Neurodegeneration in Lurcher mice occurs via multiple cell death pathways

Abstract

Lurcher (Lc) is a gain-of-function mutation in the delta2 glutamate receptor (GRID2) that results in the cell-autonomous death of cerebellar Purkinje cells in heterozygous lurcher (+/Lc) mice. This in turn triggers the massive loss of afferent granule cells during the first few postnatal weeks. Evidence suggests that the death of Purkinje cells as a direct consequence of GRID2(Lc) activation and the secondary death of granule cells because of target deprivation occur by apoptosis. We have used mice carrying null mutations of both the Bax and p53 genes to examine the roles of these genes in cell loss in lurcher animals. The absence of Bax delayed Purkinje cell death in response to the GRID2(Lc) mutation and permanently rescued the secondary death of granule cells. In contrast, the p53 deletion had no effect on either cell death pathway. Our results demonstrate that target deprivation induces a Bax-dependent, p53-independent cell death response in cerebellar granule cells in vivo. In contrast, Bax plays a minor role in GRID2(Lc)-mediated Purkinje cell death.

Fig. 1.

Bax deletion reduces +/Lc cerebellar atrophy. Posterior view of the whole cerebellum lightly stained with cresyl violet. Note the larger lobules and more pronounced fissure of the +/Lc:Bax^−/− compared with the +/Lc:Bax^+/−cerebellum. Scale bar, 1 mm.

Fig. 2.

Bax deletion rescues granule cells from secondary cell death in +/Lc mice. Midsagittal cerebellar sections of postnatal day 30 mice from +/Lc:Bax^+/−crosses stained with cresyl violet. Note the large numbers of granule cells in the well lobulated cerebellum of +/Lc:Bax^−/−mice compared with the cell-sparse, atrophic lobules of +/Lc mice containing either one or both alleles of theBax gene. Scale bar, 1 mm.

Fig. 3.

Bax deletion partially rescues +/Lc Purkinje cells from cell-autonomous death. Calbindin-immunolabeled midsagittal cerebellar sections of postnatal day 30 mice from +/Lc:Bax^+/−crosses. a, Whole cerebellum view. Note the increased numbers of immunolabeled Purkinje cells in the posterior cerebellum of +/Lc:Bax^−/−compared with the +/Lc:Bax^+/−mice. b, Higher-powered view of the posterior cerebellum showing the stunted Purkinje cell morphology characteristic of theLc mutation in the +/Lc:Bax^+/−and +/Lc:Bax^−/−mice. Scale bars: a, 1 mm; b, 100 μm.

Fig. 4.

Quantification of granule cell and Purkinje cell survival in postnatal day 30 and postnatal day 300 (Aged) mice from +/Lc:Bax^+/−crosses. a,Bax deletion permanently rescues granule cells from target-related cell death in +/Lc mice. The number of granule cells per midsagittal cerebellar section was estimated from the area and cell density of the internal granule cell layer. b,Baxdeletion delays but does not prevent cell-autonomous Purkinje cell death in +/Lc mice. The number of Purkinje cells per midsagittal cerebellar section was counted from sections processed for calbindin immunocytochemistry. The data are presented as the mean value ± SEM (n = 3–5 mice).Asterisks denote statistically significant differences:a, *p < 0.000001; b, *p < 0.05 (unpaired two-tailed Student'st test).

Fig. 5.

The effect of Bax deletion on granule cell and Purkinje cell development in +/Lc mice.a, Midsagittal cerebellar sections of postnatal day 15 mice from +/Lc:Bax^+/−crosses stained with cresyl violet. Note the already increased cerebellar size and greater area and cell density of the internal granule cell layer in the +/Lc:Bax^−/−mice. b, Calbindin-immunolabeled midsagittal cerebellar sections of postnatal day 15 mice from +/Lc:Bax^+/−crosses. Gaps in the Purkinje cell layer reveal cell death in the developing +/Lc:Bax^+/−and +/Lc:Bax^−/−mice. Scale bar: a, b, 1 mm. c,Quantification of granule cell survival in postnatal day 15 mice from +/Lc:Bax^+/−crosses. The data are the mean estimated number of granule cells per midsagittal cerebellar section ± SEM (n = 3–5 mice). The asterisk indicates a statistically significant difference (p < 0.00001; unpaired two-tailed Student's t test).d, Quantification of Purkinje cell survival in postnatal day 15 mice from +/Lc:Bax^+/−crosses. The data are the mean number of calbindin-immunolabeled Purkinje cells per midsagittal cerebellar section ± SEM (n = 3–5 mice).