Cerebellar hypoplasia in mice lacking selenoprotein biosynthesis in neurons

Abstract

Selenium exerts many, if not most, of its physiological functions as a selenocysteine moiety in proteins. Selenoproteins are involved in many biochemical processes including regulation of cellular redox state, calcium homeostasis, protein biosynthesis, and degradation. A neurodevelopmental syndrome called progressive cerebello-cortical atrophy (PCCA) is caused by mutations in the selenocysteine synthase gene, SEPSECS, demonstrating that selenoproteins are essential for human brain development. While we have shown that selenoproteins are required for correct hippocampal and cortical interneuron development, little is known about the functions of selenoproteins in the cerebellum. Therefore, we have abrogated neuronal selenoprotein biosynthesis by conditional deletion of the gene encoding selenocysteyl tRNA([Ser]Sec) (gene symbol Trsp). Enzymatic activity of cellular glutathione peroxidase and cytosolic thioredoxin reductase is reduced in cerebellar extracts from Trsp-mutant mice. These mice grow slowly and fail to gain postural control or to coordinate their movements. Histological analysis reveals marked cerebellar hypoplasia, associated with Purkinje cell death and decreased granule cell proliferation. Purkinje cell death occurs along parasagittal stripes as observed in other models of Purkinje cell loss. Neuron-specific inactivation of glutathione peroxidase 4 (Gpx4) used the same Cre driver phenocopies tRNA([Ser]Sec) mutants in several aspects: cerebellar hypoplasia, stripe-like Purkinje cell loss, and reduced granule cell proliferation. Parvalbumin-expressing GABAergic interneurons (stellate and/or basket cells) are virtually absent in tRNA([Ser]Sec)-mutant mice, while some remained in Gpx4-mutant mice. Our data show that selenoproteins are specifically required in postmitotic neurons of the developing cerebellum, thus providing a rational explanation for cerebellar hypoplasia as occurring in PCCA patients.

Fig. 1

Neuron-specific inactivation of selenoprotein expression. a Schematic drawing shows dependence of selenoprotein synthesis on selenocysteine tRNA. b Body weight in postnatal Tα1-Cre/Trsp ^fl/fl mice and controls. c Cytosolic glutathione peroxidase activity was determined with tert-butyl hydroperoxide as a substrate in P9 cerebella. d Cytosolic thioredoxin reductase activity was determined with the DTNB assay in P9 cerebella. n = 5–7, **p < 0.01; ***p < 0.001, Student’s t test

Fig. 2

Cerebellar hypoplasia and Purkinje cell loss in mice lacking neuronal selenoprotein expression. a Nissl staining reveals marked cerebellar hypoplasia and immature foliation pattern in Tα1-Cre/Trsp ^fl/fl mice on P12. The Purkinje cell layer is partially disrupted. Insets indicate stunted Purkinje cell dendrites and reduced external germinal layer (egl) thickness in Tα1-Cre/Trsp ^fl/fl mice. α-Calbindin brown. b Whole-mount immunohistochemistry α-calbindin reveals stripe-like Purkinje cell loss on P12. c Camera lucida representation of Purkinje cells. d Co-expression of zebrin II (red) and calbindin (green) on P11. e Left panel TUNEL staining shows apoptosis on P8. Cell death (arrows) occurs in the egl, during migration in the molecular layer, and in the internal granule cell layer (igl). Occasionally, TUNEL + Purkinje cells (PC) are identified based on the large size of their nuclei. Right panel quantification of TUNEL + cells per 0.35 mm² on P6 and P8. Diamonds indicate wild type and open circles mutant. f Left panel immunostaining for phosphorylated histone H3 (phospho H3+) as an indicator of proliferation. Mitosis in egl is clearly reduced in Tα1-Cre/Trsp ^fl/fl cerebellum on P8. Right panel quantification of pH3+ cells per 1-mm egl. Diamonds indicate wild type and open circles mutant. ***p < 0.001, Student’s t test. Scale bar = 50 μm

Fig. 3

Conditional inactivation of Gpx4 mimics Trsp deficiency. a Cerebellar hypoplasia and Purkinje cell loss in Gpx4-mutant mice on P12. Cresyl violet stain (blue) and α-calbindin (brown). Scale bars = 200 μm (upper panel) and 50 μm (lower panel). b Stripe-like loss (arrows) of Purkinje cells on P8. α-Calbindin (brown). c Few parvalbumin + cerebellar interneurons remain in Gpx4-mutant mice on P12. Purkinje cells are labeled yellow (parvalbumin is green, and calbindin is red), while interneurons express only parvalbumin (green)

Fig. 4

The density and orientation of radial glial fibers are not disrupted in Trsp-mutant cerebella on P12. a Staining of radial glia with an antibody against GFAP reveals intact pial contacts of radial glial fibers. b Double immunohistochemistry of calbindin and GFAP reveals that the radial glial network is not disrupted at sites of Purkinje cell loss