ EPD0630_2_B09) on a C57BL/6N genetic background, JM8A3.N1 subline) and containing a gene trap (GT) in intron 2, were acquired from EUCOMM (Helmholtz Zentrum, Munich, Germany).

Techniques: