Neuronal selenoprotein expression is required for interneuron development and prevents seizures and neurodegeneration

Abstract

Cerebral selenium (Se) deficiency is associated with neurological phenotypes including seizures and ataxia. We wanted to define whether neurons require selenoprotein expression and which selenoproteins are most important, and explore the possible pathomechanism. Therefore, we abrogated the expression of all selenoproteins in neurons by genetic inactivation of the tRNA[Ser](Sec) gene. Cerebral expression of selenoproteins was significantly diminished in the mutants, and histological analysis revealed progressive neurodegeneration. Developing interneurons failed to specifically express parvalbumin (PV) in the mutants. Electrophysiological recordings, before overt cell death, showed normal excitatory transmission, but revealed spontaneous epileptiform activity consistent with seizures in the mutants. In developing cortical neuron cultures, the number of PV(+) neurons was reduced on combined Se and vitamin E deprivation, while other markers, such as calretinin (CR) and GAD67, remained unaffected. Because of the synergism between Se and vitamin E, we analyzed mice lacking neuronal expression of the Se-dependent enzyme glutathione peroxidase 4 (GPx4). Although the number of CR(+) interneurons remained normal in Gpx4-mutant mice, the number of PV(+) interneurons was reduced. Since these mice similarly exhibit seizures and ataxia, we conclude that GPx4 is a selenoenzyme modulating interneuron function and PV expression. Cerebral SE deficiency may thus act via reduced GPx4 expression.-Wirth, E. K., Conrad, M., Winterer, J., Wozny, C., Carlson, B. A., Roth, S., Schmitz, D., Bornkamm, G. W., Coppola, V., Tessarollo, L., Schomburg, L., Köhrle, J., Hatfield, D. L., Schweizer, U. Neuronal selenoprotein expression is required for interneuron development and prevents seizures and neurodegeneration.

Figure 1.

Abrogation of neuronal selenoprotein biosynthesis leads to neurodegeneration. A) Schematic representation of selenoprotein biosynthesis. Sec is inserted in response to UGA codons in selenoprotein mRNAs, which are characterized by a selenocysteine insertion sequence (SECIS) element in the 3′-UTR. Genetic deletion (KO) of Trsp prevents translation of functional selenoproteins such as GPx and Txnrd, as indicated by the slash over tRNA[Ser]^Sec. B) Lack of postural control and growth defect in P12 Tα1-Cre/Trsp^fl/fl mice. C) Cerebral activitiy of the prototype selenoenzyme cellular GPx in P9 Tα1-Cre/Trsp^fl/fl is significantly decreased (n=5–7). **P < 0.01; Student’s t test. D) Western blot analysis of selenoprotein expression in cerebral cortex. Selenoprotein expression is significantly reduced in homogenates from P12 Tα1-Cre/Trsp^fl/fl mice, suggesting that neurons are the predominant site of selenoprotein expression in the brain. Equal amounts of protein (50 μg) were applied to each lane (n=3–4 animals).

Figure 2.

Development of cortical interneurons is disrupted in Trsp-deficient brain. Expression of interneuron markers in somatosensory cortex in CamK-Cre/Trsp^fl/fl and control mice at P8, P11, and P15. A–D) Parvalbumin expression is not detected at any time point in Trsp-deficient mice (A), whereas other interneuron markers, such as somatostatin 14 (B), neuropeptide Y (C), and calretinin (D) appear normal. E, F) Quantitification of PV⁺ (E) and CR⁺ (F) neuronal profiles in somatosensory cortex, respectively. Solid bars, control; shaded bars, CamK-Cre/Trsp^fl/fl. **P < 0.01, ***P < 0.001; 2-sided Student’s t test. Scale bars = 100 μm (A–C); 200 μm (D).

Figure 3.

Spontaneous epileptiform activity in acute hippocampal slices in vitro. A) Unaltered input/output function of the CA1-fEPSP in Trsp mutant mice. Top panel: representative fEPSP traces recorded from 2 individual control and CamK-Cre/Trsp^fl/fl slices showing similarly increased responses to higher stimulation intensity applied in the stratum radiatum. Bottom panel; summary graph showing fEPSP slope vs. FV amplitude. B) Interictal discharges in hippocampal slices of CamK-Cre/Trsp^fl/fl mice. Representative traces of local field potentials in slices of control and Trsp mutant animals, recorded in stratum pyramidale of hippocampal CA1 in 10 μM carbachol (CCh). Bottom panel: magnification of representative traces. C) Summary plot of untreated and CCh treated slices from wild-type (n=10/group) and Trsp mutant animals (untreated, n=10; CCh treated, n=6).

Figure 4.

In vitro interneuron differentiation depends on Se and vitamin E (Vit E) in the culture medium. A) Representative images of PV⁺, GAD67⁺, and CR⁺ interneurons in cortical neuron culture at 17 d in culture. B) Fraction of PV⁺ cells among GAD67⁺ interneurons is reduced in cultures deprived of both Se and Vit E. C, D) Density of GAD67⁺ interneurons (C) and CR⁺ neurons (D) does not depend on Se and Vit E. Data are means of 3 independent cultures performed in triplicate. +, 83 nM selenite or 1 μg/ml each of α-tocopherol and α-tocopherol-acetate; −, exclusion of selenite or Vit E. **P < 0.01; ANOVA with Dunnett’s post hoc test.

Figure 5.

Neuron-specific Gpx4 inactivation resembles Trsp inactivation. A) Hippocampal cell death at P13 is limited to CA3 in CamK-Cre/Gpx4^fl/fl, but widespread inCamK-Cre/Trsp^fl/fl (arrows). B) Somatosensory cortex. C) Selective reduction of the density of parvalbumin-positive interneurons in CamK-Cre/Gpx4^fl/fl mice at P13. D) magnified view of C. E) Density and survival of CR⁺ interneurons is not reduced on genetic inactivation of Gpx4. F) Quantification of PV⁺and CR⁺interneurons in somatosensory cortex at P13. Solid bars, control; open bars, CamK-Cre/Gpx4^fl/fl. ***P < 0.001; 2-sided Student’s t test. Scale bars = 200 μm (A–C, E); 50 μm (D).