Thiamine deficiency induces endoplasmic reticulum stress in neurons

Abstract

Thiamine (vitamin B1) deficiency (TD) causes region selective neuronal loss in the brain; it has been used to model neurodegeneration that accompanies mild impairment of oxidative metabolism. The mechanisms for TD-induced neurodegeneration remain incompletely elucidated. Inhibition of protein glycosylation, perturbation of calcium homeostasis and reduction of disulfide bonds provoke the accumulation of unfolded proteins in the endoplasmic reticulum (ER), and cause ER stress. Recently, ER stress has been implicated in a number of neurodegenerative models. We demonstrated here that TD up-regulated several markers of ER stress, such as glucose-regulated protein (GRP) 78, growth arrest and DNA-damage inducible protein or C/EBP-homologus protein (GADD153/Chop), phosphorylation of eIF2alpha and cleavage of caspase-12 in the cerebellum and the thalamus of mice. Furthermore, ultrastructural analysis by electron microscopic study revealed an abnormality in ER structure. To establish an in vitro model of TD in neurons, we treated cultured cerebellar granule neurons (CGNs) with amprolium, a potent inhibitor of thiamine transport. Exposure to amprolium caused apoptosis and the generation of reactive oxygen species in CGNs. Similar to the observation in vivo, TD up-regulated markers for ER stress. Treatment of a selective inhibitor of caspase-12 significantly alleviated amprolium-induced death of CGNs. Thus, ER stress may play a role in TD-induced brain damage.

Fig. 1

TD-induced ER stress in the cerebellum. A. The mice were divided into control and TD groups (n = 5 for each group). TD was induced in mice as described under the Materials and Methods. At specified times after TD, the cerebellum was removed. For each independent experiment, brain samples collected from five animals at a specified time point were mixed. The expressions of GRP78, GADD153, phospho-eIF2α, and cleaved caspase-12 were determined by immunoblotting. Each data point was the mean of three independent experiments. B. The relative amounts of GRP78, GADD153, phospho-eIF2α and pro-/cleaved-caspase-12 were measured microdensitometrically and normalized to the expression of tubulin.

Fig. 1

TD-induced ER stress in the cerebellum. A. The mice were divided into control and TD groups (n = 5 for each group). TD was induced in mice as described under the Materials and Methods. At specified times after TD, the cerebellum was removed. For each independent experiment, brain samples collected from five animals at a specified time point were mixed. The expressions of GRP78, GADD153, phospho-eIF2α, and cleaved caspase-12 were determined by immunoblotting. Each data point was the mean of three independent experiments. B. The relative amounts of GRP78, GADD153, phospho-eIF2α and pro-/cleaved-caspase-12 were measured microdensitometrically and normalized to the expression of tubulin.

Fig. 2

TD-induced ER stress in the thalamus. Notations are as Fig. 1.

Fig. 2

TD-induced ER stress in the thalamus. Notations are as Fig. 1.

Fig. 3

TD-induced alterations in the ultrastructure of the ER in neurons. After TD for 8 days, the neurons in the submedial thalamus nuclei (SmTN) of mice were processed and examined under a transmission electron microscope as described under the Materials and Methods. Arrow heads indicate ER; arrow indicates an inclusion of medium electron density in the ER lumen. Bar = 455 nm

Fig. 4

Effect of TD on the viability of cultured CGNs. A. CGNs were treated with amprolium (Amp; 0, 0.5, 1, or 1.5 mM) for specified times, and the viability of cells was determined by MTT. Each data point represents the mean of five independent experiments. B. CGNs were treated with amprolium (Amp; 0 or 1.5 mM) for 4 or 7 days. Cultures were stained with DAPI for visualizing nuclear morphology (top panel); higher magnification shows condensed or fragmented nuclei following TD treatment (arrows), bar = 20 μm. Cells with condensed or fragmented nuclei were counted (bottom panel). Y-axis indicates the percentage of cells with condensed or fragmented nuclei. Each data point represents the mean of three independent experiments.

Fig. 4

Effect of TD on the viability of cultured CGNs. A. CGNs were treated with amprolium (Amp; 0, 0.5, 1, or 1.5 mM) for specified times, and the viability of cells was determined by MTT. Each data point represents the mean of five independent experiments. B. CGNs were treated with amprolium (Amp; 0 or 1.5 mM) for 4 or 7 days. Cultures were stained with DAPI for visualizing nuclear morphology (top panel); higher magnification shows condensed or fragmented nuclei following TD treatment (arrows), bar = 20 μm. Cells with condensed or fragmented nuclei were counted (bottom panel). Y-axis indicates the percentage of cells with condensed or fragmented nuclei. Each data point represents the mean of three independent experiments.

Fig. 5

TD-induced ROS production in cultured CGNs. CGNs were exposed to amprolium (Amp; 0 or 1.5 mM) for specified times. After the exposure, cells were labeled with CM-H₂DCFDA (10 μM) as described under the Materials and Methods. The percentage of ROS producing cells (positive for DCF fluorescence) was determined by flow cytometry. Each data point represents the mean of three independent experiments.

Fig. 6

TD-induced ER stress in primary cultures of CGNs. A. CGNs were treated with amprolium (Amp; 0 or 1.5 mM) for specified times. The expression of GRP78, cytoplasmic/ nuclear GADD153, phospho-eIF2α and caspase-12 was determined with immunoblotting. The expression of tubulin and histone H1o served as an internal loading control for cytoplasmic and nuclear protein, respectively. B. The relative amounts of these proteins were measured microdensitometrically and normalized to the expression of tubulin. TD-induced alterations were controls of matched time points. Each data point represents the mean of three independent experiments.

Fig. 6

TD-induced ER stress in primary cultures of CGNs. A. CGNs were treated with amprolium (Amp; 0 or 1.5 mM) for specified times. The expression of GRP78, cytoplasmic/ nuclear GADD153, phospho-eIF2α and caspase-12 was determined with immunoblotting. The expression of tubulin and histone H1o served as an internal loading control for cytoplasmic and nuclear protein, respectively. B. The relative amounts of these proteins were measured microdensitometrically and normalized to the expression of tubulin. TD-induced alterations were controls of matched time points. Each data point represents the mean of three independent experiments.

Fig. 7

Effect of TD on the expression and localization of GRP78 and GADD153 in cultured CGNs. A. After treating with amprolium (Amp, 0 or 1.5 mM) for 24 h, the expression of GRP78 (green) was examined with immunocytochemistry as described under the Materials and Methods. The nuclei (blue) were visualized with DAPI staining. B. After treating with amprolium (Amp; 0 or 1.5 mM) for 12 and 24 h, the expression of GADD153 (green) was examined with immunocytochemistry. DAPI staining was performed to visualize nuclei (blue). Bar = 10 μm.

Fig. 7

Effect of TD on the expression and localization of GRP78 and GADD153 in cultured CGNs. A. After treating with amprolium (Amp, 0 or 1.5 mM) for 24 h, the expression of GRP78 (green) was examined with immunocytochemistry as described under the Materials and Methods. The nuclei (blue) were visualized with DAPI staining. B. After treating with amprolium (Amp; 0 or 1.5 mM) for 12 and 24 h, the expression of GADD153 (green) was examined with immunocytochemistry. DAPI staining was performed to visualize nuclei (blue). Bar = 10 μm.

Fig. 8

. Effect of caspase-12 inhibitor on the viability of CGNs. CGNs were pretreated with a selective caspase-12 inhibitor (0 or 2 μM) for 30 min and exposed to amprolium (Amp; 0 or 1.5 mM) for 5 days. The viability of cells was determined by MTT assay. Each data point represents the mean of four independent experiments. ^# p < 0.05, statistically significant difference from untreated control groups. * p < 0.05, statistically significant difference from TD-treated groups.