A cell cycle alteration precedes apoptosis of granule cell precursors in the weaver mouse cerebellum

Abstract

A missense mutation in the gene coding for the G-protein-activated inwardly rectifying potassium (GIRK) channel, GIRK2, is responsible for apoptosis in the external germinal layer (EGL) of the cerebellum and a nonapoptotic death of midbrain dopaminergic neurons in the weaver (wv) mouse. Failure of axonogenesis and migration are considered to be the primary consequences of GIRK2 channel malfunction in the cerebellum. We investigated whether a disruption of the cell cycle precedes the failure of migration and axonogenesis and leads to massive apoptosis. To this end, immunohistochemistry and immunoblotting for PCNA, Cdk4, cyclin D, cyclin A, and the Cdk inhibitor p27/kip1, as well as in situ end-labeling for apoptotic DNA fragmentation, were applied to cerebella of P7-P21+/+, wv/+, and wv/wv mice. In +/+ and wv/+ mice, the expression of cell cycle proteins was limited to the outer, premigratory zone of the EGL. Antibodies to p27, a marker of cell differentiation, gave a reverse staining pattern. Due to migration delay, patches of p27-positive cells persisted in the outer EGL in P21 wv/+ mice. On the contrary, marked cell cycle up-regulation and absence of p27 occurred throughout the EGL at all ages in wv/wv mice, indicating an inability to switch off the cell cycle. Mitotic index evaluation showed that cell cycle activation was unrelated to proliferative events. Cell cycle proteins were not expressed in the substantia nigra, suggesting that nonapoptotic death of mature dopaminergic neurons is not preceded by abortive cell cycle re-entry. Our data show that abnormalities of the cell cycle in wv/wv cerebellum represent a major and early consequence of GIRK2 channel malfunction and may strongly influence the susceptibility of EGL cells to apoptosis. These observations may help in understanding the pathogenesis of human neurological channelopathies.

Figure 1.

PCNA immunostaining in the EGL from +/+ (A, D, G), wv/+ (B, E, H), and wv/wv (C, F, I) mice at P7 (A-C), P14 (D-F), and P21 (G-I). Note the progressive dilution of labeling in migratory EGL cells in P7-P14 +/+ (A, D) and wv/+ (B, E) mice. In P21 +/+ mice (G), the EGL has disappeared, and no PCNA staining is seen in either molecular or granule cell layer. Patches of cells persist in the EGL of P21 wv/+ mice (H), but they have become PCNA-negative. On the contrary, EGL cells remain labeled at all ages in wv/wv mice (C, F, I). In addition to proliferating cells, strongly labeled apoptotic cells can be seen in A and C (arrows). All pictures were taken at 1000× except G (400×).

Figure 2.

Immunostaining for Cdk4 (A-C), cyclin D (D-F), and p27/kip1 (G-I) in +/+ (A, D, G), wv/+ (B, E, H), and wv/wv (C, F, I) mice at P7. The labeling pattern of Cdk4 and cyclin D is similar and corresponds to that of PCNA (see Figure 1, A ▶ -C). In particular, only premigratory EGL cells are labeled in +/+ (A, D) and wv/+ (B, E) mice, while migratory cells of the innermost EGL zone are no longer stained. On the contrary, diffuse staining of the whole EGL is present in wv/wv mice (C, F). Antibodies to p27 produce a reverse staining pattern, with migratory EGL cells in +/+ (G) and wv/+ (H) mice becoming progressively labeled. On the contrary, all EGL cells in wv/wv mouse remain p27-negative. All pictures were taken at 1000×.

Figure 3.

Immunostaining for cyclin A (A, B) and p27 (D, E) in P21 wv/+ (A, D) and wv/wv (B, E) mice. Note that patches of cells in the EGL have an opposite expression of cell cycle markers in the two genotypes. Absence of cyclin and presence of p27 in wv/+ cells indicates the postproliferative, differentiated nature of these cells, which are about to migrate from the EGL. On the contrary, persistence of cyclin expression and lack of p27 in wv/wv mouse indicates the proliferative, undifferentiated nature of EGL cells. C shows ISEL labeling in the EGL of P7 wv/wv mice. Note that most apoptotic cells are concentrated at the boundary with the Purkinje’s cell layer. F: Double immunostaining for tyrosine hydroxylase and PCNA in the substantia nigra of P14 wv/wv mice. PCNA strongly labels an apoptotic cell (arrow), while dopaminergic neurons are unstained. All pictures were taken at 1000×.

Figure 4.

Immunoblot analysis of PCNA (top) and p27/kip1 (bottom) protein expression in P7-P14 +/+ and wv/wv mice. PCNA is upregulated in P7 wv/wv mice and is still detectable at P14. Compared to PCNA, p27 is constantly more expressed in +/+ mice and becomes more evident in older mice.

Figure 5.

Evaluation of the mitotic index in the EGL of +/+, wv/+, and wv/wv mice between P7 and P21. No statistically significant difference was found between the three genotypes at any age. No value is given for P21 +/+ mice because the EGL is no longer represented.