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Abnormal function of astroglia lacking Abr and Bcr RacGAPs

¹ Department of Pathology and Laboratory Medicine, Childrens Hospital Los Angeles Research Institute and Keck School of Medicine of the University of Southern California, 4650 Sunset Boulevard, Los Angeles, CA 90027, USA
² Division of Hematology and Oncology, Childrens Hospital Los Angeles Research Institute and Keck School of Medicine of the University of Southern California, 4650 Sunset Boulevard, Los Angeles, CA 90027, USA

View larger version (58K): [in a new window]   Fig. 1. Double knockout (Abr-/-;Bcr-/-) mice display abnormal cerebellar development. (A) Schematic drawing of the wild-type Abr locus, the targeting vector and the mutant allele. The targeting vector was constructed by inserting a PGK-neo-pA cassette into the NcoI site of exon 3. The hatched box represents the XhoI-HindIII fragment which was used as an external 3' probe. The predicted sizes of HindIII fragments hybridizing to this probe are shown above the wild-type locus and below the targeted locus. Restriction enzymes: H, HindIII; Bg, BglII; N, NcoI; B, BamHI; Xh, XhoI. (B-E) Hematoxylin and Eosin stained midsagittal sections through wild-type (B,D) and double null mutant cerebellum (C,E). Samples were taken at 11 days (B,C; insets at postnatal day 0) and 3 months (D-I) of age. Note abnormal foliation (E, asterisk) and the presence of persistent granule cells on the surface of the molecular layer (E, arrow). (F-I) The double knockout specimens display fusion of folia (F, arrow), abnormal foliation (G) in the anterior folia (III and IV) of the cerebellum, and presence of abnormal ectopic granule cells (G-H, arrows) when compared with age-matched wild-type controls. There was a rosette-like arrangement of the ectopic granule cells around an eosinophilic fibrillary core (I, arrow). Vermal folia are indicated with roman numerals, III-X. Anterior is towards the left of each photograph. Ce, cerebellum; IC, inferior colliculus. Scale bars: 1 mm in B-E; 300 µm in insets of B,C; 100 µm in D-H; 50 µm in I.

View larger version (81K): [in a new window]   Fig. 2. Abr and Bcr are expressed in the postnatal cerebellum. (A) Bcr and Abr protein levels were examined in the cerebellum at P1-15, in adult wild-type cerebellum (a1), in adult Abr-/-;Bcr-/- cerebellum (a2) and in primary cerebellar astrocytes isolated at P0. In total cerebellar extracts, two forms (96 kDa and 91 kDa) of Abr are present, whereas cultured astrocytes express only the smaller 91 kDa form. Null mutants lacked both 96 and 91 kDa forms but the antiserum used aspecifically reacts with proteins in that size range. (B-E) In situ hybridization of wild-type midsagittal sections of the cerebellum at the age of 8 days using Abr (B,D;, left panel) and Bcr (C,E, right panel) probes. (B,C) Anti-sense probes. (D,E) Sense probes used as a negative control. Arrows in B,C point to intensely stained Purkinje cells. Scale bars: 150 µm.

View larger version (45K): [in a new window]   Fig. 3. Lack of Purkinje and granule cell abnormalities in double null mutants. (A,B) Staining for calbindin does not reveal detectable differences in Purkinje cell dendritic trees at 3 months of age between control (A) and double knockout (B) samples. (C,D) Golgi staining shows that spines in double null mutant Purkinje cells are comparable with those of control Purkinje cells (arrows, C,D). Note that the Purkinje cells in A-D are from the antrior folia, which show the most prominent ectopic granule cell accumulation. (E-F) Calbindin staining of a double null mutant sagittal section (F) demonstrates the continuous Purkinje cell layer comparable with a control (E) at 2 weeks of age (arrows in E,F). Scale bars: 50 µm in A,B; 25 µm in C,D; 150 µm in E,F. (G) Double null mutant and control samples display comparable cell proliferation (n=3). Cultured vibratome sections were treated with BrdU and after 16 hours stained for the presence of BrdU-positive cells as described (Wechsler-Reya and Scott, 1999). (H) The IGL of folia III-V covers a comparable area both in controls and double null mutants (n=3). The area encompassing the IGL layers was analyzed using MetaMorphR software. (I) Double null mutant and control granule cell cultures contain similar comparable amounts of apoptotic cells (n=3). Cultures were maintained as described (Galli et al., 1995) and treated for 24 hours either with 25 mM KCl in the presence of serum (protected against apoptosis, blue columns) or with 5 mM KCl in the absence of serum (apoptosis induced, red columns). The number of apoptotic cells was analyzed using FACS as described (Spector et al., 1997).

View larger version (161K): [in a new window]   Fig. 4. Immature Bergmann glia are indistinguishable between double null mutants and controls at p1. Immunostaining using nestin (A,B) and RC2 (C,D) antibodies shows that immature double null mutant Bergmann protrusions (B,D) do not differ from immature control protrusions (A,C) at P1. Arrows point to positively staining protrusions. PS, pial surface; PC, Purkinje cell layer. Scale bars: 20 µm.

View larger version (96K): [in a new window]   Fig. 5. Abnormal Bergmann processes and aberrant astroglia in double null mutant cerebellar cortex after P5. (A-E) Immunostaining for GFAP at P5. (A,D) In controls, radial Bergmann processes form a well-defined cerebellar boundary (arrows). (B,C,E) In double mutants, Bergmann processes often extend beyond the pial surface (arrows in B,C), or they form thick end-feet processes without reaching the pial surface (E, also see inset). Moreover, in double null mutants, Bergmann processes often originate from cells that locate underneath the Golgi-cell/Purkinje-cell layer (arrowhead in E; the broken line indicates the position of the Purkinje cell layer). (F-I) Immunostaining for S100 at p5. Both control and null mutants show Bergmann processes and somata underneath the Purkinje cell layer (brown staining). (F,H) In controls, no positively staining somata are visible either in the intrafissural space (F, inset) or on the pial surface of the anterior lobes (H). (G,I) In double null mutants, positively staining somata are present in the intrafissural space and on the pial surface (arrows, G and inset). Occasionally, these S100-positive somata displayed long processes (arrow, I). PC, Purkinje cell layer; EGL, external germinal layer. GFAP and S100 were detected using a colorimetric immunoperoxidase reaction. Cells were counter-stained with Hematoxylin. Scale bars: 30 µm in A-E (15 µm in insets); 50 µm in F,G (25 µm in insets); 30 µm in H,I.

View larger version (125K): [in a new window]   Fig. 6. Confocal microscopy analysis shows disorganization of Bergmann fibers and long processes of abnormal GFAP-positive astroglia in double null mutants. (A,C) Wild types at P5 (A) and p8 (C) display well-organized Bergmann processes with an exclusively radial orientation and well-defined end-feet (arrows). (B,D) By contrast, Abr;Bcr double null mutant Bergmann processes show disorganization close to the pial surface both at P5 (B) and P8 (D). In addition, double null mutants show the presence of aberrant glial cells on the pial surface (arrowheads in insets) which display long thin processes similar to Bergmann processes (inset D, arrowhead). Arrows point to the pial surface. Immunostaining for GFAP and detection of FITC immunofluorescence (green) was performed using confocal microscopy. Purkinje cells were stained with calbindin and detected by TRITC immunofluorescence (red) in the images on the left. Scale bar: 30 µm A,C (15 µm inset); 50 µm B,D (25 µm inset).

View larger version (130K): [in a new window]   Fig. 7. Glial hypertrophy in the anterior cerebellum and midbrain of adult Abr;Bcr double null mutants. (A,B) In the anterior cerebellum of double null mutants, the ectopic granule cells are surrounded by numerous GFAP-positive glial cells (B), whereas in controls, a similar staining is absent (A). (C,D) Glial hypertrophy is also present in the midbrain of adult Abr;Bcr mutants (D) but not in controls (C). (E) Control midbrains show no detectable glial hypertrophy upon exposure to kainic acid. (F) Treatment of double null mutants with kainic acid results in massive gliosis in the midbrain. Ce, cerebellum; Sc, superior colliculus; KA, kainic acid. Scale bars: 100 µm in A,B; 50 µm in C-F.

View larger version (54K): [in a new window]   Fig. 8. Altered response to external stimuli in double null mutant cells. (A) Primary astrocytes were stimulated with EGF (25 ng/ml; upper panel) or LPS (1 µg/ml; lower panel) and analyzed for p38 MAPK phosphorylation using anti-phospho-p38 MAPK antibodies. The filters were subsequently stripped and probed with anti-p38 MAPK antibodies. The histograms to the right represent the relative quantitation of the scanned images. In the histograms, the wild-type (control) phospho-p38/p38 ratio without stimuli was arbitrarily set at 1 (upper histogram, average of two independent experiments; lower histogram, n=4, error bars indicate s.d.). Blue bars, wild-type samples; red bars, double null mutant samples. (B,C) Comparison of serum-starved and EGF-stimulated wild-type control and double null mutant primary astrocytes. Primary astrocytes from wild-type controls and double null mutants were serum-starved for 24 hours (left panels) or stimulated with EGF (25 ng/ml) for 24 hours (right panels). Astrocytes were identified by labeling for GFAP (red-Cy3), double stained for actin (green-FITC) and analyzed using confocal microscopy. Serum-starved null mutant astrocytes were more spread than controls. When stimulated with EGF, wild-type controls showed no morphological changes, whereas some Abr-/-;Bcr-/- astrocytes (arrows in C) responded with the projection of GFAP-positive protrusions. Scale bar: 20 µm.

View larger version (32K): [in a new window]   Fig. 9. Abrogation of Abr and Bcr causes increased levels of RacGTP. RacGTP was affinity-precipitated from lysates using GST-PAK (top panel). Levels of total Rac and RacGTP were evaluated using western blotting with anti-Rac antibodies. The lower panel represents the quantitation of scanned images. When compared with two wild-type controls, non-stimulated monocytes from two independent Abr-/-;Bcr-/- mutants showed approx. threefold higher levels of RacGTP.