germline null allele  (International Mouse Phenotyping Consortium)

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

Journal: bioRxiv

Article Title: MKK4 and MKK7 control degeneration of retinal ganglion cell somas and axons after glaucoma-relevant injury

doi: 10.1101/2024.09.27.614559

Figure Lengend Snippet: A. Longitudinal intraocular pressure (IOP) measurements from WT and Ddit3/Jun -/- eyes at 5, 9, 10.5, and 12 months of age. Both genotype groups had significantly elevated IOPs at 9M, 10.5M, and 12M compared to 5M ( P< 0.001, n≥ 60 for each comparison). At these timepoints, Ddit3/Jun -/- eyes did not have a statistically significant reduction in IOP compared to WT, although Ddit3/Jun -/- eyes had slightly higher IOPs at 9M of age compared to WT ( n≥ 60, * P= 0.004, Two-way ANOVA, Holm-Sidak’s post hoc ). B. Examples of optic nerve cross sections with no or early (noe) and severe (sev) glaucomatous damage from WT and Ddit3/Jun -/- mice and percentages of optic nerves with noe, moderate (mod) and sev glaucomatous damage in 12M WT and Ddit3/Jun -/- mice. Ddit3/Jun deletion did not afford protection to RGC axons and in fact slightly worsened axonal degeneration ( n≥ 54, P= 0.049, Chi square test). C. Representative retinal flat mounts immunoassayed for RBPMS and quantification of RBPMS+ cells from 12M WT and Ddit3/Jun -/- retinas with corresponding noe or sev optic nerves. Sev Ddit3/Jun -/- retinas had 77.0±3.1% improved RGC survival compared to WT controls. RBPMS+ cells/mm 2 ±SEM for WT and Ddit3/Jun -/- respectively: Noe: 2899.6±111.0, 2765.4±49.8; Sev: 226.7±20.8, 2151.5±83.0 ( n= 6, * P <0.001, Two-way ANOVA, Holm-Sidak post-hoc ). Scale bars, 50μm.

Article Snippet: Ddit3 null alleles ( ) (Jackson Laboratory, Stock# 005530), floxed alleles of Jun ( ) ( Jun fl ), and the Six3-cre transgene ( ) (Jackson Laboratory, Stock# 019755) were backcrossed >10 times to both the C57BL/6J genetic background (>99% C57BL/6J) and the DBA/2J background (>99% DBA/2J).

Techniques: Comparison

Journal: bioRxiv

Article Title: MKK4 and MKK7 control degeneration of retinal ganglion cell somas and axons after glaucoma-relevant injury

doi: 10.1101/2024.09.27.614559

Figure Lengend Snippet: A. Representative pattern electroretinography (PERG) traces and amplitude quantification ( B ) from Gpnmb + , WT, Ddit3 -/- , Jun -/- , and Ddit3/Jun -/- mice at 5, 9, and 12M. Normotensive Gpnmb + mice did not have significant decline in PERG amplitude at 9M (n≥21, P>0.05), but had a slight but significant decline in PERG amplitude by 12M compared to 5M (n≥21, P=0.007), Ocular hypertensive mice of all genotype groups had significant PERG amplitude decline at 9M and 12M compared to 5M (n≥34, P<0.001, two-way ANOVA, Holm-Sidak’s post hoc). At 9M and 12M, each ocular hypertensive group’s PERG amplitude was significantly lower than normotensive Gpnmb + controls (n≥20, P<0.001). No increase in PERG amplitude was observed between WT and Ddit3 -/- , Jun -/- , or Ddit3/Jun -/- groups at any timepoint measured. Scale bar: Y: 5μV, X: 100ms. C. Quantification of full-field ERG a-wave and b-wave ( D ) amplitudes. By 12M, each ocular hypertensive group had a slight but significant decline of electroretinography (ERG) a and b-wave amplitudes compared to 5M (n≥34, P<0.001, two-way ANOVA, Holm-Sidak’s post hoc), but not nearly to the same extent as PERG amplitude decline ( A ). Percentage of PERG and ERG amplitude declines at 9 and 12M are listed for each group in . E. PERG amplitude quantifications from 12M WT and Ddit3/Jun -/- retinas with noe and sev optic nerve damage. Neither genotype nor optic nerve damage level influenced PERG amplitude (n≥6, P >0.05, two-way ANOVA). Of note, PERG amplitudes were significantly reduced compared with 12M Gpnmb + ( n =21, 7.2±0.6) regardless of genotype or level of axonal damage. PERG amplitude (μV)±SEM: WT noe: 2.0±0.4, Ddit3/Jun -/- noe: 1.8±0.2, WT sev: 2.3±0.3, Ddit3/Jun -/- sev: 1.9±0.2 ( n≥ 6, P< 0.001, one-way ANOVA, Holm-Sidak’s post hoc ). Scale bar: Y: 5μV, X: 100ms. F. High-resolution images of retinal flat mounts immunoassayed for RBPMS (scale bar, 50μm) and quantification of average RGC soma size from WT and Ddit3/Jun -/- retinas with noe and sev glaucomatous damage. Both genotype groups had significant reductions in RGC soma size in sev glaucoma compared to respective noe controls (* P <0.001). While Ddit3/Jun -/- noe retinas had slightly smaller RGCs (* P =0.044), Ddit3/Jun deletion did not attenuate RGC soma shrinkage in sev retinas. Soma size (μm 2 )±SEM from WT and Ddit3/Jun -/- retinas, respectively: noe: 143.1±3.9, 131.7±2.9; sev: 87.6±2.5, 78.3±3.0 (n≥5, two-way ANOVA, Holm-Sidak’s post hoc ).

Article Snippet: Ddit3 null alleles ( ) (Jackson Laboratory, Stock# 005530), floxed alleles of Jun ( ) ( Jun fl ), and the Six3-cre transgene ( ) (Jackson Laboratory, Stock# 019755) were backcrossed >10 times to both the C57BL/6J genetic background (>99% C57BL/6J) and the DBA/2J background (>99% DBA/2J).

Techniques:

Journal: bioRxiv

Article Title: MKK4 and MKK7 control degeneration of retinal ganglion cell somas and axons after glaucoma-relevant injury

doi: 10.1101/2024.09.27.614559

Figure Lengend Snippet: A. Representative PERG traces and quantification of PERG amplitudes from WT, Ddit3/Jun -/- , and Mkk4/7 -/- retinas 14 days post-CONC or Sham procedures. WT and Ddit3/Jun -/- PERG amplitudes significantly declined 14 days post-CONC (by 52.6±5.7, * P <0.001 and 57.9±7.9µV, * P =0.002, respectively), while Mkk4/7 -/- PERG amplitudes did not significantly decline ( n≥ 17, two-way ANOVA, Holm-Sidak’s post-hoc .). Scale bar: Y: 5μV, X: 100ms. B. Representative high-resolution images of retinal flat mounts immunoassayed for RBPMS (scale bar, 50μm) and quantification of RGC soma size from WT, Ddit3/Jun -/- and Mkk4/7 -/- retinas 14 days post-Sham or CONC procedures. After CONC, WT and Ddit3/Jun -/- RGC soma sizes were reduced to 78.4±0.8% (* P =0.001) and 65.5±4.6% (* P <0.001) the size of respective RGCs after Sham procedures. Mkk4/7 -/- RGC soma sizes did not change after CONC compared to Sham ( P =0.586). Mkk4/7 -/- RGCs were significantly larger compared to WT (* P =0.006) and Ddit3/Jun -/- (* P <0.001) RGCs after CONC, and Ddit3/Jun -/- RGCs were significantly smaller than WT post-CONC (* P =0.002). Soma size (μm 2 )±SEM from WT, Ddit3/Jun -/- , and Mkk4/7 -/- mice, respectively: Sham: 154.5±2.8, 137.0±7.3, 155.8±3.2; CONC: 121.2±1.1, 89.7±6.3, 147.5±7.8 ( n ≥7, two-way ANOVA, Holm-Sidak’s post hoc ).

Article Snippet: Ddit3 null alleles ( ) (Jackson Laboratory, Stock# 005530), floxed alleles of Jun ( ) ( Jun fl ), and the Six3-cre transgene ( ) (Jackson Laboratory, Stock# 019755) were backcrossed >10 times to both the C57BL/6J genetic background (>99% C57BL/6J) and the DBA/2J background (>99% DBA/2J).

Techniques: