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Partial rescue of neural apoptosis in the Lurcher mutant mouse through elimination of tissue plasminogen activator

Program in Molecular and Cellular Pharmacology and Department of Pharmacological Sciences, University Medical Center at Stony Brook, NY 11794-8651

View larger version (70K): [in a new window]   Fig. 1. (A) tPA mRNA is expressed in both Purkinje cells (P) and granule cells (G) in mouse cerebellum during development. In situ hybridization was performed on wild-type mouse cerebellum at P12 using tPA antisense mRNA probes corresponding to either nucleotides 977-1409 of the tPA-coding region (shown), or 1890-2401 of the tPA 3' untranslated region (not shown). Identical patterns of expression were observed for the probes. No specific expression above background was detected when the corresponding sense probes were used. Scale bars: 100 µm (left); 20 µm (right). (B) Increased tPA activity in Lurcher cerebella at P12 but not P30. The amidolytic assay was performed as described in Materials and Methods. Notice that the level of tPA activity in the cerebella of Lc/+ at P12 was significantly higher than in wild-type mice, but no significant difference was detected at P30. The data are presented as mean±s.e.m. (n=4 or 5 mice). *P<0.05 by ANOVA.

View larger version (56K): [in a new window]   Fig. 2. Decreased neuronal apoptosis in Lc/+; tPA–/– cerebella at P12. Cellular apoptosis was detected using the TUNEL assay as described in the Materials and Methods. (A) TUNEL assay. Anti-fluorescein antibody conjugated with peroxidase (POD) was used to analyze the section under light microscope. Notice that more TUNEL-positive cells were detected in the Lc/+ cerebella than in the Lc/+; tPA–/– cerebella. Scale bar: 20 µm. (B) Quantification of apoptotic neurons in cerebellum of wild-type, Lc/+ and Lc/+; tPA–/– mice at P12. The data presented are mean±s.e.m. (n=6 mice). *P<0.05 by ANOVA.

View larger version (69K): [in a new window]   Fig. 3. Elimination of tPA delayed and partially rescued Purkinje cell death in Lurcher mice. (A) Immunostaining of sagittal cerebella sections of wild-type, tPA–/–, Lc/+, and Lc/+; tPA–/– mice at P12 and P30, using a monoclonal antibody raised against the Purkinje cell marker Calbindin D 28K. Note that the Purkinje cells in Lc/+; tPA–/– mice displayed a more branched dendritic tree when compared with Lc/+ mice. Scale bars: 100 µm (top); 20 µm (bottom). In tPA–/– mice at P12 and P30, the scale bars represent 100 µm and 50 µm, respectively. (B) Quantification of Purkinje cell survival in the cerebellum of wild-type, Lc/+, and Lc/+; tPA–/– mice at P12 and P30. Note the significantly lower number of Purkinje cells in both Lc/+ and Lc/+; tPA–/– mice when compared with the wild-type mice, but that Purkinje cell death was clearly attenuated in Lc/+ mice that lacked tPA. The data are presented as mean±s.e.m. (n=6 mice). *P<0.05 by ANOVA.

View larger version (118K): [in a new window]   Fig. 4. Secondary death of granule cells is decreased in Lurcher mice with tPA deficiency. Sagittal cerebella sections of wild-type, tPA–/–, Lc/+, and Lc/+; tPA–/– mice at P12 and P30 were stained with Cresyl Violet. At P12, both Lc/+; tPA–/– and wild-type cerebella showed normal lobular patterns, well-developed folia and densely stained granule layers. By contrast, the Lc/+ cerebella were greatly reduced in size, with poorly formed folia and sparse granule cells. By P30, although Lc/+; tPA–/– cerebella revealed atrophic folia, more granule cells were still detected there than in the Lc/+ cerebella. Scale bars: 100 µm (top); 20 µm (bottom).

View larger version (121K): [in a new window]   Fig. 5. Elimination of tPA reduces Jun phosphorylation in Lurcher mice. Double immunofluorescence [against calbindin (red) and JunP (green)] was used to evaluate apoptosis of Purkinje cells (see quantitative data in Fig. 6A,B). Almost all of the cells were doubly-labeled in Lc/+ mice at P12, note the severe shrinkage of Purkinje cell bodies. By P30, hardly any Purkinje cells remained in Lc/+ mice. In Lc/+;tPA–/– cerebella, there were few JunP-positive Purkinje cells at P12, and at P30 the Purkinje cells observed stained only with calbindin and not with JunP. Scale bar: 20 µm.

View larger version (36K): [in a new window]   Fig. 6. Caspase 8 activation is decreased in Lurcher mice that lack tPA. (A) Western blotting was performed for wild-type (wt), Lc/+, and Lc/+; tPA–/– mice at P12 and P30, using either rabbit polyclonal caspase 8 antibody (1:500 dilution, reacts with both the p18 subunit and precursor of caspase 8). (B) Quantification of caspase 8 activation by western blot analysis. The activated caspase 8-specific bands were quantitated using a fluorimager, as described in the Materials and Methods. Note that the decreased caspase 8 activation (18 kDa band) in the Lc/+; tPA–/– cerebellum was detected at both P12 and P30. The data presented are from four different experiments. (C) Coomassie Blue staining to show protein loading. *P<0.001 by ANOVA.

View larger version (25K): [in a new window]   Fig. 7. Proposed model for tPA involvement in the Lurcher apoptotic neuronal death pathway.

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