Journal: bioRxiv

Article Title: Progressive myoclonic epilepsy-associated gene Kctd7 regulates retinal neurovascular patterning and function

doi: 10.1101/647008

Figure Lengend Snippet: A. To examine retina vascularization, 16-week-old wildtype control and Kctd7 −/− retinas were stained with an antibody to CD31. Representative images of the deep, intermediate, and superficial vascular layers are shown. Noted increases in vascular branching were observed in the intermediate and deep vessel layers. B . Differences in vessel branching were quantified in wildtype controls and Kctd7 −/− mice in each of the vascular layers by counting the number of vascular branch points following staining with CD31. Significant alterations were observed in the deep and intermediate layers. C-D . Kctd7 −/− intermediate and deep vessel layers showed an increase in the mean vessel covered area ( C ) and a decrease in lacunarity ( D ) relative to wildtype mice following automated segmentation, reconstruction, and quantification using Angiotool v0.6. n=10 wildtype and 9 Kctd7 −/− animals. Data are represented as the mean ± the s.e.m. Scale bars = 100μm. *** P<0.001, ** P<0.01, * P<0.05, 2way ANOVA and Dunnett’s multiple comparison test for significance.

Article Snippet: To examine the role of Kctd7 in retina vascular organization, we obtained Kctd7 -null animals from the International Mouse Phenotyping Consortium (IMPC, ).

Techniques: Control, Staining, Comparison

Journal: bioRxiv

Article Title: Progressive myoclonic epilepsy-associated gene Kctd7 regulates retinal neurovascular patterning and function

doi: 10.1101/647008

Figure Lengend Snippet: A. Schematic of vasculature development from P0 to P15. The primary superficial plexus begins to emerge at P0, grows laterally to the periphery, and reaches its full radial growth by P8. From day 7, capillaries ascend into the OPL to form the deep plexus, which then interdigitates this layer and is completed by P12. Capillaries from the deep layer then descend into the IPL to form the intermediate layer between P12 and P15. By P15 all three layers are present. B-C . To visualize vascular development, whole mount retinas from wildtype and Kctd7 −/− animals were stained for CD31 at P3, P8, and P12. In wildtype mice, radial development of the superficial layer is apparent at P3 and complete by P8. Vessels grow in complexity until P12 ( B , scale bars = 250μm), forming elaborate branching patterns ( C , scale bars = 100μm). In Kctd7 −/− animals, development of the nascent superficial layer is intact at P3 ( B-C ). At P8, however, Kctd7 −/− animals showed a significant reduction in blood vessel coverage but an increase in branching complexity ( D , E). n ≥ 6 wildtype and ≥ 5 Kctd7 −/− animals. Data are represented as the mean ± the s.e.m. *** P<0.001, ** P<0.01, * P<0.05, 2way ANOVA and Dunnett’s multiple comparison test for significance.

Article Snippet: To examine the role of Kctd7 in retina vascular organization, we obtained Kctd7 -null animals from the International Mouse Phenotyping Consortium (IMPC, ).

Techniques: Staining, Comparison

Journal: bioRxiv

Article Title: Progressive myoclonic epilepsy-associated gene Kctd7 regulates retinal neurovascular patterning and function

doi: 10.1101/647008

Figure Lengend Snippet: Vessel laminar targeting was visualized in wildtype (upper panel) and Kctd7 −/− mice (lower panel) at P3, P8, and P12 following generation of retinal cross sections and staining for the vessel marker CD31. The superficial layer could be observed beginning at P3 (yellow arrows), the deep layer at P8 (green arrows), and intermediate layer at P12 (cyan arrows). Occasional gaps in the deep layer were present in Kctd7 −/− mice at P8 (green *), consistent with a reduction in deep vessel complexity at this time (see ). However, no large-scale defects in layer restriction were observed in Kctd7 mutants. Scale bar = 100μm.

Article Snippet: To examine the role of Kctd7 in retina vascular organization, we obtained Kctd7 -null animals from the International Mouse Phenotyping Consortium (IMPC, ).

Techniques: Staining, Marker

Journal: bioRxiv

Article Title: Progressive myoclonic epilepsy-associated gene Kctd7 regulates retinal neurovascular patterning and function

doi: 10.1101/647008

Figure Lengend Snippet: A-B. Whole mount retinas from wildtype and Kctd7 −/− animals were stained for CD31 at P3, P8, and P12. In wildtype animals, the deep layer has not emerged at P3 but becomes visible by P8 and forms a complex and patterned plexus. In Kctd7 −/− animals, the deep layer is only sparsely present at P8 ( A , scale bars = 250μm) and displays discontinuous vessel coverage and a reduction in growth across the retina ( B , scale bars = 100μm). C-D . The defects in Kctd7 −/− deep layer vessel development were accompanied by a marked decrease in the area of the retina covered by the deep plexus ( C ) and a significant reduction in Kctd7 −/− deep layer vessel branch number ( D) n=6 wildtype and 7 Kctd7 −/− animals. Data are represented as the mean ± the s.e.m. *** P<0.001, ** P<0.01, * P<0.05, 2way ANOVA and Dunnett’s multiple comparison test for significance.

Article Snippet: To examine the role of Kctd7 in retina vascular organization, we obtained Kctd7 -null animals from the International Mouse Phenotyping Consortium (IMPC, ).

Techniques: Staining, Comparison

Journal: bioRxiv

Article Title: Progressive myoclonic epilepsy-associated gene Kctd7 regulates retinal neurovascular patterning and function

doi: 10.1101/647008

Figure Lengend Snippet: The presence and localization of Kctd7 and the pericyte vasculature marker Cspg4 were assayed in wildtype animals by florescent in situ hybridization at P3 ( A ), P8 ( B ), P12 ( C ), and 16 weeks ( D ). Consistent with the localization of retina vasculature, Cspg4 was present in bands that colocalized with the retina synaptic layers (magenta arrows). In contrast, Kctd7 was expressed broadly in retina neurons with an enrichment in the inner nuclear layer (white arrows) and did not display an expression pattern consistent with blood vessel localization. Scale bars = 50μm.

Article Snippet: To examine the role of Kctd7 in retina vascular organization, we obtained Kctd7 -null animals from the International Mouse Phenotyping Consortium (IMPC, ).

Techniques: Marker, In Situ Hybridization, Expressing

Journal: bioRxiv

Article Title: Progressive myoclonic epilepsy-associated gene Kctd7 regulates retinal neurovascular patterning and function

doi: 10.1101/647008

Figure Lengend Snippet: A . Representative schematics of the location of each major retina neuron type together with the vasculature network with which it is hypothesized to interact (yellow, RGCs; dark blue, horizontal cells; magenta, bipolars; cyan, amacrine cells; green, photoreceptors; red, blood vessels). B . Immunohistochemical images of each neuron type at P8 in wildtype and Kctd7 −/− mice: starburst amacrine cells (ChAT, cyan); retinal ganglion cells (Rbpms, yellow); horizontal cells (Calbindin, blue); cones (MCAR, green). C . Each neuron type was quantified at P8 in wildtype (n=5) and Kctd7 −/− mice (n=4) using antibodies specific for each population. No significant difference in the numbers of these neuron types were observed. D-E. Immunohistochemical images ( D ) and quantification ( E ) of bipolar cell nuclei following staining with Chx10 at P8, P12, and 16 weeks in wildtype and Kctd7 −/− mice. At P8, the number of bipolar cells was significantly increased (P=0.012), while bipolar cell numbers were not significantly altered at the other ages. F-G. The number of bipolar cells undergoing apoptosis was visualized ( F ) and quantified ( G ) in wildtype and Kctd7 −/− mice at P8 and P12 following co-staining for Chx10 and activated caspase 3. At P8, Kctd7 −/− bipolar cells showed a significant increase in the number of caspase positive nuclei relative to controls (P=0.0014, G ). Scale bars = 50μm. Data are represented as the mean ± the s.e.m. *** P<0.001, ** P<0.01, * P<0.05, unpaired two-tailed Student’s t test.

Article Snippet: To examine the role of Kctd7 in retina vascular organization, we obtained Kctd7 -null animals from the International Mouse Phenotyping Consortium (IMPC, ).

Techniques: Immunohistochemical staining, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Progressive myoclonic epilepsy-associated gene Kctd7 regulates retinal neurovascular patterning and function

doi: 10.1101/647008

Figure Lengend Snippet: Photopic and scotopic ERGs were recorded from 16-week-old wildtype (n=8) and Kctd7 mutant animals (n=7). A . Representative traces of 4 steps (0.01, 0.10, 1.00, and 5.00 cd*s/m 2 ) are shown of ERG recordings under scotopic conditions. B-C . Under scotopic conditions, ERG recordings show a significant reduction in the a-wave amplitude for flash intensities greater than 0.003 cd*s/m 2 ( B ) and in all b-wave amplitudes ( C ) relative to wildtype controls. D-E . Under photopic conditions ERG recordings trended lower for the a-wave but were not significantly different ( D ), while a significant reduction was observed in the b-wave at both 3.0 and 10.0 cd*s/m 2 ( E ). Data are represented as the mean ± the s.e.m. *** P<0.001, ** P<0.01, * P<0.05, unpaired t-test using the Holm-Sidak method to correct for multiple comparison.

Article Snippet: To examine the role of Kctd7 in retina vascular organization, we obtained Kctd7 -null animals from the International Mouse Phenotyping Consortium (IMPC, ).

Techniques: Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Employing single-stranded DNA donors for the high-throughput production of conditional knockout alleles in mice

doi: 10.1101/195651

Figure Lengend Snippet: Conditional KO ssODN (A) and lssODN (B) targeting attempts. Each donut chart represents the summation of each allele type for all F0 mice genotyped, by ODN donor. In the center of the chart is the total number of F0 mice genotyped. Percentages of each allele type of the total number of mice genotyped are listed on each segment. 2 loxP: Includes animals genotyped for both 5’ and 3’ loxP sites, irrespective of the presence of any additional alleles (e.g., animals with 5’ loxP, 3’ loxP and a null allele detected); Null Allele: Includes animals genotyped for a null allele, which may also have a single HDR and/or NHEJ indel event; Single HDR: Includes animals genotyped for a single HDR event with or without additional indel events; NHEJ indel event: Animals in which only indel alleles were observed. (A) Summary of genotypes identified from ssODN targeting attempts, and symmetric and asymmetric homology arm designs. (B) Summary of genotypes identified from 4 lssODN targeting attempts.

Article Snippet: The International Mouse Phenotyping Consortium (IMPC) and its NIH-funded Knockout Mouse Production and Phenotyping (KOMP 2 ) component is producing a null allele mouse line for every protein coding gene in the mouse genome and phenotyping each null allele line to elucidate gene function in human biology and disease ( ).

Techniques:

Journal: bioRxiv

Article Title: Employing single-stranded DNA donors for the high-throughput production of conditional knockout alleles in mice

doi: 10.1101/195651

Figure Lengend Snippet: Frequency distributions of (A) projects with conditional, null, or both allele types, and (B) projects with loxP in cis , in trans , or both, binned by loxP distance. Project results derived from genotyping information gathered from F0 (A) and F1 (B) mice. LoxP distance calculated from genomic distance between Cas9 cut site of each sgRNA. X-axis labels indicate the median value of each bin. Left Y-axis depicts the number of projects attempted for each loxP distance bin, while the right Y-axis depicts the targeting or transmission percentage for all projects within each loxP distance bin.

Article Snippet: The International Mouse Phenotyping Consortium (IMPC) and its NIH-funded Knockout Mouse Production and Phenotyping (KOMP 2 ) component is producing a null allele mouse line for every protein coding gene in the mouse genome and phenotyping each null allele line to elucidate gene function in human biology and disease ( ).

Techniques: Derivative Assay, Transmission Assay

Journal: bioRxiv

Article Title: Employing single-stranded DNA donors for the high-throughput production of conditional knockout alleles in mice

doi: 10.1101/195651

Figure Lengend Snippet: Pearson correlations for 5’ and 3’ loxP sites. Data plotted based on number of F0 mice with an HDR event (x-axis) vs. any evidence for a double-strand break generated at the respective sgRNA site, 5’ and 3’, such as a NHEJ indel, HDR event, or the formation of a null allele (y-axis).

Article Snippet: The International Mouse Phenotyping Consortium (IMPC) and its NIH-funded Knockout Mouse Production and Phenotyping (KOMP 2 ) component is producing a null allele mouse line for every protein coding gene in the mouse genome and phenotyping each null allele line to elucidate gene function in human biology and disease ( ).

Techniques: Generated

Journal: PLOS ONE

Article Title: Witch Nails ( Krt90 whnl ): A spontaneous mouse mutation affecting nail growth and development

doi: 10.1371/journal.pone.0277284

Figure Lengend Snippet: A-D: A single blot from 10% SDS-PAGE (reducing conditions) of protein extracts from haired dorsal skin (lanes 1S, 2S, 6S), left rear toes (lanes 1R, 2R, 6R) and left front toes (1F, 2F, 6F) from wildtype male mice (10 μg total protein) and positive control equine lamellar tissue (Eq; 1 μg total protein) protein extracts. Approximate locations of proteins, based on predicted relative molecular mass (M r ), indicated to the right. M r of protein standards indicated to the left. Representative images are shown from four experiments using wildtype mouse male (N = 6) and female (N = 6) dorsal skin and toe samples. A) Immunoblotting performed using the K124C monoclonal antibody against a C-terminal peptide (1:5,000) as previously described . A single band at approximately 54 kDa was detected in the positive control (Eq) and a doublet was detected at approximately 58 kDa, the predicted M r of murine KRT90 (possibly due to post-translational modification or alternate splicing) in the toe lanes, but not the dorsal skin lanes. There was non-specific detection of an approximately 50 kDa M r band in all mouse protein lanes. This blot was then stripped for 15 min at room temperature using the Restore™ Western Blot Stripping Buffer (Thermo Scientific: Rockford, IL) and re-probed with a mouse anti-human KRT14 monoclonal antibody (1:500; clone LL002, Abcam Inc., Cambridge, UK) and mouse anti-β-actin-HRP mAb (1:15,000, clone AC-15; Sigma-Aldrich: St Louis, MO) as previously described . The equine KRT124 band was still visible at M r 54 kDa due to incomplete stripping and the KRT14 band was at M r 50 kDa and the β-actin band was at approximately M r 42 kDa in both the mouse and equine lanes. The mouse KRT14 bands appeared to align with the non-specific 50 kDa band in (A) , consistent with some cross-reactivity of the equine KRT124C antibody with another murine keratin under these conditions . C) Following immunoblotting, the blot was stained for protein content using the amido black stain (Sigma-Aldrich). The major protein bands aligned with some of the detected keratin bands, as expected due to the abundance of keratin in skin and nail unit epidermal tissues. D) Prior to any immunoblotting, the blot was also probed with the secondary antibody alone and the chemiluminescence exposure was double that shown in (A) . Mouse 6 appeared to have immunoreactivity to immunoglobulin in the samples and faint background staining is present in the other lanes, consistent with some cross-reactivity of the secondary antibody with the most abundant proteins.

Article Snippet: It is interesting to note that the Krt90 em1(IMPC)J null mutant produced in the International Mouse Phenotyping consortium (IMPC) large scale mutagenesis program website did not report any significant limb, digit, or tail lesions ( https://www.mousephenotype.org/ , 17 Feb 2021).

Techniques: SDS Page, Positive Control, Western Blot, Modification, Stripping, Stripping Membranes, Staining

Journal: PLOS ONE

Article Title: Witch Nails ( Krt90 whnl ): A spontaneous mouse mutation affecting nail growth and development

doi: 10.1371/journal.pone.0277284

Figure Lengend Snippet: (A) Whole genome sequencing identified a 7-bp insertion in exon 9 of Krt90 (formerly the Riken cDNA 4732456N10Rik gene) resulting in a serine to arginine change at position 476 of the mature protein followed by a frame shift and insertion of 36 novel amino acids and a premature stop codon (left panel). (B) Amino acid sequences of mutant (MUT) and corresponding wild type (WT) C-terminal regions. Residues indicated in color are R (arginine, red), G (glycine, green), and S (serine, blue). (C) Peptide coverage of the mutant and wild type sequences in proteomic analysis, which differ in C-terminal residues (476–510 or 476–538). Yields of the C-terminal regions differed significantly by t-test (†, p = 0.04).

Article Snippet: It is interesting to note that the Krt90 em1(IMPC)J null mutant produced in the International Mouse Phenotyping consortium (IMPC) large scale mutagenesis program website did not report any significant limb, digit, or tail lesions ( https://www.mousephenotype.org/ , 17 Feb 2021).

Techniques: Sequencing, Mutagenesis

Journal: PLOS ONE

Article Title: Witch Nails ( Krt90 whnl ): A spontaneous mouse mutation affecting nail growth and development

doi: 10.1371/journal.pone.0277284

Figure Lengend Snippet: Normal wildtype ( Krt90 +/+ ; A) nails compared to the long, curved, mutant ( Krt90 whnl/whnl ; B) nails.

Article Snippet: It is interesting to note that the Krt90 em1(IMPC)J null mutant produced in the International Mouse Phenotyping consortium (IMPC) large scale mutagenesis program website did not report any significant limb, digit, or tail lesions ( https://www.mousephenotype.org/ , 17 Feb 2021).

Techniques: Mutagenesis

Journal: PLOS ONE

Article Title: Witch Nails ( Krt90 whnl ): A spontaneous mouse mutation affecting nail growth and development

doi: 10.1371/journal.pone.0277284

Figure Lengend Snippet: The wildtype control had a well-formed normal nail plate (NP, A) that covered the nail bed (NB, boxed, B) and matrix (M). By contrast, the nail unit of a 4-week old (C, D) and 24-week old (E, F) Krt90 whnl/whnl mouse had various degrees of nail dystrophy (disorganization of the cells of the nail bed) and premature cornification.

Article Snippet: It is interesting to note that the Krt90 em1(IMPC)J null mutant produced in the International Mouse Phenotyping consortium (IMPC) large scale mutagenesis program website did not report any significant limb, digit, or tail lesions ( https://www.mousephenotype.org/ , 17 Feb 2021).

Techniques: Control

Journal: PLOS ONE

Article Title: Witch Nails ( Krt90 whnl ): A spontaneous mouse mutation affecting nail growth and development

doi: 10.1371/journal.pone.0277284

Figure Lengend Snippet: A 6-week old male Krt90 whnl/whnl mutant mouse nail section (A) had separation at the dermal-epidermal junction of the nail bed (C) starting at the matrix (B). Premature cornification (*) of the hyponychium extending under the nail bed caused separation of the nail plate from the nail bed (D). Note the abnormal location of the stratum granulosum deep under the nail plate (D, *).

Article Snippet: It is interesting to note that the Krt90 em1(IMPC)J null mutant produced in the International Mouse Phenotyping consortium (IMPC) large scale mutagenesis program website did not report any significant limb, digit, or tail lesions ( https://www.mousephenotype.org/ , 17 Feb 2021).

Techniques: Mutagenesis

Journal: PLOS ONE

Article Title: Witch Nails ( Krt90 whnl ): A spontaneous mouse mutation affecting nail growth and development

doi: 10.1371/journal.pone.0277284

Figure Lengend Snippet: Mouse specific keratin 14 (KRT14) labeled normal mouse nail matrix, nail bed, and hyponychial basal equivalent cells. Those in the Krt90 whnl/whnl mutant mice had intermittent labeling of cells in the nail bed corresponding to dystrophy and premature cornification. Keratin 1 (KRT1) labeled epidermal suprabasal cells not the nail matrix or nail bed. By contrast, the hyponychium was labeled far back under the nail plate indicating abnormal differentiation. Keratin 17 (KRT17) labeled the hyponychium and part of the matrix. In the Krt90 whnl/whnl mutant mouse, the hyponychium was hyperplastic and extended under the nail bed.

Article Snippet: It is interesting to note that the Krt90 em1(IMPC)J null mutant produced in the International Mouse Phenotyping consortium (IMPC) large scale mutagenesis program website did not report any significant limb, digit, or tail lesions ( https://www.mousephenotype.org/ , 17 Feb 2021).

Techniques: Labeling, Mutagenesis

Journal: PLOS ONE

Article Title: Witch Nails ( Krt90 whnl ): A spontaneous mouse mutation affecting nail growth and development

doi: 10.1371/journal.pone.0277284

Figure Lengend Snippet: A monoclonal antibody directed at equine KRT124 (ortholog of mouse KRT90) labeled the secondary epidermal lamellae in a horse hoof (nail bed equivalent, A). The same antibody labeled the nail bed and hyponychium (B-D) of a normal mouse nail unit, the same cells affected in the Krt90 whnl/whnl mutant mouse.

Article Snippet: It is interesting to note that the Krt90 em1(IMPC)J null mutant produced in the International Mouse Phenotyping consortium (IMPC) large scale mutagenesis program website did not report any significant limb, digit, or tail lesions ( https://www.mousephenotype.org/ , 17 Feb 2021).

Techniques: Labeling, Mutagenesis

Journal: Human Molecular Genetics

Article Title: Alzheimer’s disease risk gene CD2AP is a dose-sensitive determinant of synaptic structure and plasticity

doi: 10.1093/hmg/ddae115

Figure Lengend Snippet: Cd2ap is neuronally expressed and localizes to synapses in hippocampus and cortex. (A) Cd2ap is expressed ubiquitously in the adult mouse brain (coronal slice at bregma ~ −1.58 mm), including in the hippocampus cornu ammonis (CA) and dentate gyrus (DG) and in cortex. Other areas of Cd2ap expression include thalamus [ventral posteromedial nucleus (VPM) labeled], habenula [medial (MH) and lateral (LH) habenula indicated], and amygdala [basolateral amygdala (BLA) and basomedial amygdala (BMA) indicated]. Other abbreviations: RSA = retrosplenial agranular cortex, RSG = retrosplenial granular cortex, M1 = primary motor cortex, M2 = supplementary motor cortex, S1 = primary somatosensory cortex, S2 = second somatosensory cortex, AuD = auditory cortex, TeA = temporal cortex, Ect = ectorhinal cortex, PRh = perirhinal cortex, Ent = entorhinal cortical area, Pir = piriform cortex. (B) CD2AP cortical staining with the anti-CD2AP antibody (green) in wildtype controls is mostly abolished in animal homozygous for the germline knockout allele ( Cd2ap −/− ). Nuclei are counterstained with DAPI (blue). Representative images from 5-week-old mouse brain slices stained. See also . (C) Cd2ap is expressed in neurons. Mouse brain slices were co-stained for CD2AP (green) and neuronal markers, including MAP2 (neuronal perikarya and dendrites; magenta) and NeuN (neuronal nuclei; red). Hippocampal CA1 region (indicated by dashed rectangle) shown at higher power (right). See for additional images and for complementary studies of cultured neurons. (D) CD2AP (green) shows overlapping localization with the neuronal markers MAP2 (magenta) and NeuN (red) in the hippocampus CA1 region and the cortex. Colocalization indicated by white or yellow, for CD2AP staining overlapping with MAP2 or NeuN, respectively (arrowheads). (E) CD2AP shows overlapping localization with the pre-synaptic marker, Synapsin (red) but not the post-synaptic marker, PSD95 (magenta) in the CA1 and the cortex. Colocalization of CD2AP with Synapsin is indicated by yellow (arrowheads). See also for additional images and for complementary studies of cultured neurons.

Article Snippet: Mice heterozygous for a germline null allele ( Cd2ap +/− ) have previously been evaluated by the International Mouse Phenotyping Consortium [ , ].

Techniques: Expressing, Labeling, Staining, Knock-Out, Cell Culture, Marker

Journal: Human Molecular Genetics

Article Title: Alzheimer’s disease risk gene CD2AP is a dose-sensitive determinant of synaptic structure and plasticity

doi: 10.1093/hmg/ddae115

Figure Lengend Snippet: Loss of Cd2ap disrupts neuronal proteostasis. (A) CRISPR-mediated knockout (KO) of Cd2ap in mouse primary Cas9 neurons results in increased expression of Synapsin and PSD95, and decreased expression of CD2AP compared to control neurons transfected with AAV expressing guide sequences targeting LacZ . Statistical analysis was based on t-tests with sample sizes N = 3 per genotype. Western blots were performed on homogenates from DIV21 neurons. Protein expression was normalized against tubulin expression. * P < 0.05; * * * * P < 0.0001. All error bars denote mean ± SEM. (B) Cd2ap KO primary neurons show decreased activity of the ubiquitin proteasome system. Statistical analysis based on t-test with sample size N = 10 per genotype. Proteasome activity assay was completed on homogenates from DIV21 neurons. * * P < 0.01. Error bar denotes mean ± SEM.

Article Snippet: Mice heterozygous for a germline null allele ( Cd2ap +/− ) have previously been evaluated by the International Mouse Phenotyping Consortium [ , ].

Techniques: CRISPR, Knock-Out, Expressing, Control, Transfection, Western Blot, Activity Assay, Ubiquitin Proteomics

Journal: Human Molecular Genetics

Article Title: Alzheimer’s disease risk gene CD2AP is a dose-sensitive determinant of synaptic structure and plasticity

doi: 10.1093/hmg/ddae115

Figure Lengend Snippet: Loss of Cd2ap disrupts neuronal and synaptic morphology. (A) CRISPR-mediated knockout (KO) of Cd2ap in mouse primary Cas9 neurons results in increased dendritic spine density and fewer dendritic branch points at DIV21. Control Cas9 neurons were transfected with control AAV expressing guide sequences targeting LacZ . Statistical analysis of dendritic spines and branchpoints was based on t-tests, with sample size (N) = 20 and 19 cells for Cd2ap controls, respectively. Cd2ap KO primary neurons also show a decrease in the number of dendritic intersections based on Sholl analysis, based on statistical analysis using linear mixed-effects models with N = 18 or 15 cells for Cd2ap and controls, respectively. ns, non-significant; * * * P < 0.001; * * * * P < 0.0001. All error bars denote mean ± SEM. (B) Cd2ap KO neurons show increased density of mushroom spines and filopodia, but unchanged stubby or long thin spines, based on t-tests. Density calculated as number of spines per 10 μm of dendrite. N = 12 and 13 cells for Cd2ap or control, respectively. ns, non-significant; * P < 0.05; * * P < 0.01. All error bars denote mean ± SEM. (C) Representative CA1 hippocampal pyramidal neuron traces showing the analyzed apical and basal dendritic trees of 5–6-weeks-old wildtype control (WT) and Cd2ap −/− mice. (D) Cd2ap −/− mice have increased spine density on both main and oblique apical dendrites. Arrowheads indicate representative spines. By contrast, basal dendrites show decreased spine density in both Cd2ap −/− and Cd2ap +/− mice. Statistical analysis was based on t-tests. For apical dendrites, samples sizes (number cells quantified) were WT = 15 (4F and 11M); Cd2ap +/− = 18 (10F and 8M); and Cd2ap −/− = 11 (11M). For basal dendrites samples sizes were WT = 15 (4F and 11M); Cd2ap +/− = 16 (8F and 8M); and Cd2ap −/− = 16 (16M). Cells were counted from at least 2 independent animals per genotype. ns, non-significant; * P < 0.05; * * P < 0.01. All error bars denote mean ± SEM. See also for results of Sholl analyses. (E) Dendritic branching of basal but not apical dendrites is decreased in Cd2ap −/− mice. No change was observed in Cd2ap +/− heterozygotes for either apical or basal dendrites. Statistical analysis was based on t-tests, with N = 9 cells for each group. Sex distribution of the cells’ animals of origin was as follows: WT had 2F and 7M cells; Cd2ap +/− had 3F and 6M cells; and Cd2ap −/− had all M cells. ns, non-significant; * P < 0.05. All error bars denote mean ± SEM. The color of individual data points in panel (D) and (E) bar graphs indicates the sex of the animal of origin for each cell, with teal indicating male mice and lavender indicating female mice. Samples sizes (number of mice) used for analyses in (D) and (E) were WT = 4 (1F and 3M); Cd2ap +/− = 4 (2F and 2M); and Cd2ap −/− = 2 (2M). Given the unequal distribution of males and females in the control versus Cd2ap −/− groups in (D) and (E) we cannot exclude sex effects as a potential source of variability. See also panels A-B for the animal of origin distribution for all cells analyzed in D and E and a display of potential effects of animal-dependent variability.

Article Snippet: Mice heterozygous for a germline null allele ( Cd2ap +/− ) have previously been evaluated by the International Mouse Phenotyping Consortium [ , ].

Techniques: CRISPR, Knock-Out, Control, Transfection, Expressing

Journal: Human Molecular Genetics

Article Title: Alzheimer’s disease risk gene CD2AP is a dose-sensitive determinant of synaptic structure and plasticity

doi: 10.1093/hmg/ddae115

Figure Lengend Snippet: Cd2ap is required for short-term hippocampal plasticity. (A) Graphical representation of the setup for recording from Schaffer collaterals of the hippocampal CA1 region. (B) mEPSC frequency and amplitude are not significantly changed in Cd2ap homozygous or heterozygous animals ( P > 0.05; ns, non-significant), when compared with wildtype controls (WT). All electrophysiological recordings were performed in acute coronal brain slices from 5 to 8-week old animals (mean 6.7 weeks). Statistical analysis was performed using one-way ANOVA with Dunnett’s post-hoc test. For analysis of mEPSC frequency, sample sizes (N cells recorded) were as follows: WT = 10 (7 female and 3 male); Cd2ap +/− = 7 (4F and 3M); and Cd2ap −/− = 8 (8M). For analysis of mEPSC amplitude, sample sizes were as follows: WT = 12 (8F and 4M); Cd2ap +/− = 11 (8F and 3M); and Cd2ap −/− = 8 (8M). Samples sizes (number of mice) were WT = 7 (4F and 3M); Cd2ap +/− = 5 (3F and 2M); and Cd2ap −/− = 4 (4M). All error bars denote mean ± SEM. See also for additional neurophysiology examining basal membrane properties. (C) Representative traces from paired pulse facilitation (PPF) trials, recorded from the hippocampal CA1 Schaffer collaterals in WT, Cd2ap +/− , and Cd2ap −/− animals. The top trace recorded from each genotype is the excitatory post-synaptic potential (EPSP) response to the first PPF pulse, and the bottom, larger trace is the EPSP response to the second pulse. To obtain PPF, the slope (mV/ms) of the rising phase at the initial segment of each response is measured and PPF is calculated as the ratio of EPSP slopes (second pulse/first pulse). (D) PPF is increased in both Cd2ap homozygous and heterozygous animals, when compared with WT controls for the 50 ms inter-stimulus interval (ISI) trial. Statistical analysis was performed using two-way ANOVA with Holm-Šídák’s post-hoc test, with sample sizes (slices recorded): WT = 17 (3F and 14M); Cd2ap +/− = 17 (2F and 15M); and Cd2ap −/− = 9 (6F and 3M). Samples sizes (number of mice) were WT = 10 (1F and 9M); Cd2ap +/− = 9 (1F and 8M); and Cd2ap −/− = 5 (2F and 3M). * P < 0.05. All error bars denote mean ± SEM. (E) Bar graph shows increase in PPF in Cd2ap homozygous and heterozygous animals at 50 ms ISI. Teal data points are from recording on slices from male mice and lavender data points are from recording on slices from female mice. * P < 0.05. All error bars denote mean ± SEM. The color of individual data points in panel (B) and (E) bar graphs indicates the sex of the animal of origin for each cell, with teal indicating male mice and lavender indicating female mice. Given the unequal distribution of males and females in the control versus Cd2ap −/− groups in (B) and (E) we cannot exclude sex effects as a potential source of variability. See also panels C-D for the animal of origin distribution for all cells/slices analyzed in (B) and (E) and a display of potential effects of animal-dependent variability.

Article Snippet: Mice heterozygous for a germline null allele ( Cd2ap +/− ) have previously been evaluated by the International Mouse Phenotyping Consortium [ , ].

Techniques: Membrane, Control

Journal: Human Molecular Genetics

Article Title: Alzheimer’s disease risk gene CD2AP is a dose-sensitive determinant of synaptic structure and plasticity

doi: 10.1093/hmg/ddae115

Figure Lengend Snippet: Cd2ap is largely dispensable for cognitive and motor behaviors. Representative results are shown from behavioral assessments of Cd2ap conditional knockout animals (cKO; Cd2ap f/f ;Nes-Cre ) and controls ( CD2AP f/f ). Mice were examined at either 3.5 (A) or 12 months of age (B). Overall, no significant differences were detected (all P > 0.05), except for increased locomotor activity among 3.5-month-old Cd2ap conditional knockout mice during the first 10 min of the open field assay and decreased activity for 12-month-old Cd2ap cKO during the last 10 min of the open field assay. Statistical analysis was based on t-tests. For assessments of young mice, samples sizes of N = 23 cKO (13F and 10M) and 13 control (6F and 7M) mice were used for the elevated plus maze and hot plate tests; N = 24 cKO (14F and 10M) and 12 control (5F and 7M) mice for open field; and N = 19 cKO (with 10F and 9M) and 11 (with 5F and 6M) control mice were examined for novel object preference. For studies of aged mice, sample sizes were of N = 15 cKO (9F and 6M) and 22 control (10F and 12M) mice were used for the elevated plus maze tests; N = 15 cKO (with 9F and 6M) and 23 control (11F and 12M) mice for open field; N = 12 cKO (7F and 5M) and 16 control (7F and 9M) mice for novel object preference; and N = 16 cKO (9F and 7M) and 25 controls (10F and 15M) for hotplate testing. The color of individual data points indicates the sex of each animal, with teal indicating male mice and lavender indicating female mice. ns, non-significant; * P < 0.05; * * P < 0.01. All error bars indicate mean ± SEM. See also for results of additional assays.

Article Snippet: Mice heterozygous for a germline null allele ( Cd2ap +/− ) have previously been evaluated by the International Mouse Phenotyping Consortium [ , ].

Techniques: Knock-Out, Activity Assay, Control

Journal: Human Molecular Genetics

Article Title: Alzheimer’s disease risk gene CD2AP is a dose-sensitive determinant of synaptic structure and plasticity

doi: 10.1093/hmg/ddae115

Figure Lengend Snippet: Haploinsufficient requirement of Cd2ap for pairwise visual discrimination. (A) Schematic of the behavioral chamber set-up, including an illustration of the stimuli displayed on the touchscreen. (B) Experimental design and criteria for progressing through each training module, including “must touch,” “must initiate,” and “punish incorrect”, prior to pairwise discrimination testing. (C) Cd2ap +/− heterozygous mice (N = 18, 9F and 9M) showed impaired discrimination learning compared with wildtype controls (N = 21, 10F and 11M) when tested at 2.5 months of age, with fewer animals successfully reaching the pre-specified criteria for success by day 20, based on statistical analysis using the t-test. * P < 0.05. Among those animals that successfully pass the test, there was no significant difference in the time required achieve success (see ). See also for a breakdown of the N of mice that satisfied criteria for each module of the assay.

Article Snippet: Mice heterozygous for a germline null allele ( Cd2ap +/− ) have previously been evaluated by the International Mouse Phenotyping Consortium [ , ].

Techniques:

Journal: Human Molecular Genetics

Article Title: Alzheimer’s disease risk gene CD2AP is a dose-sensitive determinant of synaptic structure and plasticity

doi: 10.1093/hmg/ddae115

Figure Lengend Snippet: Cd2ap loss triggers protein signatures of altered proteostasis, lipid dysmetabolism, and synaptic dysfunction. (A) At 5-weeks of age, Cd2ap homozygous and heterozygous animals have significantly overlapping hippocampal differential expression signatures, when compared with wildtype controls. Differentially expressed proteins were P < 0.05, and overlap for directionally concordant changes was based on Fisher’s exact test ( P = 1.58 × 10 −26 ). 130 out of 133 overlapping, differentially expressed proteins were concordant. One dissected hippocampus from 5 animals per genotype were profiled (N = 5 each for wildtype, Cd2ap −/− , and Cd2ap +/− ; with 2 female and 3 male wildtype, 3F and 2M Cd2ap −/− ; and 3F and 2M Cd2ap +/− ). See also and for full results. (B) Volcano plot highlighting results from differential expression analysis of Cd2ap −/− homozygous animals vs. wildtype controls. Significantly differentially expressed protein ( P < 0.05, dotted horizontal line) are indicated with dark gray). Proteins highlighted in black were consistently differentially expressed in Cd2ap +/− heterozygotes. The residual Cd2ap protein signal in the Cd2ap −/− germline null animal is likely attributable to background chemical noise, possibly stemming from tandem mass tag isotope impurity. See also and . (C) Gene ontology term enrichment analysis highlights significant pathways affected in Cd2ap −/− animals (Fisher’s exact test false discovery rate P < 0.05). Representative terms were selected from among the top results. Darker bars denote pathways that are consistent affected in Cd2ap +/− heterozygous animals. See also and for full results.

Article Snippet: Mice heterozygous for a germline null allele ( Cd2ap +/− ) have previously been evaluated by the International Mouse Phenotyping Consortium [ , ].

Techniques: Quantitative Proteomics