Dab2IP GTPase Activating Protein Regulates Dendrite Development and Synapse Number in Cerebellum

<div><p>DOC-2/DAB-2 interacting protein (Dab2IP) is a GTPase activating protein that binds to Disabled-1, a cytosolic adapter protein involved in Reelin signaling and brain development. Dab2IP regulates PI3K-AKT signaling and is associated with metastatic prostate cancer, abdominal aortic aneurysms and coronary heart disease. To date, the physiological function of Dab2IP in the nervous system, where it is highly expressed, is relatively unknown. In this study, we generated a mouse model with a targeted disruption of <em>Dab2IP</em> using a retrovirus gene trap strategy. Unlike <em>reeler</em> mice, Dab2IP knock-down mice did not exhibit severe ataxia or cerebellar hypoplasia. However, Dab2IP deficiency produced a number of cerebellar abnormalities such as a delay in the development of Purkinje cell (PC) dendrites, a decrease in the parallel fiber synaptic marker VGluT1, and an increase in the climbing fiber synaptic marker VGluT2. These findings demonstrate for the first time that Dab2IP plays an important role in dendrite development and regulates the number of synapses in the cerebellum.</p> </div

Dab2IP expression in brain.

<p>(A) Immunohistochemical staining of sagittal brain section of P30 mouse using rabbit polyclonal antiserum specific to Dab2IP. Dab2IP is highly expressed throughout the brain. The distance of the sections from the midline of the cerebellum is ∼0.4 mm. (B) In the cerebellum, Dab2IP is expressed in granule cell layer, Purkinje cells bodies and dendrites and molecular layer. (C) Higher magnification of boxed area in B. GL, granule cell layer. ML, molecular layer. Scale bars: 250 µm (A), 100 µm (B), 25 µm (C).</p

Changes in parallel fiber and climbing fiber synaptic markers in Dab2IP KD mice.

<p>(A–D) Confocal images of VGluT1-labeled parallel fibers terminals (red) on PCs stained for Calbindin (green) in P30 control mice (A) or (C) Dab2IP KD littermates. B and D correspond to boxed areas in A and C. Single plane confocal images were used to determine the number of VGluT1 positive varicosities per 100 µm². (E) Quantitation of VGluT1 positive puncta in WT (N = 3, 360 observations) and Dab2IP KD (N = 3, 360 observations) littermates. **, p<0.01, student’s t-test. Scale bars: 50 µm (A, C), 5 µm (B, D). (F–K) Confocal images of VGluT2-labeled climbing fibers terminals (red) on PCs stained for Calbindin (green) in P30 WT (F–H) and Dab2IP KD littermates (I–K). Scale bars: 20 µm. (G, J) Quantitation of VGluT2 positive puncta in WT (N = 3, 171 observations) and Dab2IP KD (N = 3, 195 observations) along the entire length of the molecular layer (L) or in five equal segments from PC soma to the most distal part of the molecular layer (M). **, p<0.01, *, P<0.05, student’s t-test.</p

Cellular distribution of Dab2IP in P30 cerebellar Purkinje cells and granule cell layer.

<p>(A1–A3) Double immunofluorescent labeling of Dab2IP (green) and Calbindin (red) in Purkinje cell layer. (B1–B3) Double immunofluorescent labeling of Dab2IP (green) and GluR delta2 (red) in the molecular layer of the cerebellum. (C1–C3) Double immunofluorescent labeling of Dab2IP (green) and VGluT1 (red) in the granular layer of the cerebellum. (D1–D3) Double immunofluorescent labeling of Dab2IP (green) and VGluT2 (red) in the granular layer of the cerebellum. Scale bars: 5 µm.</p

Cellular distribution of Dab2IP in the molecular layer of P30 cerebellum.

<p>(A1–A4) Double immunofluorescent labeling of Dab2IP (green) and GFAP (red) in sagittal sections of P30 mouse cerebellum. (A4) Higher magnification of boxed region in A3. (B1–B4) Double fluorescent labeling of Dab2IP (green) and Parvalbumin (red) in sagittal sections of P30 mouse cerebellum. (B4) Higher magnification of boxed region in B3. C4 is a higher magnification of the boxed area in C3. GL, granule cell layer, PCL, Purkinje cell layer; ML, molecular layer. Scale bars: 50 µm (A1–A3, B1–B3), 10 µm (A4, B4).</p

Altered Serotonin, Dopamine and Norepinepherine Levels in 15q Duplication and Angelman Syndrome Mouse Models

<div><p>Childhood neurodevelopmental disorders like Angelman syndrome and autism may be the result of underlying defects in neuronal plasticity and ongoing problems with synaptic signaling. Some of these defects may be due to abnormal monoamine levels in different regions of the brain. <em>Ube3a</em>, a gene that causes Angelman syndrome (AS) when maternally deleted and is associated with autism when maternally duplicated has recently been shown to regulate monoamine synthesis in the <em>Drosophila</em> brain. Therefore, we examined monoamine levels in striatum, ventral midbrain, frontal cerebral cortex, cerebellar cortex and hippocampus in <em>Ube3a</em> deficient and <em>Ube3a</em> duplication animals. We found that serotonin (5HT), a monoamine affected in autism, was elevated in the striatum and cortex of AS mice. Dopamine levels were almost uniformly elevated compared to control littermates in the striatum, midbrain and frontal cortex regardless of genotype in <em>Ube3a</em> deficient and <em>Ube3a</em> duplication animals. In the duplication 15q autism mouse model, paternal but not maternal duplication animals showed a decrease in 5HT levels when compared to their wild type littermates, in accordance with previously published data. However, maternal duplication animals show no significant changes in 5HT levels throughout the brain. These abnormal monoamine levels could be responsible for many of the behavioral abnormalities observed in both AS and autism, but further investigation is required to determine if any of these changes are purely dependent on Ube3a levels in the brain.</p> </div

Dopamine (DA) levels are increased in striatum, midbrain and frontal cortex under conditions of increased and decreased <i>Ube3a</i> expression.

<p>A significant increase was detected in DA levels in <i>Ube3a</i> deficient mice in both striatum and frontal cortex (p≤0.05) and a clear trend was detected in midbrain that did not reach significance. Animals with a 7p duplication encompassing the <i>Ube3a</i> gene showed a significant increase in DA levels in all three regions when the duplication was paternally inherited, but only showed a significant increase in striatum and frontal cortex when maternally inherited, although a trend towards increased DA is detected here as well. In all cases, DA levels for each genotype were compared to wild type littermates from each cross (100%). Significant differences were found at p≤0.05 for all groups (n = 6). * p≤0.05 compared to wild type littermate.</p

Serotonin (5HT) levels are elevated in <i>Ube3a</i> deficient mice in the striatum and cortex and decreased in paternal 7p duplication animals.

<p>No significant change in 5HT was seen in <i>Ube3a</i> duplication animals in the striatum. Maternal 7p duplication animals did not show any significant changes in 5HT in either striatum or frontal cortex. In all cases, 5HT levels for each genotype were compared to wild type littermates from each cross (100%). Significant differences were found (p≤0.05 and n = 6) for all groups. *p≤0.05 compared to wild type littermate.</p

Delayed Purkinje cell dendrite outgrowth in Dab2IP KD mice.

<p>(A) Immunohistochemical staining of cerebellar Purkinje cells with anti-calbindin antibody at various postnatal ages (P5, P8 and P14). Scale bar: 50 µm. (B) Measurement of PC dendritic tree length on lobule IV/V in WT (N = 3, 210 observations) and Dab2IP KD (N = 3, 210 observations) littermates at P5, P8 and P14. Values are expressed as means ± S.D. *, p<0.05; **, p<0.01, student’s t-test.</p

Histological analysis of WT and Dab2IP KD cerebellum.

<p>(A–D) Nissl staining of sagittal sections of WT and <i>Dab2IP</i> KD cerebella at P0, P8, P14 and P30. (E) Immunostaining of Purkinje cells with anti-calbindin antibodies (green) in WT and <i>Dab2IP</i> KD P8 mice. (F–G) Higher magnification views of boxed areas in B and C showing the thickness of the molecular layer at P8 and P14, respectively. (H) Immunostaining of glial fibers using anti-GFAP antibodies in P30 WT and Dab2IP KD cerebella. EGL, external granule layer; ML, molecular layer; IGL, internal granule layer; Scale bars: 500 µm (A–D), 100 µm (E–H).</p