Selenoprotein T Exerts an Essential Oxidoreductase Activity That Protects Dopaminergic Neurons in Mouse Models of Parkinson's Disease

Abstract

Aims: Oxidative stress is central to the pathogenesis of Parkinson's disease (PD), but the mechanisms involved in the control of this stress in dopaminergic cells are not fully understood. There is increasing evidence that selenoproteins play a central role in the control of redox homeostasis and cell defense, but the precise contribution of members of this family of proteins during the course of neurodegenerative diseases is still elusive.

Results: We demonstrated first that selenoprotein T (SelT) whose gene disruption is lethal during embryogenesis, exerts a potent oxidoreductase activity. In the SH-SY5Y cell model of dopaminergic neurons, both silencing and overexpression of SelT affected oxidative stress and cell survival. Treatment with PD-inducing neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or rotenone triggered SelT expression in the nigrostriatal pathway of wild-type mice, but provoked rapid and severe parkinsonian-like motor defects in conditional brain SelT-deficient mice. This motor impairment was associated with marked oxidative stress and neurodegeneration and decreased tyrosine hydroxylase activity and dopamine levels in the nigrostriatal system. Finally, in PD patients, we report that SelT is tremendously increased in the caudate putamen tissue.

Innovation: These results reveal the activity of a novel selenoprotein enzyme that protects dopaminergic neurons against oxidative stress and prevents early and severe movement impairment in animal models of PD.

Conclusions: Our findings indicate that selenoproteins such as SelT play a crucial role in the protection of dopaminergic neurons against oxidative stress and cell death, providing insight into the molecular underpinnings of this stress in PD.

FIG. 1.

Production and enzymatic activity of recombinant SelT. (A) Alignment of SelW and SelT amino acid sequences showing several similarities in secondary structures between the two proteins and the presence of several helical structures, including a hydrophobic domain, which is found in SelT, but not SelW. The latter domain (shown below the SelT sequence) was deleted from SelT by site-directed mutagenesis to allow the production and purification of recombinant SelT. (B) Recombinant SelT was produced in BL21 bacterial cells after induction by IPTG, purified on nickel affinity columns, and analyzed by Coomassie blue staining or Western blot using anti-SelT or anti-HisTag antibodies. (C) The activity of recombinant SelT (20 μg) with a Cys (C) instead of the Sec (U) residue in the redox motif (SelT CVSU/C) was tested using a TrxR assay and compared with that of a liver native TrxR (20 μl of the sample supplied by the manufacturer) or SelT mutant (20 μg) where the Cys and Sec residues were replaced by Ser (S) residues (SelT C/SVSU/S) in the presence (20 μM) or absence of ATM. The fluorescence of the reduced substrate was measured at 412 nm. One representative experiment is shown of three different experiments with similar results. ATM, aurothiomalate; IPTG, isopropyl β-D-1-thiogalactopyranoside; Sec, selenocysteine; SelT, selenoprotein T; TrxR, thioredoxin reductase. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

MPP⁺ stimulates SelT expression in SH-SY5Y cells. (A) SH-SY5Y cells were treated or not with MPP⁺ (50 μM) for 36 h, and SelT or TH immunoreactivity was analyzed by confocal microscopy. Scale bar: 50 μm. (B) Quantification of the immunoreactive signals of SelT and TH in control and MPP⁺-stimulated conditions. Data are expressed as mean ± SEM and are compared using Student's t-test, ***p < 0.001 (n = 3 per group). (C) Western blot analysis of SelT and TH levels in control and MPP⁺-stimulated conditions. (D) The data from the Western blot were quantified, presented as mean ± SEM, and are compared using Student's t-test, *p < 0.05 (n = 3 per group). (E) SelT mRNA levels were determined by quantitative PCR in control or MPP⁺-stimulated conditions, and the data are expressed as mean ± SEM and are compared using Student's t-test, *p < 0.05 (n = 3 per group). MPP⁺, 1-methyl-4-phenylpyridinium; PCR, polymerase chain reaction; SEM, standard error of the mean; TH, tyrosine hydroxylase. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 3.

SelT promotes survival and inhibits oxidative stress in SH-SY5Y cells. (A) Confocal microscopic images of SH-SY5Y cells that were transfected with the pCiG2 vector expressing SelT (pCiG2-SelT) or a mutant version where the Sec residue was replaced by the Ser residue (pCiG2-SelTm), or with the empty vector (pCiG2), and challenged by MPP⁺ (100 μM) for 36 h. Transfected cells are stained in green due to eGFP expression thanks to an IRES sequence present in the pCiG2 vector. Nuclei are stained in blue by DAPI. Scale bar: 50 μm. (B) Quantification of the number of eGFP-positive cells in control and MPP⁺-treated conditions. The data are expressed as mean ± SEM and are compared using Student's t-test, **p < 0.01 (n = 3 independent experiments with three determinations per condition in each experiment). (C) Transfected SH-SY5Y cells were treated or not with MPP⁺ (100 μM) and incubated with the DCFDA probe for 45 min. The fluorescence of ROS-oxidized DCF probe was measured. The data are expressed as mean ± SEM and are compared using Student's t-test, *p < 0.05 (n = 3 independent experiments with six determinations per condition in each experiment). (D) Photomicrographs of SH-SY5Y cells that were transduced with lentiviral vectors for sh-SelT or scrambled shRNA and treated or not with MPP⁺ (100 μM) for 24 h. Scale bar = 50 μm. (E) Cell viability was assessed after transduction with sh-SelT or scrambled shRNA and treatment with MPP⁺. The data are expressed as mean ± SEM and compared using Student's t-test, *p < 0.05 (n = 3 independent experiments with six determinations per condition in each experiment). (F) SH-SY5Y cells that were transduced with lentiviral vectors for sh-SelT or a scrambled shRNA and treated or not with MPP⁺ (100 μM) for 24 h were incubated with the DCFDA probe for 45 min, and the fluorescence of ROS-oxidized DCF probe was measured. The data are expressed as mean ± SEM and compared using Student's t-test, **p < 0.001 (n = 4 independent experiments with six determinations per condition in each experiment). (G) Western blot analysis of phosphoTH-Ser31 in SH-SY5Y cells that were transduced with lentiviral vectors for sh-SelT or a scrambled shRNA and treated or not with MPP⁺ (100 μM) for 24 h. Alpha-tubulin (α-Tub) signal was used to ensure equal protein loading. (H) Quantification of phosphoTH-Ser31 signal observed by Western blot after normalization by quantified α-Tub signal. The data are expressed as mean ± SEM and are compared using Student's t-test, *p < 0.05 (n = 2 independent experiments with four determinations per condition in each experiment). DAPI, 4,6-diamino-2-phenylindole; DCF, 2′,7′-dichlorofluorescein; ROS, reactive oxygen species. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

Induction of SelT expression in the nigrostriatal pathway following MPTP administration. (A) Exposure to MPTP (four doses of 15 mg/kg) provokes a progressive degeneration of dopaminergic neurons in the SNc and fibers in the striatum (Str) as revealed by TH immunolabeling of tissue sections at 2, 4, and 8 days post-treatment. Note that the cortex (Cox) region is devoid of labeling. (B) The number of TH-positive neurons in the SNc at the different times of treatment with MPTP (five sections from each animal were counted, n = 5 per animal group). (C) SelT and TH immunoreactivity in the SNc of control and MPTP-treated (8 days) mice. (D) SelT and TH immunoreactivity in the Str of control and MPTP-treated (8 days) mice. SelT was detected in dopaminergic fibers and astrocytes (Ast) in the Str. (E) SelT and GFAP immunoreactivity in the SNc of control and MPTP-treated mice at 2 (d2), 4 (d4), and 8 days (d8) post-treatment. (F) SelT and GFAP immunoreactivity in the Str of control and MPTP-treated mice at 2 (d2), 4 (d4), and 8 days (d8) post-treatment. The insets depict the colocalization of SelT with GFAP in Ast. Nuclei are stained in blue with DAPI. Similar results were obtained using several animals (n = 5 per animal group) in two different experiments. Scale bars = 50 μm. GFAP, glial fibrillary acidic protein; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; SNc, substantia nigra compacta. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 5.

Disruption of the SelT gene provokes early lethality and higher vulnerability to MPTP in the brain. (A) Schematic representation of WT, floxed, and mutant mouse SelT alleles showing the location of the LoxP sites introduced by homologous recombination to be able to delete exons 2–3, which contain the SelT redox center, including the Sec residue. The location of the primers used (arrows) and the size of the DNA fragments characteristic of the different alleles are also shown. (B) PCR analysis on tail DNA samples showing the different alleles (WT: wild-type; HT: heterozygous; KO: null) obtained after breeding of SelT^fl animals with a second mice strain expressing Cre recombinase under the control of the CMV promoter (CMV-Cre) or the nerve cell-specific promoter and enhancer of the rat nestin gene (Nes-Cre). (C) The percentage of pups with different genotypes in 240 born animals was determined after inbreeding of CMV-Cre/SelT^WT/fl (SelT^+/−). No homozygous mice for the SelT⁻ allele were found after total SelT gene knockout. (D) Western blot analysis of SelT expression in different tissues from Nes-Cre/SelT^fl/fl. α-Tub was used as an internal control to ensure equal protein loading. (E) Western blot analysis of the Str showing the very low expression of SelT and its induction in WT, but not Nes-Cre/SelT^fl/fl (KO) mice, following MPTP administration (15 mg/kg). α-Tub was used as an internal control to ensure equal protein loading. (F) Quantification of SelT signal observed by Western blot after normalization by quantified α-Tub signal. The data are expressed as mean ± SEM and are compared using Student's t-test, **p < 0.01 (n = 3). (G) Motor skills of WT and Nes-Cre/SelT^fl/fl (KO) mice were tested between 15 and 60 min after injection of the vehicle or MPTP (15 mg/kg). The forelimbs of the animals were placed on a horizontal bar, and the time spent on the bar was registered for each group. The data are expressed as mean ± SEM and are compared using Student's t-test, **p < 0.01 (n = 6–7 per animal group). (H) A Kaplan–Meier curve for animal survival in the two groups in control or MPTP-treated conditions. The data were compared between groups using the Mantel–Cox test. **p < 0.01 (n = 7–8 per animal group). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

Exposure to MPTP increases 3-NT levels and decreases active TH and dopamine levels in Nes-Cre/SelT^fl/fl. (A) 3-NT and TH immunolabeling was performed in the substantia nigra from Nes-Cre/SelT^fl/fl (KO) and WT mice after MPTP treatment (15 mg/kg, 2 h). Note that there is no 3-NT signal in nondopaminergic cells (arrows). Scale bar: 50 μm. (B) Quantification of the immunoreactive signals in the substantia nigra. The data are expressed as mean ± SEM and are compared using the Mann–Whitney U-test, **p < 0.01; ns, not significant (n = 3–4). (C) 3-NT and TH immunolabeling was performed in the Str from Nes-Cre/SelT^fl/fl (KO) and WT mice after MPTP treatment. Note that there is no 3-NT signal in nondopaminergic cells (arrows). Scale bar = 50 μm. (D) Quantification of the immunoreactive signals in the Str. The data are expressed as mean ± SEM and are compared using the Mann–Whitney U-test, *p < 0.05; ns, not significant (n = 3–4 per animal group). (E) Western blot analysis of phosphoTH-Ser31, phosphoTH-Ser40, and total TH in the Str of WT and Nes-Cre/SelT^fl/fl (KO) mice with or without MPTP treatment (15 mg/kg, 2 h). α-Tub signal was used to ensure equal protein loading. (F) Quantification of phosphoTH-Ser31 and phosphoTH-Ser40 signals observed by Western blot after normalization by quantified TH and α-Tub signals. The data are expressed as mean ± SEM and are compared using Student's t-test for phosphoTH-Ser31, ^§§§p < 0.001; ^§p < 0.05 (n = 3), and for phosphoTH-Ser40, ***p < 0.001; **p < 0.01; *p < 0.05 (n = 3). (G) Dopamine measurement in the Str. The data are expressed as mean ± SEM and are compared using the Mann–Whitney U-test, *p < 0.05 (n = 5–6). 3-NT, 3-nitrotyrosine. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 7.

Exposure to low doses of rotenone altered rearing behavior in Nes-Cre/SelT^fl/fl mice. (A) WT and Nes-Cre/SelT^fl/fl (KO) mice were injected with the vehicle or rotenone (ROT) (1.5 mg/kg), and a Kaplan–Meier curve for animal survival in control and ROT-treated conditions is shown. The data are compared between groups using the Mantel–Cox test (*p < 0.05, n = 4 per animal group). (B) Motor skills were tested after treatment with low doses of ROT (three successive doses of 0.25, 0.5, and 1 mg/kg over 3 weeks), using the rearing test. The data are expressed as percent ± SEM of initial rearing levels for each group and were compared using Student's t-test (*p < 0.05, **p < 0.01, n = 3–4 per animal group). (C) A Kaplan–Meier curve for animal survival in the different groups of animals. The data are compared between groups using the Mantel–Cox test (ns, not significant, n = 4 per animal group). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 8.

Exposure to rotenone increases dopaminergic neuron degeneration and decreases active TH and dopamine levels in Nes-Cre/SelT^fl/fl. (A) TH immunolabeling was performed in the SNc from WT and Nes-Cre/SelT^fl/fl (KO) mice in control and ROT-treated conditions (three successive doses of 0.25, 0.5, and 1 mg/kg over 3 weeks). Scale bar = 50 μm. (B) TH immunolabeling was performed in the Str from WT and Nes-Cre/SelT^fl/fl (KO) mice in control and ROT-treated conditions. Scale bar: 50 μm. (C) Dopaminergic neurons (TH⁺) were quantified after analysis of anti-TH immunohistochemistry in 5 midbrain serial sections of control and ROT-treated mice. (D) The total number of dopaminergic neurons (TH⁺) in five midbrain serial sections was compared between control and ROT-treated mice (*p < 0.01, n = 3–4). (E) Western blot analysis of total TH in the Str of WT and Nes-Cre/SelT^fl/fl (KO) mice with or without ROT treatment. α-Tub signal was used to ensure equal protein loading. (F) Western blot analysis of phosphoTH-Ser31 in the Str of WT and Nes-Cre/SelT^fl/fl (KO) mice with or without ROT treatment. (G) Nondopaminergic neurons, stained by DAPI, but not by TH antibody, were counted in the SNc of the different animal groups (five sections per animal, n = 3). (H) Quantification of TH (^§p < 0.05, n = 3) and phosphoTH-Ser31 (**p < 0.01, *p < 0.05; n = 3) signals observed by Western blot after normalization by quantified α-Tub signals. (I) Dopamine levels in the Str of WT and Nes-Cre/SelT^fl/fl (KO) mice with or without ROT treatment (**p < 0.01, *p < 0.05, n = 3–4). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 9.

SelT expression is increased in the caudate putamen (CPu) of PD patients. (A) SelT mRNA levels were measured by quantitative PCR in the mesencephalon (Mes), CPu, and subthalamic nucleus (STN) of control subjects and/or Parkinson's patients. Median values are indicated by the bars and are presented as percent of the control CPu value. (B) Schematic drawing of the CPu projections from the substantia nigra [adapted from Schultz (46)]. (C) Tissue sections at the level of the CPu as shown in (B) from control subjects or PD patients were labeled by TH or SelT antibodies and counterstained by hematoxylin and shown in the middle images. Regions indicated by squares in these images within the putamen (PUT) and caudate (CD) were magnified ×10, and the resulting images are shown on the left or the right, respectively. Scale bar = 50 μM. PD, Parkinson's disease. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars